Stripping Voltammetric Methods For The Determination of Aflatoxin Compounds

STRIPPING VOLTAMMETRIC METHODS FOR
THE DETERMINATION OF AFLATOXIN COMPOUNDS
MOHAMAD HADZRI BIN YAACOB
UNIVERSITI TEKNOLOGI MALAYSIA

BAHAGIAN A – Pengesahan Kerjasama *
Adalah disahkan bahawa projek penyelidikan tesis ini telah dilaksanakan melalui
kerjasama antara _____________________ dengan _________________________
Disahkan oleh:
Tandatangan : .......................................................... Tarikh : ..........................
Nama : ..........................................................
Jawatan:...........................................................
(Cop rasmi)
* Jika penyediaan tesis/projek melibatkan kerjasama.
BAHAGIAN B – Untuk Kegunaan Pejabat Sekolah Pengajian Siswazah
Tesis ini telah diperiksa dan diakui oleh:
Nama dan Alamat

Pemeriksa Luar : Prof. Dr. Noor Azhar Bin Mohd Shazili
Pengarah
Institut Oseanografi,
Kolej Universiti Sains dan Teknologi Malaysia,
Mengabang Telipot
21030 Kuala Terengganu
Nama dan Alamat

Pemeriksa Dalam I : Prof. Madya Dr. Razali Bin Ismail
Fakulti Sains,
UTM, Skudai
Pemeriksa Dalam II :
Nama Penyelia Lain

(jika ada) :
Disahkan oleh Penolong Pendaftar di Sekolah Pengajian Siswazah:
Nama : .GANESAN A/L ANDIMUTHU

A thesis submitted in fulfilment of the

requirements for the award of the degree of
Doctor of Philosophy
Faculty of Science
Universiti Teknologi Malaysia
APRIL 2006
ii
iii
Specially dedicated to:
My mother, wife, sons, daughters and all families for

all the love, support and continuous prayer
for my success in completing this work.
iv
ACKNOWLEDGEMENT
All praise be to ALLAH SWT and blessing be upon His Prophet SAW whose
ultimate guidance creates a more meaningful purpose to this work.
I wish to express my sincere gratitude and appreciation to the people who have both
directly and indirectly contributed to this thesis. The following are those to whom I
am particularly indebted:
My supervisors A.P. Dr. Abdull Rahim bin Hj. Mohd. Yusoff and Prof. Dr.
Rahmalan Ahamad for their invaluable guidance, freedom of work and
constant encouragement throughout the course of this work.
School Of Health Sciences, USM, Health Campus, Kubang Krian Kelantan

for awarding study leave together with scholarship in completing the work.
Prof Baharuddin Saad from USM Penang, AP Dr. Razali Ismail from
Chemistry Department, Faculty of Science, UTM and Prof Barek from
Charles University, Prague, Czeck Republic for their useful discussion and
suggestion. Also to Mr Radwan Ismail from Department of Chemistry,
Penang Branch, Mrs Marpongahtun Misni and Mr Wan Kamaruzaman Wan
Ahmad for their friendship, ideas and continuous support in carrying out this
work. Also to Mr Mat Yasin bin Sirin, Mrs Ramlah binti Husin and Mr Azmi
Mahmud for their assistance throughout the work.
UTM for awarding Short Term Grant No: 75152 / 2004
My mother, wife and all families for their encouragements, supports, patience,
tolerance and understanding.
v
ABSTRACT
Aflatoxin, which is produced by Aspergillus flavus and Aspergillus parasiticus

fungi is one of the compounds in the mycotoxin group. The main types of aflatoxins
are AFB1, AFB2, AFG1 and AFG2 which have carcinogenic properties and are
dangerous to human health. Various techniques have been used for their measurements
such as the high performance liquid chromatography (HPLC), enzyme linked
immunosorbant assay (ELISA) and radioimmunoassay (RIA) but all these methods
have disadvantages such as long analysis time, consume a lot of reagents and
expensive. To overcome these problems, the voltammetric technique was proposed in
this study using controlled growth mercury drop (CGME) as the working electrode and
Britton Robinson buffer (BRB) as the supporting electrolyte. The voltammetric
methods were used for investigating the electrochemical properties and the quantitative
analysis of aflatoxins at the mercury electrode. The experimental conditions were
optimised to obtain the best characterised peak in terms of peak height with analytical
validation of the methods for each aflatoxin. The proposed methods were applied for
the analysis of aflatoxins in groundnut samples and the results were compared with
those obtained by the HPLC technique. All aflatoxins were found to adsorb and
undergo irreversible reduction reaction at the working mercury electrode. The
optimum experimental parameters for the differential pulse cathodic stripping
voltammetry (DPCSV) method were the BRB at pH 9.0 as the supporting electrolyte,
initial potential (Ei): -0.1 V, final potential (Ef): -1.4 V, accumulation potential (Eacc): -
0.6 V, accumulation time (tacc): 80 s, scan rate: 50 mV/s and pulse amplitude: 80 mV.
The optimum parameters for the square wave stripping voltammetry (SWSV) method
were Ei = -0.1 V, Ef = -1.4 V, Eacc: -0.8 V, tacc: 100 s, scan rate: 3750 mV/s, frequency:
125 Hz and voltage step: 30 V. At the concentration of 0.10 µM, using DPCSV
method with the optimum parameters, AFB1, AFB2, AFG1 and AFG2 produced a
single peak at -1.21 V, -1.23 V, -1.17 V and -1.15 V (versus Ag/AgCl) respectively.
Using the SWSV method, a single peak appeared at -1.30 V for AFB1 and AFB2 while
-1.22 V for AFG1 and AFG2. The calibration curves for all aflatoxins were linear with
the limit of detection (LOD) of approximately 2.0 ppb and 0.50 ppb obtained by the
DPCSV and SWSV methods respectively. The results of aflatoxins content in
individual groundnut samples do not vary significantly when compared with those
obtained by the HPLC technique. Finally, it can be concluded that both proposed
methods which are accurate, precise, robust, rugged, fast and low cost were
successfully developed and are potential alternative methods for routine analysis of
aflatoxins in groundnut samples.
vi
ABSTRAK
Aflatoksin adalah sejenis sebatian yang dihasilkan oleh kulat Aspergillus flavus
dan Aspergillus parasiticus yang digolongkan di dalam kumpulan mikotoksin. Jenis
utama aflatoksin adalah AFB1, AFB2, AFG1 dan AFG2 yang bersifat karsinogen serta
merbahaya kepada kesihatan manusia. Pelbagai teknik telah digunakan untuk
menentukan aflatoksin seperti kromatografi cecair prestasi tinggi (HPLC), asai serapan
imuno berikatan enzim (ELISA) dan radioamunoasai (RIA) tetapi teknik-teknik ini
mempunyai kelemahan seperti masa analisis yang panjang, melibatkan reagen yang
banyak dan kos yang mahal. Untuk mengatasi masalah ini, teknik voltammetri telah
dicadangkan untuk kajian aflatoksin menggunakan titisan raksa pembesaran terkawal
(CGME) sebagai elektrod bekerja dan larutan penimbal Britton-Robinson (BRB)
sebagai elektrolit penyokong. Pelbagai kaedah voltammetri telah digunakan untuk
mengkaji sifat elektrokimia aflatoksin pada elektrod raksa dan analisis kuantitatifnya.
Parameter kajian telah dioptimumkan untuk memperolehi puncak yang elok
berdasarkan ketinggian puncak serta pengesahan analisis untuk kaedah yang
dibangunkan bagi setiap aflatoksin. Kaedah ini telah digunakan untuk menentukan
kandungan aflatoksin di dalam sampel kacang tanah di mana keputusan yang diperolehi
telah dibandingkan dengan keputusan HPLC. Semua aflatoksin yang dikaji didapati
terjerap dan menjalani proses tindakbalas penurunan tidak berbalik pada elektrod raksa.
Parameter optimum untuk kaedah voltammetri perlucutan kathodik denyut pembeza
(DPCSV) adalah larutan BRB pada pH 9.0 sebagai larutan elektrolit, keupayaan awal
(Ei): -1.0 V, keupayaan akhir (Ef): -1.4 V, keupayaan pengumpulan (Eacc): -0.6 V, masa
pengumpulan (tacc): 80 s, kadar imbasan: 50 mV/s dan amplitud denyut: 80 mV. Untuk
kaedah voltammetri perlucutan gelombang bersegi (SWSV), parameter optimum adalah
Ei : -1.0 V, Ef : -1.4 V, Eacc: -0.8 V, tacc: 100 s, kadar imbasan: 3750 mV/s, frekuensi:
125 Hz dan beza keupayaan: 30 mV. Menggunakan parameter optimum untuk DPCSV,
0.10 µM AFB1, AFB2, AFG1 dan AFG2 menghasilkan puncak tunggal pada
keupayaan -1.21 V, -1.23 V, -1.17 V dan -1.15 V (melawan Ag/AgCl) masing-
masingnya. Menggunakan kaedah SWSV, puncak terhasil pada -1.30 V untuk AFB1
dan AFB2, -1.22 V untuk AFG1 dan AFG2. Keluk kalibrasi adalah linear untuk semua
aflatoksin dengan had pengesanan (LOD) pada 2.0 dan 0.5 ppb diperolehi dari kaedah
DPCSV dan SWSV masing-masingnya. Keputusan analisis kandungan aflatoksin di
dalam sampel kacang tanah tidak memberi perbezaan ketara berbanding dengan yang
diperolehi menggunakan teknik HPLC. Kesimpulannya, kedua-dua kaedah yang dikaji
yang merupakan kaedah yang tepat, jitu, cepat, sesuai digunakan dengan pelbagai
model voltammetri dan kos yang murah telah berjaya dibangunkan dan berpotensi besar
menjadi kaedah alternatif untuk analisis kandungan aflatoksin di dalam kacang tanah
secara berkala.
vii
TABLE OF CONTENTS
CHAPTER TITLE PAGE
TITLE i
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES xiii
LIST OF FIGURES xvi
ABBREVATIONS xxix
LIST OF APPENDICES xxxiii
1 LITERATURE REVIEW 1
1.1 Overview 1
1.2 Aflatoxins 3
1.2.1 Aflatoxins in general 3
1.2.2 Chemistry of aflatoxins 5
1.2.3 Health aspects of aflatoxins 12
1.2.4 Analytical methods for the determination of 16
aflatoxins
1.2.5 Electrochemical properties of aflatoxins 28
1.3. Voltammetric technique 30
1.3.1 Voltammetric techniques in general 30
viii
1.3.2 Voltammetric measurement 31

1.3.2.1 Instrumentation 31
1.3.2.2 Solvent and supporting electrolyte 44
1.3.2.3 Current in voltammetry 46
1.3.2.4 Quantitative and quantitative aspects of 48
voltammetry
1.3.3 Type of voltammetric techniques 49
1.3.3.1 Polarography 49
1.3.3.2 Cyclic voltammetry 51
1.3.3.3 Stripping voltammetry 54
1.3.3.3a Anodic stripping voltammetry 56
1.3.3.3b Cathodic stripping voltammetry 57
1.3.3.3c Adsorptive stripping voltammetry 58
1.3.3.4 Pulse voltammetry 59
1.3.3.4a Differential pulse voltammetry 60
1.3.3.4b Square wave voltammetry 61
1.4 Objective and scope of study 64
1.4.1 Objective of study 64
1.4.2 Scope of study 67
2 RESEARCH METHODOLOGY 70
2.1 Apparatus, material and reagents 70

2.1.1 Apparatus 70
2.1.2 Materials 72
2.1.2.1 Aflatoxin stock and standard solutions 72
2.1.2.2 Real samples 73
2.1.3 Reagents 73
2.1.3.1 Britton Robinson buffer, 0.04 M 73
2.1.3.2 Carbonate buffer, 0.04 M 74
2.1.3.3 Phosphate buffer, 0.04 M 74
ix
2.1.3.4 Ascorbic acid 74

2.1.3.5 β-cyclodextrin solution, 1.0 mM 75
-5
2.1.3.6 L-Cysteine, 1.0 x 10 M 75
2.1.3.7 2,4-dihydrofuran, 0.15 M 75
-2
2.1.3.8 Coumarin, 3.0 x 10 M 75
2.1.3.9 Poly-L-lysine, 10 ppm 75
2.1.3.10 Standard aluminium (II) solution, 1.0 mM 75
2.1.3.11 Standard plumbum(II) solution, 1.0 mM 76
2.1.3.12 Standard zinc (II) solution, 1.0 mM 76
2.1.3.13 Standard copper (II) solution, 1.0 mM 76
2.1.3.14 Standard nickel (II) solution, 1.0 mM 76
2.1.3.15 Methanol: 0.1 N HCl solution, 95% 76
2.1.3.16 Zinc sulphate solution, 15% 76
2.2 Analytical Technique 77

2.2.1 General procedure for voltammetric analysis 77
2.2.2 Cyclic voltammetry (Anodic and cathodic 77
directions)
2.2.2.1 Standard addition of sample 77
2.2.2.2 Repetitive cyclic voltammetry 78
2.2.2.3 Effect of scan rate 78
2.2.3 Differential pulse cathodic stripping 78
voltammetric determination of AFB2
2.2.3.1 Effect of pH 79
2.2.3.2 Method optimisation for the determination 79
of AFB2
2.2.3.2a Effect of scan rate 79
2.2.3.2b Effect of accumulation potential 80
2.2.3.2c Effect of accumulation time 80
2.2.3.2d Effect of initial potential 80
2.2.3.2e Effect of pulse amplitude 80
x
2.2.3.3 Method validation 80

2.2.3.4 Interference studies 81
2.2.3.4a Effect of Cu(II), Ni (II), Al(III), 81
Pb(II) and Zn(II)
2.2.3.4b Effect of ascorbic acid, 82
β-cyclodextrin and L-cysteine
2.2.3.5 Modified mercury electrode with PLL 82
2.2.4 Square wave cathodic stripping voltammetry 82
(SWSV)
2.2.4.1 SWSV parameters optimisation 82
2.2.4.2 SWSV determination of all aflatoxins 82
2.2.5 Stability studies of aflatoxins 83
2.2.5.1 Stability of 10 ppm aflatoxins 83
2.2.5.3 Stability of 0.1 µM aflatoxins exposed 83
to ambient temperature
2.2.5.4 Stability of 0.1 µM aflatoxins in different 84
pH of BRB
2.2.6 Application to food samples 84
2.2.6.1 Technique 1 84
2.2.6.4 Blank measurement 85
2.2.6.5 Recovery studies 85
2.2.6.6 Voltammetric analysis 86
3 RESULTS AND DISCUSSION 88
3.1 Cyclic voltammetric studies of aflatoxins 88

3.1.1 Cathodic and anodic cyclic voltammetric 89
of aflatoxins
xi
3.2 Differential pulse cathodic stripping voltammetry 102

of AFB2
3.2.1 Optimisation of conditions for the stripping 104
analysis
3.2.1.1 Effect of pH and type of supporting 104
electrolyte
3.2.1.2 Optmisation of instrumental conditions 117
3.2.1.2b Effect of accumulation time 119
3.2.1.2c Effect of accumulation 120
potential
3.2.2 Analysis of aflatoxins 127
3.2.2.1 Calibration curves of aflatoxins and 129
validation of the proposed method
3.2.2.1a Calibration curve of AFB2 129
3.2.2.1b Calibration curve of AFB1 134
3.2.2.1c Calibration curve of AFG1 137
3.2.2.1d Calibration curve of AFG2 140
3.2.2.2 Determination of limit of detection 143
3.2.2.3 Determination of limit of quantification 147
3.2.2.4 Inteference studies 150
3.3 Square-wave stripping voltammetry (SWSV) of 157
aflatoxins
3.3.1 SWSV determination of AFB2 158
3.3.1.1 Optimisation of experimental and 159
instrumental SWSV parameters
3.3.3.1a Influence of pH of BRB 159
3.3.3.1b Effect of instrumental 160
variables
xii
3.3.2 SWSV determination of other aflatoxins 166

3.3.3 Calibration curves and method validation 168
3.4 Stability studies of aflatoxins 175
3.4.1 10 ppm aflatoxin stock solutions 175
3.4.2 1 ppm aflatoxins in BRB at pH 9.0 179
3.4.2.1 Month to month stability studies 179
3.4.2.2 Hour to hour stability studies 181
3.4.2.3 Stability studies in different pH 186
of BRB
3.4.2.4 Stability studies in 1.0 M HCl and 191
1.0 M NaOH
3.5 Voltammetric analysis of aflatoxins in real samples 192
3.5.1 Study on the extraction techniques 193
3.5.2 Analysis of blank 194
3.5.3 Recovery studies of aflatoxins in real samples 196
3.5.4 Analysis of aflatoxins in real samples 199
4 CONCLUSIONS AND RECOMMENDATIONS 204
4.1 Conclusions 204

4.2 Recommendations 206
REFERENCES 208
Appendices A – AM 255 - 317
xiii
LIST OF TABLES
TABLE NO. TITLE PAGE
1.0 Scientific name for aflatoxin compounds 7
1.1 Chemical and physical properties of aflatoxin 10

compounds
1.2 Summary of analysis methods used for 19

determination of aflatoxins in various samples
1.3 Working electrode and limit of detection for 32

modern polarographic and voltammetric
techniques.
1.4 The application range of various analytical 33

techniques and their concentration limits when
compared with the requirements in different
fields of chemical analysis
1.5 List of different type of working electrodes and 36

its potential windows
1.6 Electroreducible and electrooxidisable organic 50

functional groups
1.7 The characteristics of different type of 53

electrochemical reaction.
1.8 Application of Square Wave Voltammetry 62

technique
2.0 List of aflatoxins and their batch numbers 72

used in this experiment
2.1 Injected volume of aflatoxins into eluate of 86

groundnut and the final concentrations obtained
in voltammetric cell
3.0 The dependence of current peaks of aflatoxins to 97

their concentrations obtained by cathodic cyclic
measurements in BRB at 9.0.
xiv
3.1 Effect of buffer constituents on the peak height 109

of 2.0 µM AFB2 at pH 9.0. Experimental
conditions are the same as Figure 3.21
3.2 Compounds reduced at the mercury electrode 116
3.3 Optimum parameters for 0.06 µM and 2.0 µM 126

AFB2 in BRB at pH 9.0.
3.4 The peak height and peak potential of aflatoxins 127

obtained by optimised parameters in BRB at
pH 9.0 using DPCSV technique.
3.5 Peak height (in nA) obtained for intra-day and 131
inter-day precision studies of 0.10 µM and 0.20
µM by the proposed voltammetric procedure (n=8).
3.6 Mean values for recovery of AFB2 standard 132

solution (n=3).
3.7 Influence of small variation in some of the 133

assay condition of the proposed procedure on its
suitability and sensitivity using 0.10 µM AFB2.
3.8 Results of ruggedness test for proposed method 134

using 0.10 µM AFB2.
µM AFB1 by proposed voltammetric procedure
(n=5).

solution (n=3).
µM AFG1 by proposed voltammetric procedure
(n=5).
3.12 Mean values for recovery of AFG1 standard 139

solution (n=3).
µM AFG2 by proposed voltammetric procedure.
xv

solution (n=5).
3.15 Peak height and peak potential of 0.10 µM 143

aflatoxins obtained by BAS and Metrohm
voltammetry analysers under optimised
operational parameters for DPCSV method.
3.16 Analytical parameters for calibration curves 145

for AFB1,AFB2, AFG1 and AFG2 obtained by
DPCSV technique using BRB at pH 9.0 as the
supporting electrolyte.
3.17 LOD values for determination of aflatoxins 148

obtained by various methods.
3.18 LOQ values for determination of aflatoxins 149

3.19 Peak current and peak potential for all aflatoxins 167
obtained by SWSV in BRB at pH 9.0 (n=5).
3.20 Analytical parameters for calibration curves for 172

AFB1, AFB2, AFG1 and AFG2 obtained by
SWSV technique in BRB pH 9.0 as the
3.21 Result of reproducibility study (intra-day and inter- 173

day measurements) for 0.1 µM aflatoxins in
BRB at pH 9.0 obtained by SWSV method.
3.22 Application of the proposed method in evaluation 174

of the SWSV method by spiking the aflatoxin
standard solutions.
3.23 Average concentration of all aflatoxins within a 175

year stability studies.
3.24 The peak current and peak potential of 10 ppb 196

AFB2 in presence and absence of a blank sample.
3.25 Total aflatoxin contents in real samples which 203

were obtained by DPCSV and HPLC
techniques (average of duplicate analysis)
xvi
LIST OF FIGURES
FIGURE NO. TITLE PAGE
1.0 Aspergillus flavus seen under an electron 4

microscope.
1.1 Chemical structure of coumarin 6
1.2 Chemical structures of (a) AFB1, (b) AFB2, 8

(c) AFG1, (d) AFG2, (e) AFM1 and (f) AFM2
1.3 Hydration of (a) AFB1 and (b) AFG1 by TFA 9

produces (c) AFB2a and (d) AFG2a
1.4 Transformation of toxic (a) AFB1 to non-toxic 11

(b) aflatoxicol A.
1.5 Major DNA adducts of AFB1; (a) 8,9-Dihydro-8- 13

(N7-guanyl)-9-hydroxy-aflatoxin B1 (AFB1-Gua)
and (b) 8,9-Dihydro-8-(N5-Formyl-2’.5’.6’-
triamino-4’-oxo-N5-pyrimidyl)-9-hydroxy-Aflatoxin
B1 (AFB1-triamino-Py)
1.6 Metabolic pathways of AFB1 by cytochrom P-450 14

enzymes; B1-epoxide = AFB1 epoxide, M1=
aflatoxin M1, P1= aflatoxin P1 and Q1 = aflatoxin
Q1.
1.7 A typical arrangement for a voltammetric 34

electrochemical cell (RE: reference electrode,
WE: working electrode. AE: auxiliary electrode)
1.8 A diagram of the Hanging Mercury Drop Electrode 37

(HMDE)
1.9 A diagram of the Controlled Growth Mercury 38

Electrode (CGME)
1.10 Cyclic voltammograms of (a) reversible, 52

(b) irriversible and (c) quasireversible reaction at
mercury electrode (O = oxidised form and R = reduced
form)
xvii
1.11 The potential-time sequence in stripping analysis 55
1.12 Schematic drawing showing the Faradaic current 59

and charging current versus pulse time course
1.13 Schematic drawing of steps in DPV by 60

superimposing a periodic pulse on a linear scan
1.14 Waveform for square-wave voltammetry 61
2.0 BAS CGME stand (a) which is connected to 71

CV-50W voltammetric analyser and interface
with computer (b) for data processing
2.1 VA757 Computrace Metrohm voltammetric 71

analyser with 663 VA stand (consists of Multi
Mode (MME))
3.0 Cathodic peak current of 0.6 µM AFB1 in various 89

pH of BRB obtained in cathodic cyclic voltammetry.
Ei = 0, Elow = -1.5 V, Ehigh = 0 and scan rate = 200
mV/s)
3.1 Shifting of peak potential of AFB1 with increasing 90

pH of BRB. Parameter conditions are the same
as in Figure 3.0.
3.2 Mechanism of reduction of AFB1 in BRB at pH 6.0 90

to 8.0.

to 11.0.
3.4 Cathodic cyclic voltammogram for 1.3 µM AFB1 92

obtained at scan rate of 200 mV/s, Ei = 0, Elow =
-1.5 V and Ehigh = 0 in BRB solution at pH 9.0.

in BRB solution at pH 9.0. All parameter conditions
are the same as in Figure 3.4.
3.6 Cathodic cyclic voltammogram for 1.3 µM AFG1 93

xviii

3.8 Effect of Ei to the Ip of 0.6 µM AFB1 in BRB at pH 94

9.0 obtained by cathodic cyclic voltammetry. All
parameter conditions are the same as in Figure 3.4.
3.9 Effect of Ei to the Ip of 0.1 µM Zn2+ in BRB at pH 94

3.10 Anodic cyclic voltammogram of 1.3 µM AFB2 95

obtained at scan rate of 200 mV/s, Ei = -1.5 V,
Elow = -1.5 V and Ehigh = 0 in BRB at pH 9.0.
3.11 Effect of increasing AFB2 concentration on the 96

peak height of cathodic cyclic voltammetrc curve
in BRB at pH = 9.0. (1.30 µM, 2.0 µM, 2.70 µM
and 3.40 µM). All parameter conditions are the
same as in Figure 3.4.
3.12 Peak height of reduction peak of AFB2 with 96

increasing concentration of AFB2. All parameter
conditions are the same as in Figure 3.4.
3.13 Repetitive cathodic cyclic voltammograms of 1.3 98

µM AFB2 in BRB solution at pH 9.0. All
experimental conditions are the same as in Figure 3.4.
3.14 Increasing Ip of 1.3 µM AFB2 cathodic peak 98

obtained from repetitive cyclic voltammetry. All
3.15 Peak potential of 1.3 µM AFB2 with increasing 99

number of cycle obtained by repetitive cyclic
voltammetry.
xix
3.16 Plot of log Ip versus log υ for 1.3 µM AFB2 in 100

BRB solution at pH 9.0. All experimental conditions
3.17 Plot of Ep versus log υ for 1.3 µM AFB2 in BRB 101

solution at pH 9.0.
3.18 Plot of Ip versus υ for 1.3 µM AFB2 in BRB 102

solution at pH 9.0. All parameter conditions
3.19 DPCS voltammograms of 1.0 µM AFB2 (Peak I) 103

in BRB at pH 9.0 (a) at tacc = 0 and 30 s. Other
parameter conditions; Ei = 0, Ef = -1.50 V,
Eacc = 0, υ =50 mV/s and pulse amplitude =
100 mV. Peak II is the Zn peak.
3.20 DPCS voltammograms of 2.0 µM AFB2 in BRB 104

in BRB (Peak I) at different pH values; 6.0, 7.0,
8.0, 9.0, 11.0and 13.0. Other parameter conditions;
Ei = 0, Ef =-1.50 V, Eacc = 0, υ = 50 mV/s and pulse
amplitude =100 mV. Peak II is the Zn peak.
3.21 Dependence of the Ip for AFB2 on the pH of 105

0.04 M BRB solution. AFB2 concentration:
2.0 µM, Ei =0, Ef = -1.5 V, E acc = 0, tacc = 30 sec,
υ = 50 mV/s and pulse amplitude = 100 mV.
3.22 Ip of 2.0 µM AFB2 obtained in BRB (a) at pH 106

from 9.0 decreases to 4.0 and re-increase to 9.0 and
(b) at pH from 9.0 increase to 13.0 and re-decrease
to 9.0.
3.23 UV-VIS spectrums of 1 ppm AFB2 in BRB at 107

pH (a) 6.0, (b) 9.0 and (c) 13.0.
3.24 Opening of lactone ring by strong alkali caused no 108

peak to be observed for AFB2 in BRB at pH 13.0.
3.25 Ip of 2.0 µM AFB2 in different concentration of 109

BRB at pH 9.0. Experimental conditions are
the same as in Figure 3.20.
3.26 Ip of 2.0 µM AFB2 in different pH and concentrations 110

of BRB.
xx
3.27 DPCS voltammograms of 2.0 AFB2 (Peak I) 110

in (a) 0.04 M, (b) 0.08 M and (c) 0.08 M BRB
at pH 9.0 as the blank. Ei = 0 V, Ef = -1.5 V,
Eacc = 0 V, tacc = 30 sec, υ = 50mV/s and pulse
amplitude = 100 mV. Peak II is the Zn peak.
3.28 Chemical structures of (a) 2,3-dihydrofuran, 111

(b) tetrahydrofuran and (c) coumarin.
3.29 Voltammograms of 2,3-dihydrifuran (peak I) for 112

concentrations from (b) 0.02 to 0.2 µM in (a)
BRB at pH 9.0.
3.30 Dependence of Ip of coumarin to its concentrations 112
3.31 Voltammograms of coumarin at concentration of 113

(b) 34 µM, (c) 68 µM, (d) 102 µM, (e) 136 µM
and (f) 170 µM in (a) BRB at pH 9.0.
3.32 Chemical structures of (a) cortisone and (b) 114

testosterone.
3.33 Chemical structures of (a) digoxin and (b) digitoxin 115
3.34 Effect of pH of BRB solution on the Ep for AFB2. 117

AFB2 concentration: 2.0 µM. Ei = 0, Ef = -1.5 V,
Eacc = 0, t acc = 30 s and υ = 50 mV/s.
3.35 Effect of various υ to the (a) Ip and (b) Ep of 2.0 µM 118

AFB2 peak in BRB at pH 9.0. Ei = 0, Ef = 1.50 V,
Eacc = 0, t acc = 15 s and pulse amplitude = 100 mV.
3.36 Effect of tacc on (a) Ip and (b) Ep of 2.0 µM AFB2 119

peak in BRB at pH 9.0. Ei = 0, Ef = 1.50 V, Eacc = 0,
3.37 The relationship between (a) Ip and (b) Ep with 120

Eacc for 2.0 µM AFB2 in BRB at pH 9.0.
Ei = 0, Ef = -1.5 V, tacc = 15 s, υ = 40 mV/s and
pulse amplitude = 100 mV.
3.38 Effect of Ei on (a) Ip and (b) Ep of 2.0 µM AFB2 121

in BRB at pH 9.0. Ef = 1.50 V, Eacc = -0.80 V,
tacc = 40 s,υ = 40 mV/s and pulse amplitude =
100 mV.
xxi
3.39 Effect of pulse amplitude on (a) Ip and (b) Ep 122

of 2.0 µM AFB2 in BRB at pH 9.0. Ei = -1.0 V,
Ef = 1.50 V, Eacc = -0.80 V, tacc = 40 s and υ =
40 mV/s.
3.40 Effect of Eacc on Ip of 0.06 µM AFB2. Ei = -1.0 V, 123

Ef = -1.50 V, tacc = 40 s, υ = 40 mV/s and pulse
amplitude = 100 mV.

in BRB at pH 9.0. Ei = -1.0 V, Ef = -1.50 V, Eacc =
-0.6 V, υ = 40 mV/s and pulse amplitude = 100 mV.
3.42 The effect υ on (a) Ip and (b) Ep of 0.06 µM AFB2 125

In BRB at pH 9.0. Ei = -1.0 V, E f = -1.50 V, Eacc =
-0.6 V, tacc = 80 s and pulse amplitude = 100 mV.
3.43 Voltammograms of 0.06 µM AFB2 obtained under 126

(a) optimised and (b) unoptimised parameters in BRB
at pH 9.0.
3.44 Voltammograms of 0.1 µM (a) AFB1, (b) AFG1, 128

(c) AFB2 and (d) AFG2 in BRB at pH 9.0. Ei = 1.0
(except for AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.6 V,
tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.
3.45 Voltammograms of (b) mixed aflatoxins in (a) BRB at 129

pH 9.0 as the blank. Parameters condition: Ei = -0.95 V,
Ef = -1.4 V, Eacc = -0.6 V, t acc = 80 s, υ = 50 mV/s and
Pulse amplitude = 80 mV.
3.46 Increasing concentration of AFB2 in BRB at pH 9.0. 130

The parameter conditions: Ei = -1.0 V, Ef = -1.4 V,
Eacc =-0.6 V, t acc = 80 s, υ = 50 mV/s and
3.47 Linear plot of Ip versus concentration of AFB2 130

in BRB at pH 9.0. The parameter conditions are
3.48 Standard addition of AFB1 in BRB at pH 9.0. 135

xxii

3.50 Effect of concentration to Ip of AFG1 in BRB at 138

pH 9.0. Ei = -0.95 V, Ef = -1.40 V, Eacc =-0.8 V,
3.51 Linear plot of Ip versus concentration of AFG1 138


pH 9.0. Ei = -1.0 V, Ef = -1.40 V, Eacc =-0.8 V,

3.54 Voltammograms of 0.1 µM (a) AFB1, (b) AFB2, 144

(c) AFG1 and (d) AFG2 in BRB at pH 9.0
(d) obtained by 747 VA Metrohm. Ei = -1.0 V (except
for AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.6 V,
3.55 Ip of all aflatoxins with increasing concentration of 150

Zn 2+ up to 1.0 µM.
3.56 Voltammograms of (i) 0.1µM AFB2 and (ii) AFB2-Zn 151

complex with increasing concentration of Zn2+ (a = 0,
b = 0.75 µM, c = 1.50 µM, d = 2.25 µM and e = 3.0 µM).
Blank = BRB at pH 9.0. Experimental conditions;
Ei = -1.0 V, Ef = -1.40 V, Eacc = -0.6 V, tacc = 80 s,
3.57 Ip of all aflatoxins after reacting with increasing 151

concentration of Zn2+ in BRB at pH 3.0.
Measurements were made in BRB at pH 9.0 within
15 minutes of reaction time. Ei = -1.0 V (except
xxiii
3.58 Absorbance of all aflatoxins with increasing 152

concentration of Zn2+ in BRB at pH 3.0 within 15
minutes of reaction time
3.59 Voltammograms of 0.1µM AFB2 with increasing 153

concentration of Zn2+ (from 0.10 to 0.50 µM) in
BRB at pH 9.0. Ei = -0.25 V, Ef = -1.4 V, Eacc =
-0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplirude =
80 mV. .
3.60 Chemical structure of ascorbic acid. 153
3.61 Ip of all aflatoxins with increasing concentration 154

of ascorbic acid up to 1.0 µM. Concentrations of
all aflatoxins are 0.1µM.

concentration of ascorbic acid.

of β-cyclodextrin up to 1.0 µM.

concentration of β-cyclodextrin.
3.65 Chemical structure of L-cysteine. 156

of cysteine up to 1.0 µM.
3.67 Voltammograms of 0.1 µM AFB2 obtained by 158

(a) DPCSV and (b) SWSV techniques in BRB at
pH 9.0. Parameters for DPCSV: Ei = -1.0 V, Ef =
-1.40V, Eacc = -0.6 V, t acc = 80 s, υ = 50 mV/s and
pulse amplitude = 80 mV and for SWSV: Ei = -1.0 V,
Ef = -1.40V, Eacc = -0.6 V, tacc = 80 s, frequency =
50 Hz, voltage step = 0.02 V, amplitude = 80 mV
and υ = 1000 mV/s.
3.68 Influence of pH of BRB on the Ip of 0.10 µM 159

AFB2 using SWSV technique. The instrumental
Parameters are the same as in Figure 3.67.
3.69 Voltammograms of 0.1 µM AFB2 in different pH 160

of BRB. Parameter conditions: Ei = -1.0 V, E f =
-1.40 V, Eacc = -0.6 V, tacc = 80 s, voltage step =
xxiv
0.02 V, amplitude = 50 mV, frequency = 50 Hz

and υ = 1000 mV/s.
3.70 Effect of Ei to the Ip of 0.10 µM AFB2 in BRB at 160

pH 9.0.
3.71 Effect of Eaccto the Ip of 0.10 µM AFB2 in BRB at 161

pH 9.0.
3.72 Relationship between Ip of 0.10 µM AFB2 while 162

increasing tacc.
3.73 Effect of frequency to the Ip of 0.10 µM AFB2 in 162

BRB at pH 9.0.
3.74 Linear relatioship between Ip of AFB2 and square 163

root of frequency.
3.75 Influence of square-wave voltage step to Ip of 163

0.10 µM AFB2.
3.76 Influence of square-wave amplitude to Ip of 0.10 µM 164

AFB2.
3.77 Relationship of SWSV Ep of AFB2 with increasing 164

amplitude.
3.78 Ip of 0.10 µM AFB2 obtained under (a) non-optimised 165

and (b) optimised SWSV parameters compared with
that obtained under (c) optimised DPCSV parameters.

(a) non-optimised and (b) optimised SWSV
parameters compared with that obtained under (c)
optimised DPCSV parameters.
3.80 Ip of 0.10 µM aflatoxins obtained using two 167

different stripping voltammetric techniques under
their optimum paramter conditions in BRB at pH 9.0.
3.81 Voltammograms of (i) AFB1, (ii) AFB2, (iii) AFG1 168

and (iV) AFG2 obtained by (b) DPCSV compared
with that obtained by (c) SWSV in (a) BRB at pH 9.0.
3.82 SWSV voltammograms for different concentrations 169

of AFB1 in BRB at pH 9.0. The broken line
xxv
represents the blank: (a) 0.01 µM, (b) 0.025 µM,

(c) 0.05 µM, (d) 0.075 µM, (e) 0.10 µM, (f) 0.125
µM and (g) 0.150 µM. Parameter conditions:
Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.8 V, t acc = 100 s,
frequency = 125 Hz, voltage step = 0.03 V, pulse
amplitude = 75 mV and scan rate = 3750 mV/s.
3.83 Calibration curve for AFB1 obtained by SWSV 170

method.

method.
3.85 Calibration curve for AFG1 obtained by SWSV 170

method.

method.
3.87 LOD for determination of aflatoxins obtained by 171

two different stripping methods.
3.88 UV-VIS spectrums of 10 ppm of all aflatoxins in 176

benzene: acetonitrile (98%) at preparation date.

benzene: acetonitrile (98%) after 6 months of
storage time.

storage time.
3.91 UV-VIS spectrums of 10 ppm AFB1 (a) kept in 178

the cool and dark conditions and (b) exposed to
ambient conditions for 3 days.
3.92 UV-VIS spectrums of 1 ppm AFB1 in BRB 178

solution prepared from (a) good and (b) damaged
10 ppm AFB1 stock solution.

10 ppm AFB1 stock solution after 2 week stored in
the cool and dark conditions.
xxvi
3.94 Percentage of Ip of 0.10 µM aflatoxins in BRB at pH 180

9.0 at different storage time in the cool and dark
conditions.
3.95 Percentage of Ip of all aflatoxins in BRB at pH 9.0 181

exposed to ambient conditions up to 8 hours of
exposure time.
3.96 Reaction of 8,9 double bond furan rings in AFB1 182

with TFA, iodine and bromine under special
conditions (Kok, et al., 1986).
3.97 Voltammograms of 0.10 µM AFB1 obtained in (a) 183

BRB at pH 9.0 which were prepared from (b)
damaged and (c) fresh stock solutions.
3.98 Ip of 0.10 µM AFB1 obtained in BRB at pH 9.0 184

from 0 to 8 hours in (a) light exposed and (b)
light protected.

light protected.
3.100 Ip of 0.10 µM AFG1 obtained in BRB at pH 9.0 185

light protected.

light protected.
3.102 Peak heights of 0.10 µM aflatoxins in BRB at pH 187

(a) 6.0, (b) 7.0, (c) 9.0 and (d) 11.0 exposed to
ambient conditions up to 3 hours of exposure time.
3.103 Resonance forms of the phenolate ion 187

(Heathcote, 1984).
3.104 Voltammograms of 0.10 µM AFB2 in BRB at pH 188

6.0 from 0 to 3 hrs exposure time.

11.0 from 0 to 3 hrs of exposure time.
xxvii
3.106 Voltammograms of 0.10 µM AFG2 in BRB at pH 189


3.108 Absorbance of 1.0 ppm aflatoxins in BRB at pH 190

(a) 6.0, (b) 7.0, (c) 9.0 and (d) 11.0 from 0 to 3
hours of exposure time.
3.109 The peak heights of aflatoxins in 1.0 M HCl from 191

0 to 6 hours of reaction time.
3.110 The peak heights of aflatoxins in 1.0 M NaOH from 192

3.111 Voltammograms of real samples after extraction by 193

Technique (a) 1, (b) 2 and (c) 3 with addition of
AFB1 standard solution in BRB at pH 9.0 as a
blank.
3.112 Voltammograms of blank in BRB at pH 9.0 obtained 194

by (a) DPCSV and (b) SWSV methods.
3.113 DPCSV (a) and SWSV (b) voltammograms of 10 195

ppb AFB2 (i) in present of blank sample (ii) obtained
in BRB at pH 9.0 (iii) as the supporting electrolyte.
3.114 DPCSV voltammograms of real samples (b) added 197

with 3 ppb (i), 9 ppb (ii) and 15 ppb (iii) AFB1
obtained in BRB at pH 9.0 (a) as the blank on the
first day measurement.
3.115 Percentage of recoveries of (a) 3 ppb, (b) 9 ppb, 198

(c) 15 ppb of all aflatoxins in real samples
obtained by DPCSV methods for one to three
days of measurements.
3.116 DPCSV voltammograms of real sample, S11 (b) 199

with the addition of 10 ppb AFB1 (c) in BRB at pH
9.0 (a) as the blank. Parameter conditions: Eacc =
-0.6 V, tacc = 80 s, scan rate = 50 mV/s and pulse
amplitude = 80 mV.
xxviii
3.117 SWSV voltammograms of real sample, S11 (b) 200

-0.8 V, tacc = 100 s, scan rate = 3750 mV/s,
frequency = 125 Hz, voltage step = 0.03 V and

amplitude = 80 mV.


amplitude = 80 mV.

xxix
ABBREVIATIONS
AAS Atomic absorption spectrometry

Abs Absorbance
ACP Alternate current polarography
ACV Alternate current voltammetry
AD Amperometric detector
AdCSV Adsorptive cathodic stripping voltammetry
AE Auxiliary electrode
AFB1 Aflatoxin B1
AFB2 Aflatoxin B2
AFG1 Aflatoxin G1
AFG2 Aflatoxin G2
AFM1 Aflatoxin M1
AFM2 Aflatoxin M2
AFP1 Aflatoxin P1
AFQ1 Aflatoxin Q1
Ag/AgCl Silver/silver chloride
ASV Anodic stripping voltammetry
β-CD β-cyclodextrin
BFE Bismuth film electrode
BLMs Bilayer lipid membranes
BRB Britton Robinson Buffer
CA Concentration of analyte
CE Capillary electrophoresis
CGME Controlled growth mercury electrode
CME Chemically modified electrode
CPE Carbon paste electrode
CSV Cathodic stripping voltammetry
CV Cyclic voltammetry
xxx
DC Direct current
DCP Direct current polarography
DME Dropping mercury electrode
DMSO Dimethyl sulphonic acid
DNA Deoxyribonucliec acid
DPCSV Differential pulse cathodic stripping voltammetry
DPP Differential pulse polarography
DPV Differential pulse voltammetry
Eacc Accumulation potential
Ei Initial potential
Ef Final potential
Ehigh High potential
Elow Low potential
Ep Peak potential
ECS Electrochemical sensing
Et4NH4 OH Tetraethyl ammonium hydroxide
ELISA Enzyme linked immunosorbant assay
FD Fluorescence detector
FDA Food and Drug Administration
GCE Glassy carbon electrode
GC-FID Gas chromatography with flame ionisation detector
HMDE Hanging mercury drop electrode
HPLC High performance liquid chromatography
HPTLC High pressure thin liquid chromatography
IAC Immunoaffinity chromatography
IACLC Immunoaffinity column liquid chromatography
IAFB Immunoaffinity fluorometer biosensor
IARC International Agency for Research Cancer
Ic Charging current
Id Diffusion current
If Faradaic current
xxxi
Ip Peak height
ICP-MS Induced coupled plasma-mass spectrometer
IR Infra red
IUPAC International Union of Pure and Applied Chemistry
KGy Kilogray
LD50 Lethal dose 50
LOD Limit of detection
LOQ Limit of quantification
LSV Linear sweep voltammetry
MFE Mercury film electrode
MS Mass spectrometer
MECC Micellar electrokinetic capillary chromatography
MOPS 3-(N-morpholino)propanesulphonic
MOSTI Ministry of Science, Technology and Innovation
NP Normal polarography
NPP Normal pulse polarography
NPV Normal pulse voltammetry
OPLC Over pressured liquid chromatography
PAH Polycyclic aromatic hydrocarbon
PLL Poly-L-lysine
ppb part per billion
ppm part per million
PSA Potentiometric stripping analysis
RDX Hexahydro-1,3,5-trinitro-1,3,5-triazine
RE Reference electrode
RIA Radioimmunoassay
RNA Ribonucleic acid
RSD Relative standard deviation
S/N Signal to noise ratio
SCE Standard calomel electrode
SCV Stair case voltammetry
xxxii
SDS Sodium dodecyl sulphate

SHE Standard hydrogen electrode
SIIA Sequential injection immunoassay
SMDE Static mercury drop electrode
SPE Solid phase extraction
SPCE Screen printed carbon electrode
SWP Square-wave polarography
SWV Square-wave voltammetry
SWSV Square-wave stripping voltammetry
SV Stripping voltammetry
tacc Accumulation time
TBS Tris buffered saline
TEA Triethylammonium
TLC Thin layer chromatography
TFA Trifluoroacetic acid
UME Ultra microelectrode
ν Scan rate
v/v Volume per volume
UVD Ultraviolet-Visible detector
UV-VIS Ultraviolet-Visible
WE Working electrode
WHO World Health Organisation
λmax Maximum wavelenght
εmax Maximum molar absorptivity
xxxiii
LIST OF APPENDICES
APPENDIX TITLE PAGE
A Relative fluorescence of aflatoxins in different 255

solvent s
B UV spectra of the principal aflatoxins (in methanol) 256
C Relative intensities of principal bands in the IR 257

spectra of the aflatoxins
D Calculation of concentration of aflatoxin stock 258

solution
E Extraction procedure for aflatoxins in real samples 259
F Calculation of individual aflatoxin in groundnut 260

samples.
G Cyclic voltammograms of AFB1, AFB2 and AFG2 262

with increasing of their concentrations.
H Dependence if the peak heights of AFB1, AFG1 264

and AFG2 on their concentrations.
I Repetitive cyclic voltammograms and their peak 266

heights of AFB1, AFG1 and AFG2 in BRB at pH 9.0
J Plot Ep – log scan rate for the reduction of AFB1, 270

AFG1 and AFG2 in BRB at pH 9.0
K Plot of peak height versus scan rate for 1.3 µM of 272

AFB1, AFG1 and AFG2 in BRB at pH 9.0
L Voltammograms of AFB2 with increasing 274

concentration.
M Voltammograms of 0.1 µM and 0.2 µM AFB2 275

obtained on the same day measurements
N Voltammograms of AFB2 at inter-day 276

measurements
xxxiv
O F test for robustness and ruggedness tests 278
P Voltammograms of AFB1 with increasing 280

concentration
Q Voltammograms of AFG1 with increasing 281

concentration
R Voltammograms of AFG2 with increasing 282

concentration
S LOD determination according to Barek et al. (2001a) 283
T LOD determination according to Barek et al. (1999) 286
U LOD determination according to Zhang et al. (1996) 287
V LOD determination according to Miller and 288

Miller (1993)
W ANOVA test 290
X Peak height of aflatoxins in presence and absence of PLL 292
Y SWSV voltammograms of AFB1, AFB2, AFG1 293

and AFG2 in BRB at pH 9.0
Z SWSV voltammograms of 0.10 µM AFB1, AFB2, 295

AA UV-VIS spectrums of 10 ppm AFB1, AFB2, AFG1 297

and AFG2 stock solutions
AB Voltammograms of AFB1, AFB2, AFG1 and AFG2 299

obtained from 0 to 6 months of storage time in the
cool and dark conditions.
AC Voltammograms of AFB1, AFB2, AFG1 and 302

AFG2 in BRB at pH 9.0 from 0 to 8 hours of
exposure time.
AD UV-VIS spectrums of AFB2 in BRB at pH 6.0 305

and 11.0.
xxxv
AE Voltammograms of AFB1 and AFG1 in 1.0 M HCl 307

and 1.0 M NaOH
AF DPCSV voltammograms of real samples added with 309

various concentrations of AFG1
AG SWSV voltammograms of real samples added with 310

various concentrations of AFB1.
AH Percentage of recoveries of various concentrations 311

of all aflatoxins (3.0 and 9.0 ppb) in real samples
obtained by SWSV method.
AI Calculation of percentage of recovery for 3.0 ppb 312

AFG1 added into real samples.
AJ HPLC chromatograms of real samples: S10 and S07 313
AK Calculation of aflatoxin in real sample, S13 314
AL List of papers presented or published to date 315

resulting from this study.
AM ICP-MS results for analysis of BRB at pH 9.0 317

CHAPTER I
LITERATURE REVIEW
1.1 Overview
Humans are continuously exposed to varying amounts of chemicals that have

been shown to have carcinogenic or mutagenic properties in environmental systems.
Exposure can occur exogenously when these agents are present in food, air or water, and
also endogenously when they are products of metabolism or pathophysiologic states such
as inflammation. Great attention is focused on environmental health in the past two
decades as a consequence of the increasing awareness over the quality of life due to
major environment pollutants that affect it. Studies have shown that exposure to
environmental chemical carcinogens have contributed significantly to cause human
cancers, when exposures are related to life style factors such as diet (Wogan et al., 2004).
The contamination of food is part of the global problem of environmental

pollution. Foodstuffs have been found contaminated with substances having
carcinogenic, mutagenic, teratogenic and allergenic properties. As these substances can
be supplied with food throughout the entire life-time of a person, it is necessary to deal
with the chronic action of trace amounts of such substances. Hence the systematic
determination of the foreign substances in nutritional products and feedstock plays an
important role. The determination of trace impurities presents considerable difficulties
owing to the fact that food is a complex system containing thousands of major and minor
compounds (Nilufer and Boyacio, 2002). Increasing environment pollution by toxic
substances such as toxic metals, organometallic and organic pollutants in air,
2
water, soil and food, calls for reliable analytical procedures for their control in
environmental samples which needs reliable and sensitive methods (Fifield and Haines,
2000). The choice of the method of analysis depends on the sample, the analyte to be
assayed, accuracy, limit of detection, cost and time to complete the analysis (Aboul-
Eneim et a., 2000). For development of this method, emphasis should be on
development of simplified, cost-effective and efficient method that complies with the
legislative requirements (Stroka and Anklam, 2002; Enker, 2003).
The widespread occurrence of aflatoxins producing fungi in our environment and

the reported naturally occurring of toxin in a number of agricultural commodities has led
the investigator to develop a new method for aflatoxin analysis (Creepy, 2002). An
accurate and sensitive method of analysis is therefore required for the determination of
these compounds in foodstuffs that have sustained mould growth.
Numerous articles concerning methods for determination of aflatoxins have been

published. However, with regard to electroanalytical technique, only one method of
determination was reported using the differential pulse pulse polarographic (DPP)
technique which was developed by Smyth et al. (1979). In this experiment, the obtained
limit of detection of aflatoxin B1 was 25 ppb which was higher as compared to the
common amount of aflatoxin in contaminated food samples which is 10 ppb as reported
by Pare (1997) or even less. In Malaysia, the regulatory limit for total aflatoxins in
groundnut is 15 ppb. The regulatory for other foods and milk is 10 ppb and 0.05 ppb
respectively (Malaysian Food Act, 1983).
1.2 AFLATOXINS
1.2.1 Aflatoxins in General
Aflatoxins are a group of heterocyclic, oxygen-containing mycotoxins that

possess the bisdifuran ring system. It was discovered some 43 years ago in England
3
following a poisoning outbreak causing 100,000 turkeys death (Miller, 1987 and
Cespedez and Diaz, 1997). The aflatoxins are the most widely distributed fungal toxins
in food. The occurrence of the aflatoxins in food products demonstrated that the high
levels of aflatoxins are significant concern both for food traders and food consumers
(Tozzi et al. 2003; Herrman, 2004; Haberneh, 2004). Aflatoxin is a by-product of mold
growth in a wide range of agriculture commodities such as peanuts (Urano et al., 1993),
maize and maize based food (Papp et al., 2002; Mendez-Albores et al., 2004),
cottonseeds (Pons and Franz, 1977), cocoa (Jefferey et al., 1982), coffee beans (Batista et
al., 2003), medical herbs ( Reif and Metzger, 1998; Rizzo et al., 2004), spices
(Erdogen, 2004; Garner et al., 1993; Akiyama et al., 2001; Aziz et al., 1998), melon
seeds (Bankole et al., 2004) and also in human food such as rice (Shotwell et al., 1966;
Begum and Samajpati, 2000), groundnut (Bankole et al., 2005), peanut products ( Patey
et al., 1990), corn ( Shotwell and Goulden, 1977; Urano et al., 1993 ), vegetable oil
(Miller et al., 1985), beer (Scott and Lawrence, 1997), dried fruits ( Abdul Kadar et al.,
2004, Arrus et al. (2004), milk and dairy products (Kamkar, 2004; Aycicek et al. 2005;
Sarimehmetoglu et al., 2004; Martin and Martin, 2004 ). Meat and meat products are
also contaminated with alfatoxins when farm animals are fed with aflatoxin contaminated
feed (Miller, 1987 and Chiavaro et al., 2001).
The molds that are major producers of aflatoxin are Aspergillus flavus
(Bankole et al. 2004) and Aspergillus parasiticus (Begum and Samajpati, 2000;
Setamou et al., 1997; Erdogen, 2004; Gourama and Bullerman, 1995). Aspergillus
flavus, which is ubiquitous, produces B aflatoxins (Samajphati, 1979) while Aspergillus
parasiticus, which produces both B and G aflatoxins, has more limited distribution
(Garcia-Villanova et al., 2004). A picture of Aspergillus flavus seen under an electron
microscope is shown in Figure 1.0.
Black olive is one of the substrate for Aspergillus parasiticus growth and
aflatoxin B1 production as reported by Leontopoulos et al., (2003). Biosynthesis of
aflatoxins by this fungi depends on the environmental condition such as temperature and
humidity during crop growth and storage (Leszczynska et al., 2000; Tarin et al., 2004
4
and Pildain et al., 2004). The optimum temperatures for aflatoxins growth are 27.84 0 C
and 27.30 0 C at pH=5.9 and 5.5 respectively.
Figure 1.0: Aspergillus flavus seen under an electron microscope
Before harvest, the risk for the development of aflatoxins is greatest during major
drought (Turner et al., 2005). When soil moisture is below normal and temperature is
high, the number of Aspergillus spores in the air increases. These spores infect crops
through areas of damage caused by insects and inclement weather. Once infected, plant
stress occurs, which favor the production of aflatoxins. Fungal growth and aflatoxins
contamination are the sequence of interactions among the fungus, the host and the
environment. The appropriate combination of water stress, high temperature stress and
insect damage of the host plant are major determining factors in mold infestation and
toxin production (Faraj et al., 1991; Koehler, 1985; Park and Bullerman, 1983).
Additional factors such as heat treatment, modified-atmosphere packaging or the
presence of preservative, also contribute in increasing growth rate of the aflatoxins.
Farmers have minimal control over some of these environmental factors.

However appropriate pre-harvest and post-harvest management and good agricultural
practice, including crop rotation, irrigation, timed planting and harvesting and the use of
pesticides are the best methods for preventing or controlling aflatoxins contamination
5
(Turner et al., 2005). Timely harvesting could reduce crop moisture to a point where the
formation of the mould would not occur. For example harvesting corn early when
moisture is above 20 percent and then quickly drying it to a moisture level of at least 15
percent will keep the Aspergillus flavus from completing its life cycle, resulting in lower
aflatoxin concentration. Aflatoxins are to be found in agricultural products as a
consequence of unprosperous storage conditions where humidity of 70 -90 % and a
minimum temperature of about 10° C. Commodities that have been dried to about 12 to
0.5 % moisture are generally considered stable, and immune to any risk of additional
aflatoxins development. Moreover, the minimum damage of shells during mechanized
harvesting of crop reduces significantly the mould contamination. Biocontrol of
aflatoxin contamination is another way to reduce this contamination. The natural ability
of many microorganisms including bacteria, actinomycetes, yeasts, moulds and algae has
been a source for bacteriological breakdown of mycotoxins. The most active organism
such as Flavobacterium aurantiacum which in aqueous solution can take up and
metabolise aflatoxins B1, G1 and M1 (Smith and Moss, 1985).
Production of aflatoxins is greatly inhibited by propionic acid as revealed by

Molina and Gianuzzi (2002) when they studied the production of aflatoxins in solid
medium at different temperature, pH and concentration of propionic acid. It also can be
inhibited by essential oil extracted from thyme as found by Rasooli and Abnayeh (2004).
Other chemicals that can inhibit the growth of this fungus are ammonia, copper sulphate
and acid benzoic (Gowda et al., 2004).
1.2.2 Chemistry of Aflatoxins
Aflatoxins can be classified into two broad groups according to chemical

structure which are difurocoumarocyclopentenone series and ifurocoumarolactone
(Heathcote, 1984). They are highly substituted coumarin derivatives that contain a fused
dihydrofuran moiety. The chemical structure of coumarin is shown in Figure 1.1.
6
O O
Figure 1.1 Chemical structure of coumarin
There are six major compounds of aflatoxin such as aflatoxin B1 (AFB1),

aflatoxin B2 (AFB2), aflatoxin G1 (AFG2), aflatoxin G2 (AFG2), aflatoxin M1 (AFM1)
and aflatoxin M2 (AFM2) (Goldblatt, 1969). The former four are naturally found
aflatoxins and the AFM1 and AFM2 are produced by biological metabolism of AFB1
and AFB2 from contaminated feed used by animals. They are odorless, tasteless and
colorless. The scientific name for these aflatoxin compounds are listed in Table 1.0.
Aflatoxins have closely similar structures and form a unique group of highly oxygenated,
naturally occurring heterocyclic compounds. The chemical structures of these aflatoxins
are shown in Figure 1.2. The G series of aflatoxin differs chemically from B series by
the presence of a β-lactone ring, instead of a cyclopentanone ring. Also a double bond
that may undergo reduction reaction is found in the form of vinyl ether at the terminal
furan ring in AFB1 and AFG1 but not in AFB2 and AFG2. However this small
difference in structure at the C-2 and C-3 double bond is associated with a very
significant change in activity, whereby AFB1 and AFG1 are carcinogenic and
considerably more toxic than AFB2 and AFG2. The dihydrofuran moiety in the structure
is said to be of primary importance in producing biological effects. Hydroxylation of the
bridge carbon of the furan rings for AFM1 does not significantly alter the effects of the
compounds. The absolute configuration of AFB2 and AFG2 follows from the fact that it
is derived from the reduction of AFB1 and AFG1 respectively.
AFB is the aflatoxin which produces a blue color under ultraviolet while AFG
produces the green color. AFM produces a blue-violet fluorescence while AFM2
produces a violet fluorescence (Goldblatt, 1969). Relative fluorescence of aflatoxins in
several organic solvents are shown in Appendix A (White and Afgauer, 1970). The
7
Table 1.0 Scientific name for aflatoxin compounds
Aflatoxin B1 2,3,6a,9a-tetrahydro-4-methoxycyclopenta[c]
(AFB1) furo[3’,2’:4,5]furo[2,3-h][l] benzopyran-1,11-dione
Aflatoxin B2 2,3,6a,8,9,9a-Hexahydro-4-methoxycyclopenta[c]
Aflatoxin G1 3,4, 7a,10a-tetrahydro-5-methoxy-1H, 12H

(AFG1) furo[3’,2’:4,5]furo[2,3-h]pyrano[3,4-c]l]- benzopyran-
1,12-dione
Aflatoxin G2 3,4,7a,8,9,10, 10a-Hexahydro-5-methoxy-1H,12H-

(AFG2) furo[3’,2’:4,5]furo[2,3-h]pyrano[3,4-c][l]- benzopyran-
1,12-dione
Aflatoxin 4-Hydroxy AFB1

M1
(AFM1)

M2
natural fluorescence of aflatoxins arises from their oxygenated pentaheterocyclic

structure. The fluorescence capacity of AFB2 and AFG2 is ten times larger than that of
AFB1 and AFG1, probably owing to the structural difference, namely double bond on the
furanic ring. Such a double bond seems to be very important for the photophysical
8
O O
O O
O
O
O O
O O
O
O
(a) (b)
O O O O
O O O O
O O
O O
O O
(c) (d)
O
O O
O
O
O
OH
OH
O O
O O
O
O
(e) (f)
Figure 1.2 Chemical structures of (a) AFB1, (b) AFB2, (c) AFG1, (d) AFG2,
(e) AFM1 and (f) AFM2.
properties of these derivatives measured just after spectroscopic studies (Cepeda et al.,
1996). The excitation of the natural fluorescence of AFB1 and AFG1 can be promoted
9
in many different ways such as post-column iodination (Tuinstra and Haasnoot, 1983;
Davis and Diener, 1980), post-column bromination (Kok et al.,1986; Kok, 1994 ;
Versantroort et al., 2005 ), use of cyclodextrin compound (Cepeda et al., 1996; Chiavaro
et al., 2001; Franco et al., 1998) and trifluoroacetic acid, TFA (Stack and Pohland,
1975; Takahashi, 1977a; Haghighi et al., 1981; Nieduetzki et al., 1994 ).
AFB1 and AFG1 form hemiacetals, AFB2a and AFG2a when reacted with acidic
solution such as triflouroacetic acid (TFA) as represented in Figure 1.3 (Joshua, 1993).
The hydroxyaflatoxins are unstable and tend to decompose to yellow products in the
presence of air, light and alkali. Their UV and visible spectra are similar to those of the
major aflatoxins.
O O
O O
O O
TFA
HO
O O O
O
O
O
(a) (c)
O O
O O
O O TFA O O
HO
O O
O O O O
(b) (d)
Figure 1.3 Hydration of AFB1 (a) and AFG1 (b) by TFA produces AFB2a (c) and
AFG2a (d) (Joshua, 1993).
The close relationship between AFB1, AFG1, AFB2a and AFG2a was shown by the
similarities in their IR and UV spectra. The main difference between AFB2a and AFG2a
with AFB1 and AFG1 are found in the IR spectra, where an additional band at 3620 cm-1
10
indicates the presence of a hydroxyl group in AFB2a and AFG2a. The absence of bands
at 3100, 1067 and 722 cm-1 (which arise in AFB1 and AFG1 from the vinyl ether group)
indicates that the compounds are hydroxyl derivatives of AFB2 and AFG2. Some
chemical and physical properties of aflatoxin compounds are listed in Table 1.1
(Heathcote and Hibbert, 1978; Weast and Astle, 1987). The close relationship between
these aflatoxins was shown by the similarities in their UV and IR spectra as shown in
Appendix B and C respectively.
Table 1.1 Chemical and physical properties of aflatoxin compounds
AFB1 AFB2 AFG1 AFG2
Molecular C17H12O6 C17H14O6 C17H12O7 C17H14O7

formula
Molecular weight 312 314 328 330
Crystals Pale yellow Pale yellow Colorless Colorless
Melting point 268.9 286.9 244.6 237.40

(°C)
Fluorescence Blue Blue Green Green

under UV light
Solubility Soluble in water and polar organic solvent. Normal solvents

are: methanol, water: acetonitrile (9:1), trifluoroacetic acid,
methanol: 0.1N HCl (4:1), DMSO and acetone
Other properties Odorless, colorless and tasteless in solution form.

Incompatible with strong acids, strong oxidising agents and
strong bases. Soluble in water, DMSO, 95% acetone or
ethanol for 24 hours under ambient temperature
UNIVERSITI TEKNOLOGI MALAYSIA

BAHAGIAN A – Pengesahan Kerjasama *
Adalah disahkan bahawa projek penyelidikan tesis ini telah dilaksanakan melalui
kerjasama antara _____________________ dengan _________________________
Disahkan oleh:
Nama : ..........................................................
Jawatan:...........................................................
(Cop rasmi)
* Jika penyediaan tesis/projek melibatkan kerjasama.
BAHAGIAN B – Untuk Kegunaan Pejabat Sekolah Pengajian Siswazah
Tesis ini telah diperiksa dan diakui oleh:
Nama dan Alamat

Pemeriksa Luar : Prof. Dr. Noor Azhar Bin Mohd Shazili
Pengarah
Institut Oseanografi,
Kolej Universiti Sains dan Teknologi Malaysia,
Mengabang Telipot
21030 Kuala Terengganu
Nama dan Alamat

Pemeriksa Dalam I : Prof. Madya Dr. Razali Bin Ismail
Fakulti Sains,
UTM, Skudai
Pemeriksa Dalam II :
Nama Penyelia Lain

(jika ada) :
Disahkan oleh Penolong Pendaftar di Sekolah Pengajian Siswazah:
Nama : .GANESAN A/L ANDIMUTHU

A thesis submitted in fulfilment of the

requirements for the award of the degree of
Doctor of Philosophy
Faculty of Science
Universiti Teknologi Malaysia
APRIL 2006
ii
iii
Specially dedicated to:
My mother, wife, sons, daughters and all families for

all the love, support and continuous prayer
for my success in completing this work.
iv
ACKNOWLEDGEMENT
All praise be to ALLAH SWT and blessing be upon His Prophet SAW whose
ultimate guidance creates a more meaningful purpose to this work.
I wish to express my sincere gratitude and appreciation to the people who have both
directly and indirectly contributed to this thesis. The following are those to whom I
am particularly indebted:
My supervisors A.P. Dr. Abdull Rahim bin Hj. Mohd. Yusoff and Prof. Dr.
Rahmalan Ahamad for their invaluable guidance, freedom of work and
constant encouragement throughout the course of this work.
School Of Health Sciences, USM, Health Campus, Kubang Krian Kelantan

for awarding study leave together with scholarship in completing the work.
Prof Baharuddin Saad from USM Penang, AP Dr. Razali Ismail from
Chemistry Department, Faculty of Science, UTM and Prof Barek from
Charles University, Prague, Czeck Republic for their useful discussion and
suggestion. Also to Mr Radwan Ismail from Department of Chemistry,
Penang Branch, Mrs Marpongahtun Misni and Mr Wan Kamaruzaman Wan
Ahmad for their friendship, ideas and continuous support in carrying out this
work. Also to Mr Mat Yasin bin Sirin, Mrs Ramlah binti Husin and Mr Azmi
Mahmud for their assistance throughout the work.
UTM for awarding Short Term Grant No: 75152 / 2004
My mother, wife and all families for their encouragements, supports, patience,
tolerance and understanding.
v
ABSTRACT
Aflatoxin, which is produced by Aspergillus flavus and Aspergillus parasiticus

fungi is one of the compounds in the mycotoxin group. The main types of aflatoxins
are AFB1, AFB2, AFG1 and AFG2 which have carcinogenic properties and are
dangerous to human health. Various techniques have been used for their measurements
such as the high performance liquid chromatography (HPLC), enzyme linked
immunosorbant assay (ELISA) and radioimmunoassay (RIA) but all these methods
have disadvantages such as long analysis time, consume a lot of reagents and
expensive. To overcome these problems, the voltammetric technique was proposed in
this study using controlled growth mercury drop (CGME) as the working electrode and
Britton Robinson buffer (BRB) as the supporting electrolyte. The voltammetric
methods were used for investigating the electrochemical properties and the quantitative
analysis of aflatoxins at the mercury electrode. The experimental conditions were
optimised to obtain the best characterised peak in terms of peak height with analytical
validation of the methods for each aflatoxin. The proposed methods were applied for
the analysis of aflatoxins in groundnut samples and the results were compared with
those obtained by the HPLC technique. All aflatoxins were found to adsorb and
undergo irreversible reduction reaction at the working mercury electrode. The
optimum experimental parameters for the differential pulse cathodic stripping
voltammetry (DPCSV) method were the BRB at pH 9.0 as the supporting electrolyte,
initial potential (Ei): -0.1 V, final potential (Ef): -1.4 V, accumulation potential (Eacc): -
0.6 V, accumulation time (tacc): 80 s, scan rate: 50 mV/s and pulse amplitude: 80 mV.
The optimum parameters for the square wave stripping voltammetry (SWSV) method
were Ei = -0.1 V, Ef = -1.4 V, Eacc: -0.8 V, tacc: 100 s, scan rate: 3750 mV/s, frequency:
125 Hz and voltage step: 30 V. At the concentration of 0.10 µM, using DPCSV
method with the optimum parameters, AFB1, AFB2, AFG1 and AFG2 produced a
single peak at -1.21 V, -1.23 V, -1.17 V and -1.15 V (versus Ag/AgCl) respectively.
Using the SWSV method, a single peak appeared at -1.30 V for AFB1 and AFB2 while
-1.22 V for AFG1 and AFG2. The calibration curves for all aflatoxins were linear with
the limit of detection (LOD) of approximately 2.0 ppb and 0.50 ppb obtained by the
DPCSV and SWSV methods respectively. The results of aflatoxins content in
individual groundnut samples do not vary significantly when compared with those
obtained by the HPLC technique. Finally, it can be concluded that both proposed
methods which are accurate, precise, robust, rugged, fast and low cost were
successfully developed and are potential alternative methods for routine analysis of
aflatoxins in groundnut samples.
vi
ABSTRAK
Aflatoksin adalah sejenis sebatian yang dihasilkan oleh kulat Aspergillus flavus
dan Aspergillus parasiticus yang digolongkan di dalam kumpulan mikotoksin. Jenis
utama aflatoksin adalah AFB1, AFB2, AFG1 dan AFG2 yang bersifat karsinogen serta
merbahaya kepada kesihatan manusia. Pelbagai teknik telah digunakan untuk
menentukan aflatoksin seperti kromatografi cecair prestasi tinggi (HPLC), asai serapan
imuno berikatan enzim (ELISA) dan radioamunoasai (RIA) tetapi teknik-teknik ini
mempunyai kelemahan seperti masa analisis yang panjang, melibatkan reagen yang
banyak dan kos yang mahal. Untuk mengatasi masalah ini, teknik voltammetri telah
dicadangkan untuk kajian aflatoksin menggunakan titisan raksa pembesaran terkawal
(CGME) sebagai elektrod bekerja dan larutan penimbal Britton-Robinson (BRB)
sebagai elektrolit penyokong. Pelbagai kaedah voltammetri telah digunakan untuk
mengkaji sifat elektrokimia aflatoksin pada elektrod raksa dan analisis kuantitatifnya.
Parameter kajian telah dioptimumkan untuk memperolehi puncak yang elok
berdasarkan ketinggian puncak serta pengesahan analisis untuk kaedah yang
dibangunkan bagi setiap aflatoksin. Kaedah ini telah digunakan untuk menentukan
kandungan aflatoksin di dalam sampel kacang tanah di mana keputusan yang diperolehi
telah dibandingkan dengan keputusan HPLC. Semua aflatoksin yang dikaji didapati
terjerap dan menjalani proses tindakbalas penurunan tidak berbalik pada elektrod raksa.
Parameter optimum untuk kaedah voltammetri perlucutan kathodik denyut pembeza
(DPCSV) adalah larutan BRB pada pH 9.0 sebagai larutan elektrolit, keupayaan awal
(Ei): -1.0 V, keupayaan akhir (Ef): -1.4 V, keupayaan pengumpulan (Eacc): -0.6 V, masa
pengumpulan (tacc): 80 s, kadar imbasan: 50 mV/s dan amplitud denyut: 80 mV. Untuk
kaedah voltammetri perlucutan gelombang bersegi (SWSV), parameter optimum adalah
Ei : -1.0 V, Ef : -1.4 V, Eacc: -0.8 V, tacc: 100 s, kadar imbasan: 3750 mV/s, frekuensi:
125 Hz dan beza keupayaan: 30 mV. Menggunakan parameter optimum untuk DPCSV,
0.10 µM AFB1, AFB2, AFG1 dan AFG2 menghasilkan puncak tunggal pada
keupayaan -1.21 V, -1.23 V, -1.17 V dan -1.15 V (melawan Ag/AgCl) masing-
masingnya. Menggunakan kaedah SWSV, puncak terhasil pada -1.30 V untuk AFB1
dan AFB2, -1.22 V untuk AFG1 dan AFG2. Keluk kalibrasi adalah linear untuk semua
aflatoksin dengan had pengesanan (LOD) pada 2.0 dan 0.5 ppb diperolehi dari kaedah
DPCSV dan SWSV masing-masingnya. Keputusan analisis kandungan aflatoksin di
dalam sampel kacang tanah tidak memberi perbezaan ketara berbanding dengan yang
diperolehi menggunakan teknik HPLC. Kesimpulannya, kedua-dua kaedah yang dikaji
yang merupakan kaedah yang tepat, jitu, cepat, sesuai digunakan dengan pelbagai
model voltammetri dan kos yang murah telah berjaya dibangunkan dan berpotensi besar
menjadi kaedah alternatif untuk analisis kandungan aflatoksin di dalam kacang tanah
secara berkala.
vii
TABLE OF CONTENTS
CHAPTER TITLE PAGE
TITLE i
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES xiii
LIST OF FIGURES xvi
ABBREVATIONS xxix
LIST OF APPENDICES xxxiii
1 LITERATURE REVIEW 1
1.1 Overview 1
1.2 Aflatoxins 3
1.2.1 Aflatoxins in general 3
1.2.2 Chemistry of aflatoxins 5
1.2.3 Health aspects of aflatoxins 12
1.2.4 Analytical methods for the determination of 16
aflatoxins
1.2.5 Electrochemical properties of aflatoxins 28
1.3. Voltammetric technique 30
1.3.1 Voltammetric techniques in general 30
viii
1.3.2 Voltammetric measurement 31

1.3.2.1 Instrumentation 31
1.3.2.2 Solvent and supporting electrolyte 44
1.3.2.3 Current in voltammetry 46
1.3.2.4 Quantitative and quantitative aspects of 48
voltammetry
1.3.3 Type of voltammetric techniques 49
1.3.3.1 Polarography 49
1.3.3.2 Cyclic voltammetry 51
1.3.3.3 Stripping voltammetry 54
1.3.3.3a Anodic stripping voltammetry 56
1.3.3.3b Cathodic stripping voltammetry 57
1.3.3.3c Adsorptive stripping voltammetry 58
1.3.3.4 Pulse voltammetry 59
1.3.3.4a Differential pulse voltammetry 60
1.3.3.4b Square wave voltammetry 61
1.4 Objective and scope of study 64
1.4.1 Objective of study 64
1.4.2 Scope of study 67
2 RESEARCH METHODOLOGY 70
2.1 Apparatus, material and reagents 70

2.1.1 Apparatus 70
2.1.2 Materials 72
2.1.2.1 Aflatoxin stock and standard solutions 72
2.1.2.2 Real samples 73
2.1.3 Reagents 73
2.1.3.1 Britton Robinson buffer, 0.04 M 73
2.1.3.2 Carbonate buffer, 0.04 M 74
2.1.3.3 Phosphate buffer, 0.04 M 74
ix
2.1.3.4 Ascorbic acid 74

2.1.3.5 β-cyclodextrin solution, 1.0 mM 75
-5
2.1.3.6 L-Cysteine, 1.0 x 10 M 75
2.1.3.7 2,4-dihydrofuran, 0.15 M 75
-2
2.1.3.8 Coumarin, 3.0 x 10 M 75
2.1.3.9 Poly-L-lysine, 10 ppm 75
2.1.3.10 Standard aluminium (II) solution, 1.0 mM 75
2.1.3.11 Standard plumbum(II) solution, 1.0 mM 76
2.1.3.12 Standard zinc (II) solution, 1.0 mM 76
2.1.3.13 Standard copper (II) solution, 1.0 mM 76
2.1.3.14 Standard nickel (II) solution, 1.0 mM 76
2.1.3.15 Methanol: 0.1 N HCl solution, 95% 76
2.1.3.16 Zinc sulphate solution, 15% 76
2.2 Analytical Technique 77

2.2.1 General procedure for voltammetric analysis 77
2.2.2 Cyclic voltammetry (Anodic and cathodic 77
directions)
2.2.2.1 Standard addition of sample 77
2.2.2.2 Repetitive cyclic voltammetry 78
2.2.2.3 Effect of scan rate 78
2.2.3 Differential pulse cathodic stripping 78
voltammetric determination of AFB2
2.2.3.1 Effect of pH 79
2.2.3.2 Method optimisation for the determination 79
of AFB2
2.2.3.2b Effect of accumulation potential 80
2.2.3.2c Effect of accumulation time 80
x
2.2.3.3 Method validation 80

2.2.3.4 Interference studies 81
2.2.3.4a Effect of Cu(II), Ni (II), Al(III), 81
Pb(II) and Zn(II)
2.2.3.4b Effect of ascorbic acid, 82
β-cyclodextrin and L-cysteine
2.2.3.5 Modified mercury electrode with PLL 82
2.2.4 Square wave cathodic stripping voltammetry 82
(SWSV)
2.2.4.1 SWSV parameters optimisation 82
2.2.4.2 SWSV determination of all aflatoxins 82
2.2.5 Stability studies of aflatoxins 83
2.2.5.3 Stability of 0.1 µM aflatoxins exposed 83
to ambient temperature
2.2.5.4 Stability of 0.1 µM aflatoxins in different 84
pH of BRB
2.2.6 Application to food samples 84
2.2.6.4 Blank measurement 85
2.2.6.5 Recovery studies 85
2.2.6.6 Voltammetric analysis 86
3 RESULTS AND DISCUSSION 88
3.1 Cyclic voltammetric studies of aflatoxins 88

3.1.1 Cathodic and anodic cyclic voltammetric 89
of aflatoxins
xi
3.2 Differential pulse cathodic stripping voltammetry 102

of AFB2
3.2.1 Optimisation of conditions for the stripping 104
analysis
3.2.1.1 Effect of pH and type of supporting 104
electrolyte
3.2.1.2 Optmisation of instrumental conditions 117
3.2.1.2b Effect of accumulation time 119
3.2.1.2c Effect of accumulation 120
potential
3.2.2 Analysis of aflatoxins 127
3.2.2.1 Calibration curves of aflatoxins and 129
validation of the proposed method
3.2.2.1a Calibration curve of AFB2 129
3.2.2.1b Calibration curve of AFB1 134
3.2.2.1c Calibration curve of AFG1 137
3.2.2.1d Calibration curve of AFG2 140
3.2.2.2 Determination of limit of detection 143
3.2.2.3 Determination of limit of quantification 147
3.2.2.4 Inteference studies 150
3.3 Square-wave stripping voltammetry (SWSV) of 157
aflatoxins
3.3.1 SWSV determination of AFB2 158
3.3.1.1 Optimisation of experimental and 159
instrumental SWSV parameters
3.3.3.1a Influence of pH of BRB 159
3.3.3.1b Effect of instrumental 160
variables
xii
3.3.2 SWSV determination of other aflatoxins 166

3.3.3 Calibration curves and method validation 168
3.4 Stability studies of aflatoxins 175
3.4.1 10 ppm aflatoxin stock solutions 175
3.4.2 1 ppm aflatoxins in BRB at pH 9.0 179
3.4.2.1 Month to month stability studies 179
3.4.2.2 Hour to hour stability studies 181
3.4.2.3 Stability studies in different pH 186
of BRB
3.4.2.4 Stability studies in 1.0 M HCl and 191
1.0 M NaOH
3.5 Voltammetric analysis of aflatoxins in real samples 192
3.5.1 Study on the extraction techniques 193
3.5.2 Analysis of blank 194
3.5.3 Recovery studies of aflatoxins in real samples 196
3.5.4 Analysis of aflatoxins in real samples 199
4 CONCLUSIONS AND RECOMMENDATIONS 204
4.1 Conclusions 204

4.2 Recommendations 206
REFERENCES 208
Appendices A – AM 255 - 317
xiii
LIST OF TABLES
TABLE NO. TITLE PAGE
1.0 Scientific name for aflatoxin compounds 7
1.1 Chemical and physical properties of aflatoxin 10

compounds
1.2 Summary of analysis methods used for 19

determination of aflatoxins in various samples
1.3 Working electrode and limit of detection for 32

modern polarographic and voltammetric
techniques.
1.4 The application range of various analytical 33

techniques and their concentration limits when
compared with the requirements in different
fields of chemical analysis
1.5 List of different type of working electrodes and 36

its potential windows
1.6 Electroreducible and electrooxidisable organic 50

functional groups
1.7 The characteristics of different type of 53

electrochemical reaction.
1.8 Application of Square Wave Voltammetry 62

technique
2.0 List of aflatoxins and their batch numbers 72

used in this experiment
2.1 Injected volume of aflatoxins into eluate of 86

groundnut and the final concentrations obtained
in voltammetric cell
3.0 The dependence of current peaks of aflatoxins to 97

their concentrations obtained by cathodic cyclic
measurements in BRB at 9.0.
xiv
3.1 Effect of buffer constituents on the peak height 109

of 2.0 µM AFB2 at pH 9.0. Experimental
conditions are the same as Figure 3.21
3.2 Compounds reduced at the mercury electrode 116
3.3 Optimum parameters for 0.06 µM and 2.0 µM 126

AFB2 in BRB at pH 9.0.
3.4 The peak height and peak potential of aflatoxins 127

obtained by optimised parameters in BRB at
pH 9.0 using DPCSV technique.
µM by the proposed voltammetric procedure (n=8).

solution (n=3).
3.7 Influence of small variation in some of the 133

assay condition of the proposed procedure on its
suitability and sensitivity using 0.10 µM AFB2.
3.8 Results of ruggedness test for proposed method 134

using 0.10 µM AFB2.
µM AFB1 by proposed voltammetric procedure
(n=5).

solution (n=3).
µM AFG1 by proposed voltammetric procedure
(n=5).

solution (n=3).
µM AFG2 by proposed voltammetric procedure.
xv

solution (n=5).
3.15 Peak height and peak potential of 0.10 µM 143

aflatoxins obtained by BAS and Metrohm
voltammetry analysers under optimised
operational parameters for DPCSV method.
3.16 Analytical parameters for calibration curves 145

for AFB1,AFB2, AFG1 and AFG2 obtained by
DPCSV technique using BRB at pH 9.0 as the
3.17 LOD values for determination of aflatoxins 148

3.18 LOQ values for determination of aflatoxins 149

3.19 Peak current and peak potential for all aflatoxins 167
obtained by SWSV in BRB at pH 9.0 (n=5).
3.20 Analytical parameters for calibration curves for 172

AFB1, AFB2, AFG1 and AFG2 obtained by
SWSV technique in BRB pH 9.0 as the
3.21 Result of reproducibility study (intra-day and inter- 173

day measurements) for 0.1 µM aflatoxins in
BRB at pH 9.0 obtained by SWSV method.
3.22 Application of the proposed method in evaluation 174

of the SWSV method by spiking the aflatoxin
standard solutions.
3.23 Average concentration of all aflatoxins within a 175

year stability studies.
3.24 The peak current and peak potential of 10 ppb 196

AFB2 in presence and absence of a blank sample.
3.25 Total aflatoxin contents in real samples which 203

were obtained by DPCSV and HPLC
techniques (average of duplicate analysis)
xvi
LIST OF FIGURES
FIGURE NO. TITLE PAGE
1.0 Aspergillus flavus seen under an electron 4

microscope.
1.1 Chemical structure of coumarin 6
1.2 Chemical structures of (a) AFB1, (b) AFB2, 8

(c) AFG1, (d) AFG2, (e) AFM1 and (f) AFM2
1.3 Hydration of (a) AFB1 and (b) AFG1 by TFA 9

produces (c) AFB2a and (d) AFG2a
1.4 Transformation of toxic (a) AFB1 to non-toxic 11

(b) aflatoxicol A.
1.5 Major DNA adducts of AFB1; (a) 8,9-Dihydro-8- 13

(N7-guanyl)-9-hydroxy-aflatoxin B1 (AFB1-Gua)
and (b) 8,9-Dihydro-8-(N5-Formyl-2’.5’.6’-
triamino-4’-oxo-N5-pyrimidyl)-9-hydroxy-Aflatoxin
B1 (AFB1-triamino-Py)
1.6 Metabolic pathways of AFB1 by cytochrom P-450 14

enzymes; B1-epoxide = AFB1 epoxide, M1=
aflatoxin M1, P1= aflatoxin P1 and Q1 = aflatoxin
Q1.
1.7 A typical arrangement for a voltammetric 34

electrochemical cell (RE: reference electrode,
WE: working electrode. AE: auxiliary electrode)
1.8 A diagram of the Hanging Mercury Drop Electrode 37

(HMDE)
1.9 A diagram of the Controlled Growth Mercury 38

Electrode (CGME)
1.10 Cyclic voltammograms of (a) reversible, 52

(b) irriversible and (c) quasireversible reaction at
mercury electrode (O = oxidised form and R = reduced
form)
xvii
1.11 The potential-time sequence in stripping analysis 55
1.12 Schematic drawing showing the Faradaic current 59

and charging current versus pulse time course
1.13 Schematic drawing of steps in DPV by 60

superimposing a periodic pulse on a linear scan
1.14 Waveform for square-wave voltammetry 61
2.0 BAS CGME stand (a) which is connected to 71

CV-50W voltammetric analyser and interface
with computer (b) for data processing
2.1 VA757 Computrace Metrohm voltammetric 71

analyser with 663 VA stand (consists of Multi
Mode (MME))
3.0 Cathodic peak current of 0.6 µM AFB1 in various 89

pH of BRB obtained in cathodic cyclic voltammetry.
Ei = 0, Elow = -1.5 V, Ehigh = 0 and scan rate = 200
mV/s)
3.1 Shifting of peak potential of AFB1 with increasing 90

pH of BRB. Parameter conditions are the same
as in Figure 3.0.

to 8.0.

to 11.0.

obtained at scan rate of 200 mV/s, Ei = 0, Elow =
-1.5 V and Ehigh = 0 in BRB solution at pH 9.0.


xviii

3.8 Effect of Ei to the Ip of 0.6 µM AFB1 in BRB at pH 94

3.9 Effect of Ei to the Ip of 0.1 µM Zn2+ in BRB at pH 94

3.10 Anodic cyclic voltammogram of 1.3 µM AFB2 95

obtained at scan rate of 200 mV/s, Ei = -1.5 V,
Elow = -1.5 V and Ehigh = 0 in BRB at pH 9.0.
3.11 Effect of increasing AFB2 concentration on the 96

peak height of cathodic cyclic voltammetrc curve
in BRB at pH = 9.0. (1.30 µM, 2.0 µM, 2.70 µM
and 3.40 µM). All parameter conditions are the
same as in Figure 3.4.
3.12 Peak height of reduction peak of AFB2 with 96

increasing concentration of AFB2. All parameter
conditions are the same as in Figure 3.4.
3.13 Repetitive cathodic cyclic voltammograms of 1.3 98

µM AFB2 in BRB solution at pH 9.0. All
3.14 Increasing Ip of 1.3 µM AFB2 cathodic peak 98

obtained from repetitive cyclic voltammetry. All
3.15 Peak potential of 1.3 µM AFB2 with increasing 99

number of cycle obtained by repetitive cyclic
voltammetry.
xix
3.16 Plot of log Ip versus log υ for 1.3 µM AFB2 in 100

BRB solution at pH 9.0. All experimental conditions
3.17 Plot of Ep versus log υ for 1.3 µM AFB2 in BRB 101

solution at pH 9.0.
3.18 Plot of Ip versus υ for 1.3 µM AFB2 in BRB 102

solution at pH 9.0. All parameter conditions
3.19 DPCS voltammograms of 1.0 µM AFB2 (Peak I) 103

in BRB at pH 9.0 (a) at tacc = 0 and 30 s. Other
parameter conditions; Ei = 0, Ef = -1.50 V,
Eacc = 0, υ =50 mV/s and pulse amplitude =
100 mV. Peak II is the Zn peak.
3.20 DPCS voltammograms of 2.0 µM AFB2 in BRB 104

in BRB (Peak I) at different pH values; 6.0, 7.0,
8.0, 9.0, 11.0and 13.0. Other parameter conditions;
Ei = 0, Ef =-1.50 V, Eacc = 0, υ = 50 mV/s and pulse
amplitude =100 mV. Peak II is the Zn peak.
3.21 Dependence of the Ip for AFB2 on the pH of 105

0.04 M BRB solution. AFB2 concentration:
2.0 µM, Ei =0, Ef = -1.5 V, E acc = 0, tacc = 30 sec,
3.22 Ip of 2.0 µM AFB2 obtained in BRB (a) at pH 106

from 9.0 decreases to 4.0 and re-increase to 9.0 and
(b) at pH from 9.0 increase to 13.0 and re-decrease
to 9.0.
3.23 UV-VIS spectrums of 1 ppm AFB2 in BRB at 107

pH (a) 6.0, (b) 9.0 and (c) 13.0.
3.24 Opening of lactone ring by strong alkali caused no 108

peak to be observed for AFB2 in BRB at pH 13.0.
3.25 Ip of 2.0 µM AFB2 in different concentration of 109

BRB at pH 9.0. Experimental conditions are
3.26 Ip of 2.0 µM AFB2 in different pH and concentrations 110

of BRB.
xx
3.27 DPCS voltammograms of 2.0 AFB2 (Peak I) 110

in (a) 0.04 M, (b) 0.08 M and (c) 0.08 M BRB
at pH 9.0 as the blank. Ei = 0 V, Ef = -1.5 V,
Eacc = 0 V, tacc = 30 sec, υ = 50mV/s and pulse
amplitude = 100 mV. Peak II is the Zn peak.
3.28 Chemical structures of (a) 2,3-dihydrofuran, 111

(b) tetrahydrofuran and (c) coumarin.
3.29 Voltammograms of 2,3-dihydrifuran (peak I) for 112

concentrations from (b) 0.02 to 0.2 µM in (a)
BRB at pH 9.0.
3.30 Dependence of Ip of coumarin to its concentrations 112
3.31 Voltammograms of coumarin at concentration of 113

(b) 34 µM, (c) 68 µM, (d) 102 µM, (e) 136 µM
and (f) 170 µM in (a) BRB at pH 9.0.
3.32 Chemical structures of (a) cortisone and (b) 114

testosterone.
3.33 Chemical structures of (a) digoxin and (b) digitoxin 115
3.34 Effect of pH of BRB solution on the Ep for AFB2. 117

AFB2 concentration: 2.0 µM. Ei = 0, Ef = -1.5 V,
Eacc = 0, t acc = 30 s and υ = 50 mV/s.
3.35 Effect of various υ to the (a) Ip and (b) Ep of 2.0 µM 118

AFB2 peak in BRB at pH 9.0. Ei = 0, Ef = 1.50 V,
Eacc = 0, t acc = 15 s and pulse amplitude = 100 mV.

peak in BRB at pH 9.0. Ei = 0, Ef = 1.50 V, Eacc = 0,
3.37 The relationship between (a) Ip and (b) Ep with 120

Eacc for 2.0 µM AFB2 in BRB at pH 9.0.
Ei = 0, Ef = -1.5 V, tacc = 15 s, υ = 40 mV/s and
3.38 Effect of Ei on (a) Ip and (b) Ep of 2.0 µM AFB2 121

in BRB at pH 9.0. Ef = 1.50 V, Eacc = -0.80 V,
tacc = 40 s,υ = 40 mV/s and pulse amplitude =
100 mV.
xxi
3.39 Effect of pulse amplitude on (a) Ip and (b) Ep 122

of 2.0 µM AFB2 in BRB at pH 9.0. Ei = -1.0 V,
Ef = 1.50 V, Eacc = -0.80 V, tacc = 40 s and υ =
40 mV/s.
3.40 Effect of Eacc on Ip of 0.06 µM AFB2. Ei = -1.0 V, 123

Ef = -1.50 V, tacc = 40 s, υ = 40 mV/s and pulse
amplitude = 100 mV.

in BRB at pH 9.0. Ei = -1.0 V, Ef = -1.50 V, Eacc =
-0.6 V, υ = 40 mV/s and pulse amplitude = 100 mV.
3.42 The effect υ on (a) Ip and (b) Ep of 0.06 µM AFB2 125

In BRB at pH 9.0. Ei = -1.0 V, E f = -1.50 V, Eacc =
-0.6 V, tacc = 80 s and pulse amplitude = 100 mV.

(a) optimised and (b) unoptimised parameters in BRB
at pH 9.0.
3.44 Voltammograms of 0.1 µM (a) AFB1, (b) AFG1, 128

(c) AFB2 and (d) AFG2 in BRB at pH 9.0. Ei = 1.0
(except for AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.6 V,
3.45 Voltammograms of (b) mixed aflatoxins in (a) BRB at 129

pH 9.0 as the blank. Parameters condition: Ei = -0.95 V,
Ef = -1.4 V, Eacc = -0.6 V, t acc = 80 s, υ = 50 mV/s and
Pulse amplitude = 80 mV.
3.46 Increasing concentration of AFB2 in BRB at pH 9.0. 130


3.48 Standard addition of AFB1 in BRB at pH 9.0. 135

xxii


pH 9.0. Ei = -0.95 V, Ef = -1.40 V, Eacc =-0.8 V,


pH 9.0. Ei = -1.0 V, Ef = -1.40 V, Eacc =-0.8 V,

3.54 Voltammograms of 0.1 µM (a) AFB1, (b) AFB2, 144

(c) AFG1 and (d) AFG2 in BRB at pH 9.0
(d) obtained by 747 VA Metrohm. Ei = -1.0 V (except
3.55 Ip of all aflatoxins with increasing concentration of 150

Zn 2+ up to 1.0 µM.
3.56 Voltammograms of (i) 0.1µM AFB2 and (ii) AFB2-Zn 151

complex with increasing concentration of Zn2+ (a = 0,
b = 0.75 µM, c = 1.50 µM, d = 2.25 µM and e = 3.0 µM).
Blank = BRB at pH 9.0. Experimental conditions;
Ei = -1.0 V, Ef = -1.40 V, Eacc = -0.6 V, tacc = 80 s,
3.57 Ip of all aflatoxins after reacting with increasing 151

concentration of Zn2+ in BRB at pH 3.0.
Measurements were made in BRB at pH 9.0 within
15 minutes of reaction time. Ei = -1.0 V (except
xxiii
3.58 Absorbance of all aflatoxins with increasing 152

concentration of Zn2+ in BRB at pH 3.0 within 15
minutes of reaction time

concentration of Zn2+ (from 0.10 to 0.50 µM) in
BRB at pH 9.0. Ei = -0.25 V, Ef = -1.4 V, Eacc =
-0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplirude =
80 mV. .
3.60 Chemical structure of ascorbic acid. 153

of ascorbic acid up to 1.0 µM. Concentrations of
all aflatoxins are 0.1µM.

concentration of ascorbic acid.

of β-cyclodextrin up to 1.0 µM.

concentration of β-cyclodextrin.
3.65 Chemical structure of L-cysteine. 156

of cysteine up to 1.0 µM.
3.67 Voltammograms of 0.1 µM AFB2 obtained by 158

(a) DPCSV and (b) SWSV techniques in BRB at
pH 9.0. Parameters for DPCSV: Ei = -1.0 V, Ef =
-1.40V, Eacc = -0.6 V, t acc = 80 s, υ = 50 mV/s and
pulse amplitude = 80 mV and for SWSV: Ei = -1.0 V,
Ef = -1.40V, Eacc = -0.6 V, tacc = 80 s, frequency =
50 Hz, voltage step = 0.02 V, amplitude = 80 mV
and υ = 1000 mV/s.
3.68 Influence of pH of BRB on the Ip of 0.10 µM 159

AFB2 using SWSV technique. The instrumental
Parameters are the same as in Figure 3.67.
3.69 Voltammograms of 0.1 µM AFB2 in different pH 160

of BRB. Parameter conditions: Ei = -1.0 V, E f =
-1.40 V, Eacc = -0.6 V, tacc = 80 s, voltage step =
xxiv
0.02 V, amplitude = 50 mV, frequency = 50 Hz

and υ = 1000 mV/s.
3.70 Effect of Ei to the Ip of 0.10 µM AFB2 in BRB at 160

pH 9.0.
3.71 Effect of Eaccto the Ip of 0.10 µM AFB2 in BRB at 161

pH 9.0.
3.72 Relationship between Ip of 0.10 µM AFB2 while 162

increasing tacc.
3.73 Effect of frequency to the Ip of 0.10 µM AFB2 in 162

BRB at pH 9.0.
3.74 Linear relatioship between Ip of AFB2 and square 163

root of frequency.
3.75 Influence of square-wave voltage step to Ip of 163

0.10 µM AFB2.
3.76 Influence of square-wave amplitude to Ip of 0.10 µM 164

AFB2.
3.77 Relationship of SWSV Ep of AFB2 with increasing 164

amplitude.
3.78 Ip of 0.10 µM AFB2 obtained under (a) non-optimised 165

and (b) optimised SWSV parameters compared with
that obtained under (c) optimised DPCSV parameters.

(a) non-optimised and (b) optimised SWSV
parameters compared with that obtained under (c)
optimised DPCSV parameters.
3.80 Ip of 0.10 µM aflatoxins obtained using two 167

different stripping voltammetric techniques under
their optimum paramter conditions in BRB at pH 9.0.
3.81 Voltammograms of (i) AFB1, (ii) AFB2, (iii) AFG1 168

and (iV) AFG2 obtained by (b) DPCSV compared
with that obtained by (c) SWSV in (a) BRB at pH 9.0.
3.82 SWSV voltammograms for different concentrations 169

of AFB1 in BRB at pH 9.0. The broken line
xxv
represents the blank: (a) 0.01 µM, (b) 0.025 µM,

(c) 0.05 µM, (d) 0.075 µM, (e) 0.10 µM, (f) 0.125
µM and (g) 0.150 µM. Parameter conditions:
Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.8 V, t acc = 100 s,
frequency = 125 Hz, voltage step = 0.03 V, pulse
amplitude = 75 mV and scan rate = 3750 mV/s.

method.

method.

method.

method.
3.87 LOD for determination of aflatoxins obtained by 171

two different stripping methods.

benzene: acetonitrile (98%) at preparation date.

storage time.

storage time.
3.91 UV-VIS spectrums of 10 ppm AFB1 (a) kept in 178

the cool and dark conditions and (b) exposed to
ambient conditions for 3 days.

10 ppm AFB1 stock solution.

10 ppm AFB1 stock solution after 2 week stored in
the cool and dark conditions.
xxvi
3.94 Percentage of Ip of 0.10 µM aflatoxins in BRB at pH 180

9.0 at different storage time in the cool and dark
conditions.
3.95 Percentage of Ip of all aflatoxins in BRB at pH 9.0 181

exposed to ambient conditions up to 8 hours of
exposure time.
3.96 Reaction of 8,9 double bond furan rings in AFB1 182

with TFA, iodine and bromine under special
conditions (Kok, et al., 1986).
3.97 Voltammograms of 0.10 µM AFB1 obtained in (a) 183

BRB at pH 9.0 which were prepared from (b)
damaged and (c) fresh stock solutions.

light protected.

light protected.

light protected.

light protected.
3.102 Peak heights of 0.10 µM aflatoxins in BRB at pH 187

(a) 6.0, (b) 7.0, (c) 9.0 and (d) 11.0 exposed to
ambient conditions up to 3 hours of exposure time.
3.103 Resonance forms of the phenolate ion 187

(Heathcote, 1984).

6.0 from 0 to 3 hrs exposure time.

xxvii


3.108 Absorbance of 1.0 ppm aflatoxins in BRB at pH 190

(a) 6.0, (b) 7.0, (c) 9.0 and (d) 11.0 from 0 to 3
hours of exposure time.
3.109 The peak heights of aflatoxins in 1.0 M HCl from 191

3.110 The peak heights of aflatoxins in 1.0 M NaOH from 192

3.111 Voltammograms of real samples after extraction by 193

Technique (a) 1, (b) 2 and (c) 3 with addition of
AFB1 standard solution in BRB at pH 9.0 as a
blank.
3.112 Voltammograms of blank in BRB at pH 9.0 obtained 194

by (a) DPCSV and (b) SWSV methods.
3.113 DPCSV (a) and SWSV (b) voltammograms of 10 195

ppb AFB2 (i) in present of blank sample (ii) obtained
in BRB at pH 9.0 (iii) as the supporting electrolyte.
3.114 DPCSV voltammograms of real samples (b) added 197

with 3 ppb (i), 9 ppb (ii) and 15 ppb (iii) AFB1
obtained in BRB at pH 9.0 (a) as the blank on the
first day measurement.
3.115 Percentage of recoveries of (a) 3 ppb, (b) 9 ppb, 198

(c) 15 ppb of all aflatoxins in real samples
obtained by DPCSV methods for one to three
days of measurements.

amplitude = 80 mV.
xxviii


amplitude = 80 mV.


amplitude = 80 mV.

xxix
ABBREVIATIONS
AAS Atomic absorption spectrometry

Abs Absorbance
ACP Alternate current polarography
ACV Alternate current voltammetry
AD Amperometric detector
AdCSV Adsorptive cathodic stripping voltammetry
AE Auxiliary electrode
AFB1 Aflatoxin B1
AFB2 Aflatoxin B2
AFG1 Aflatoxin G1
AFG2 Aflatoxin G2
AFM1 Aflatoxin M1
AFM2 Aflatoxin M2
AFP1 Aflatoxin P1
AFQ1 Aflatoxin Q1
Ag/AgCl Silver/silver chloride
ASV Anodic stripping voltammetry
β-CD β-cyclodextrin
BFE Bismuth film electrode
BLMs Bilayer lipid membranes
BRB Britton Robinson Buffer
CA Concentration of analyte
CE Capillary electrophoresis
CGME Controlled growth mercury electrode
CME Chemically modified electrode
CPE Carbon paste electrode
CSV Cathodic stripping voltammetry
CV Cyclic voltammetry
xxx
DC Direct current
DCP Direct current polarography
DME Dropping mercury electrode
DMSO Dimethyl sulphonic acid
DNA Deoxyribonucliec acid
DPCSV Differential pulse cathodic stripping voltammetry
DPP Differential pulse polarography
DPV Differential pulse voltammetry
Eacc Accumulation potential
Ei Initial potential
Ef Final potential
Ehigh High potential
Elow Low potential
Ep Peak potential
ECS Electrochemical sensing
Et4NH4 OH Tetraethyl ammonium hydroxide
ELISA Enzyme linked immunosorbant assay
FD Fluorescence detector
FDA Food and Drug Administration
GCE Glassy carbon electrode
GC-FID Gas chromatography with flame ionisation detector
HMDE Hanging mercury drop electrode
HPLC High performance liquid chromatography
HPTLC High pressure thin liquid chromatography
IAC Immunoaffinity chromatography
IACLC Immunoaffinity column liquid chromatography
IAFB Immunoaffinity fluorometer biosensor
IARC International Agency for Research Cancer
Ic Charging current
Id Diffusion current
If Faradaic current
xxxi
Ip Peak height
ICP-MS Induced coupled plasma-mass spectrometer
IR Infra red
IUPAC International Union of Pure and Applied Chemistry
KGy Kilogray
LD50 Lethal dose 50
LOD Limit of detection
LOQ Limit of quantification
LSV Linear sweep voltammetry
MFE Mercury film electrode
MS Mass spectrometer
MECC Micellar electrokinetic capillary chromatography
MOPS 3-(N-morpholino)propanesulphonic
MOSTI Ministry of Science, Technology and Innovation
NP Normal polarography
NPP Normal pulse polarography
NPV Normal pulse voltammetry
OPLC Over pressured liquid chromatography
PAH Polycyclic aromatic hydrocarbon
PLL Poly-L-lysine
ppb part per billion
ppm part per million
PSA Potentiometric stripping analysis
RDX Hexahydro-1,3,5-trinitro-1,3,5-triazine
RE Reference electrode
RIA Radioimmunoassay
RNA Ribonucleic acid
RSD Relative standard deviation
S/N Signal to noise ratio
SCE Standard calomel electrode
SCV Stair case voltammetry
xxxii
SDS Sodium dodecyl sulphate

SHE Standard hydrogen electrode
SIIA Sequential injection immunoassay
SMDE Static mercury drop electrode
SPE Solid phase extraction
SPCE Screen printed carbon electrode
SWP Square-wave polarography
SWV Square-wave voltammetry
SWSV Square-wave stripping voltammetry
SV Stripping voltammetry
tacc Accumulation time
TBS Tris buffered saline
TEA Triethylammonium
TLC Thin layer chromatography
TFA Trifluoroacetic acid
UME Ultra microelectrode
ν Scan rate
v/v Volume per volume
UVD Ultraviolet-Visible detector
UV-VIS Ultraviolet-Visible
WE Working electrode
WHO World Health Organisation
λmax Maximum wavelenght
εmax Maximum molar absorptivity
xxxiii
LIST OF APPENDICES
APPENDIX TITLE PAGE
A Relative fluorescence of aflatoxins in different 255

solvent s
B UV spectra of the principal aflatoxins (in methanol) 256
C Relative intensities of principal bands in the IR 257

spectra of the aflatoxins
D Calculation of concentration of aflatoxin stock 258

solution
E Extraction procedure for aflatoxins in real samples 259
F Calculation of individual aflatoxin in groundnut 260

samples.
G Cyclic voltammograms of AFB1, AFB2 and AFG2 262

with increasing of their concentrations.
H Dependence if the peak heights of AFB1, AFG1 264

and AFG2 on their concentrations.
I Repetitive cyclic voltammograms and their peak 266

heights of AFB1, AFG1 and AFG2 in BRB at pH 9.0
J Plot Ep – log scan rate for the reduction of AFB1, 270

K Plot of peak height versus scan rate for 1.3 µM of 272

AFB1, AFG1 and AFG2 in BRB at pH 9.0
L Voltammograms of AFB2 with increasing 274

concentration.
M Voltammograms of 0.1 µM and 0.2 µM AFB2 275

obtained on the same day measurements
N Voltammograms of AFB2 at inter-day 276

measurements
xxxiv
O F test for robustness and ruggedness tests 278
P Voltammograms of AFB1 with increasing 280

concentration
Q Voltammograms of AFG1 with increasing 281

concentration
R Voltammograms of AFG2 with increasing 282

concentration
S LOD determination according to Barek et al. (2001a) 283
T LOD determination according to Barek et al. (1999) 286
U LOD determination according to Zhang et al. (1996) 287
V LOD determination according to Miller and 288

Miller (1993)
W ANOVA test 290
X Peak height of aflatoxins in presence and absence of PLL 292
Y SWSV voltammograms of AFB1, AFB2, AFG1 293

and AFG2 in BRB at pH 9.0
Z SWSV voltammograms of 0.10 µM AFB1, AFB2, 295

AA UV-VIS spectrums of 10 ppm AFB1, AFB2, AFG1 297

and AFG2 stock solutions
AB Voltammograms of AFB1, AFB2, AFG1 and AFG2 299

obtained from 0 to 6 months of storage time in the
cool and dark conditions.
AC Voltammograms of AFB1, AFB2, AFG1 and 302

AFG2 in BRB at pH 9.0 from 0 to 8 hours of
exposure time.
AD UV-VIS spectrums of AFB2 in BRB at pH 6.0 305

and 11.0.
xxxv
AE Voltammograms of AFB1 and AFG1 in 1.0 M HCl 307

and 1.0 M NaOH
AF DPCSV voltammograms of real samples added with 309

various concentrations of AFG1
AG SWSV voltammograms of real samples added with 310

various concentrations of AFB1.
AH Percentage of recoveries of various concentrations 311

of all aflatoxins (3.0 and 9.0 ppb) in real samples
AI Calculation of percentage of recovery for 3.0 ppb 312

AFG1 added into real samples.
AJ HPLC chromatograms of real samples: S10 and S07 313
AK Calculation of aflatoxin in real sample, S13 314
AL List of papers presented or published to date 315

resulting from this study.
AM ICP-MS results for analysis of BRB at pH 9.0 317

CHAPTER I
LITERATURE REVIEW
1.1 Overview
Humans are continuously exposed to varying amounts of chemicals that have

been shown to have carcinogenic or mutagenic properties in environmental systems.
Exposure can occur exogenously when these agents are present in food, air or water,
and also endogenously when they are products of metabolism or pathophysiologic
states such as inflammation. Great attention is focused on environmental health in the
past two decades as a consequence of the increasing awareness over the quality of life
due to major environment pollutants that affect it. Studies have shown that exposure to
environmental chemical carcinogens have contributed significantly to cause human
cancers, when exposures are related to life style factors such as diet (Wogan et al.,
2004).
The contamination of food is part of the global problem of environmental

pollution. Foodstuffs have been found contaminated with substances having
carcinogenic, mutagenic, teratogenic and allergenic properties. As these substances can
be supplied with food throughout the entire life-time of a person, it is necessary to deal
with the chronic action of trace amounts of such substances. Hence the systematic
determination of the foreign substances in nutritional products and feedstock plays an
important role. The determination of trace impurities presents considerable difficulties
owing to the fact that food is a complex system containing thousands of major and
minor compounds (Nilufer and Boyacio, 2002). Increasing environment pollution by
toxic substances such as toxic metals, organometallic and organic pollutants in air,
2
water, soil and food, calls for reliable analytical procedures for their control in
environmental samples which needs reliable and sensitive methods (Fifield and Haines,
2000). The choice of the method of analysis depends on the sample, the analyte to be
assayed, accuracy, limit of detection, cost and time to complete the analysis (Aboul-
Eneim et a., 2000). For development of this method, emphasis should be on
development of simplified, cost-effective and efficient method that complies with the
legislative requirements (Stroka and Anklam, 2002; Enker, 2003).
The widespread occurrence of aflatoxins producing fungi in our environment

and the reported naturally occurring of toxin in a number of agricultural commodities
has led the investigator to develop a new method for aflatoxin analysis (Creepy, 2002).
An accurate and sensitive method of analysis is therefore required for the determination
of these compounds in foodstuffs that have sustained mould growth.
Numerous articles concerning methods for determination of aflatoxins have

been published. However, with regard to electroanalytical technique, only one method
of determination was reported using the differential pulse pulse polarographic (DPP)
technique which was developed by Smyth et al. (1979). In this experiment, the
obtained limit of detection of aflatoxin B1 was 25 ppb which was higher as compared
to the common amount of aflatoxin in contaminated food samples which is 10 ppb as
reported by Pare (1997) or even less. In Malaysia, the regulatory limit for total
aflatoxins in groundnut is 15 ppb. The regulatory for other foods and milk is 10 ppb
and 0.05 ppb respectively (Malaysian Food Act, 1983).
1.2 AFLATOXINS
1.2.1 Aflatoxins in General
Aflatoxins are a group of heterocyclic, oxygen-containing mycotoxins that

possess the bisdifuran ring system. It was discovered some 43 years ago in England
3
following a poisoning outbreak causing 100,000 turkeys death (Miller, 1987 and
Cespedez and Diaz, 1997). The aflatoxins are the most widely distributed fungal toxins
in food. The occurrence of the aflatoxins in food products demonstrated that the high
levels of aflatoxins are significant concern both for food traders and food consumers
(Tozzi et al. 2003; Herrman, 2004; Haberneh, 2004). Aflatoxin is a by-product of mold
growth in a wide range of agriculture commodities such as peanuts (Urano et al., 1993),
maize and maize based food (Papp et al., 2002; Mendez-Albores et al., 2004),
cottonseeds (Pons and Franz, 1977), cocoa (Jefferey et al., 1982), coffee beans (Batista
et al., 2003), medical herbs ( Reif and Metzger, 1998; Rizzo et al., 2004), spices
(Erdogen, 2004; Garner et al., 1993; Akiyama et al., 2001; Aziz et al., 1998), melon
seeds (Bankole et al., 2004) and also in human food such as rice (Shotwell et al., 1966;
Begum and Samajpati, 2000), groundnut (Bankole et al., 2005), peanut products (
Patey et al., 1990), corn ( Shotwell and Goulden, 1977; Urano et al., 1993 ), vegetable
oil (Miller et al., 1985), beer (Scott and Lawrence, 1997), dried fruits ( Abdul Kadar et
al., 2004, Arrus et al. (2004), milk and dairy products (Kamkar, 2004; Aycicek et al.
2005; Sarimehmetoglu et al., 2004; Martin and Martin, 2004 ). Meat and meat
products are also contaminated with alfatoxins when farm animals are fed with
aflatoxin contaminated feed (Miller, 1987 and Chiavaro et al., 2001).
The molds that are major producers of aflatoxin are Aspergillus flavus
(Bankole et al. 2004) and Aspergillus parasiticus (Begum and Samajpati, 2000;
Setamou et al., 1997; Erdogen, 2004; Gourama and Bullerman, 1995). Aspergillus
flavus, which is ubiquitous, produces B aflatoxins (Samajphati, 1979) while Aspergillus
parasiticus, which produces both B and G aflatoxins, has more limited distribution
(Garcia-Villanova et al., 2004). A picture of Aspergillus flavus seen under an electron
microscope is shown in Figure 1.0.
Black olive is one of the substrate for Aspergillus parasiticus growth and
aflatoxin B1 production as reported by Leontopoulos et al., (2003). Biosynthesis of
aflatoxins by this fungi depends on the environmental condition such as temperature
and humidity during crop growth and storage (Leszczynska et al., 2000; Tarin et al.,
4
2004 and Pildain et al., 2004). The optimum temperatures for aflatoxins growth are
27.84 0 C and 27.30 0 C at pH=5.9 and 5.5 respectively.
Figure 1.0: Aspergillus flavus seen under an electron microscope
Before harvest, the risk for the development of aflatoxins is greatest during
major drought (Turner et al., 2005). When soil moisture is below normal and
temperature is high, the number of Aspergillus spores in the air increases. These spores
infect crops through areas of damage caused by insects and inclement weather. Once
infected, plant stress occurs, which favor the production of aflatoxins. Fungal growth
and aflatoxins contamination are the sequence of interactions among the fungus, the
host and the environment. The appropriate combination of water stress, high
temperature stress and insect damage of the host plant are major determining factors in
mold infestation and toxin production (Faraj et al., 1991; Koehler, 1985; Park and
Bullerman, 1983). Additional factors such as heat treatment, modified-atmosphere
packaging or the presence of preservative, also contribute in increasing growth rate of
the aflatoxins.
Farmers have minimal control over some of these environmental factors.

However appropriate pre-harvest and post-harvest management and good agricultural
practice, including crop rotation, irrigation, timed planting and harvesting and the use
5
of pesticides are the best methods for preventing or controlling aflatoxins

contamination (Turner et al., 2005). Timely harvesting could reduce crop moisture to a
point where the formation of the mould would not occur. For example harvesting corn
early when moisture is above 20 percent and then quickly drying it to a moisture level
of at least 15 percent will keep the Aspergillus flavus from completing its life cycle,
resulting in lower aflatoxin concentration. Aflatoxins are to be found in agricultural
products as a consequence of unprosperous storage conditions where humidity of 70 -
90 % and a minimum temperature of about 10° C. Commodities that have been dried
to about 12 to 0.5 % moisture are generally considered stable, and immune to any risk
of additional aflatoxins development. Moreover, the minimum damage of shells during
mechanized harvesting of crop reduces significantly the mould contamination.
Biocontrol of aflatoxin contamination is another way to reduce this contamination. The
natural ability of many microorganisms including bacteria, actinomycetes, yeasts,
moulds and algae has been a source for bacteriological breakdown of mycotoxins. The
most active organism such as Flavobacterium aurantiacum which in aqueous solution
can take up and metabolise aflatoxins B1, G1 and M1 (Smith and Moss, 1985).
Production of aflatoxins is greatly inhibited by propionic acid as revealed by

Molina and Gianuzzi (2002) when they studied the production of aflatoxins in solid
medium at different temperature, pH and concentration of propionic acid. It also can be
inhibited by essential oil extracted from thyme as found by Rasooli and Abnayeh
(2004). Other chemicals that can inhibit the growth of this fungus are ammonia, copper
sulphate and acid benzoic (Gowda et al., 2004).
1.2.2 Chemistry of Aflatoxins
Aflatoxins can be classified into two broad groups according to chemical

structure which are difurocoumarocyclopentenone series and ifurocoumarolactone
(Heathcote, 1984). They are highly substituted coumarin derivatives that contain a
fused dihydrofuran moiety. The chemical structure of coumarin is shown in Figure 1.1.
6
O O
Figure 1.1 Chemical structure of coumarin
There are six major compounds of aflatoxin such as aflatoxin B1 (AFB1),

aflatoxin B2 (AFB2), aflatoxin G1 (AFG2), aflatoxin G2 (AFG2), aflatoxin M1
(AFM1) and aflatoxin M2 (AFM2) (Goldblatt, 1969). The former four are naturally
found aflatoxins and the AFM1 and AFM2 are produced by biological metabolism of
AFB1 and AFB2 from contaminated feed used by animals. They are odorless, tasteless
and colorless. The scientific name for these aflatoxin compounds are listed in Table 1.0.
Aflatoxins have closely similar structures and form a unique group of highly
oxygenated, naturally occurring heterocyclic compounds. The chemical structures of
these aflatoxins are shown in Figure 1.2. The G series of aflatoxin differs chemically
from B series by the presence of a β-lactone ring, instead of a cyclopentanone ring.
Also a double bond that may undergo reduction reaction is found in the form of vinyl
ether at the terminal furan ring in AFB1 and AFG1 but not in AFB2 and AFG2.
However this small difference in structure at the C-2 and C-3 double bond is associated
with a very significant change in activity, whereby AFB1 and AFG1 are carcinogenic
and considerably more toxic than AFB2 and AFG2. The dihydrofuran moiety in the
structure is said to be of primary importance in producing biological effects.
Hydroxylation of the bridge carbon of the furan rings for AFM1 does not significantly
alter the effects of the compounds. The absolute configuration of AFB2 and AFG2
follows from the fact that it is derived from the reduction of AFB1 and AFG1
respectively.
AFB is the aflatoxin which produces a blue color under ultraviolet while AFG
produces the green color. AFM produces a blue-violet fluorescence while AFM2
produces a violet fluorescence (Goldblatt, 1969). Relative fluorescence of aflatoxins in
several organic solvents are shown in Appendix A (White and Afgauer, 1970). The
7
Table 1.0 Scientific name for aflatoxin compounds
Aflatoxin B1 2,3,6a,9a-tetrahydro-4-methoxycyclopenta[c]
Aflatoxin B2 2,3,6a,8,9,9a-Hexahydro-4-methoxycyclopenta[c]
Aflatoxin G1 3,4, 7a,10a-tetrahydro-5-methoxy-1H, 12H

(AFG1) furo[3’,2’:4,5]furo[2,3-h]pyrano[3,4-c]l]- benzopyran-
1,12-dione
Aflatoxin G2 3,4,7a,8,9,10, 10a-Hexahydro-5-methoxy-1H,12H-

(AFG2) furo[3’,2’:4,5]furo[2,3-h]pyrano[3,4-c][l]- benzopyran-
1,12-dione

M1
(AFM1)

M2
natural fluorescence of aflatoxins arises from their oxygenated pentaheterocyclic

structure. The fluorescence capacity of AFB2 and AFG2 is ten times larger than that of
AFB1 and AFG1, probably owing to the structural difference, namely double bond on
the furanic ring. Such a double bond seems to be very important for the photophysical
8
O O
O O
O
O
O O
O O
O
O
(a) (b)
O O O O
O O O O
O O
O O
O O
(c) (d)
O
O O
O
O
O
OH
OH
O O
O O
O
O
(e) (f)
Figure 1.2 Chemical structures of (a) AFB1, (b) AFB2, (c) AFG1, (d) AFG2,
(e) AFM1 and (f) AFM2.
properties of these derivatives measured just after spectroscopic studies (Cepeda et al.,
1996). The excitation of the natural fluorescence of AFB1 and AFG1 can be promoted
9
in many different ways such as post-column iodination (Tuinstra and Haasnoot, 1983;
Davis and Diener, 1980), post-column bromination (Kok et al.,1986; Kok, 1994 ;
Versantroort et al., 2005 ), use of cyclodextrin compound (Cepeda et al., 1996;
Chiavaro et al., 2001; Franco et al., 1998) and trifluoroacetic acid, TFA (Stack and
Pohland, 1975; Takahashi, 1977a; Haghighi et al., 1981; Nieduetzki et al., 1994 ).
AFB1 and AFG1 form hemiacetals, AFB2a and AFG2a when reacted with
acidic solution such as triflouroacetic acid (TFA) as represented in Figure 1.3 (Joshua,
1993). The hydroxyaflatoxins are unstable and tend to decompose to yellow products
in the presence of air, light and alkali. Their UV and visible spectra are similar to those
of the major aflatoxins.
O O
O O
O O
TFA
HO
O O O
O
O
O
(a) (c)
O O
O O
O O TFA O O
HO
O O
O O O O
(b) (d)
Figure 1.3 Hydration of AFB1 (a) and AFG1 (b) by TFA produces AFB2a (c) and
AFG2a (d) (Joshua, 1993).
The close relationship between AFB1, AFG1, AFB2a and AFG2a was shown by the
similarities in their IR and UV spectra. The main difference between AFB2a and
AFG2a with AFB1 and AFG1 are found in the IR spectra, where an additional band at
10
3620 cm-1 indicates the presence of a hydroxyl group in AFB2a and AFG2a. The
absence of bands at 3100, 1067 and 722 cm-1 (which arise in AFB1 and AFG1 from the
vinyl ether group) indicates that the compounds are hydroxyl derivatives of AFB2 and
AFG2. Some chemical and physical properties of aflatoxin compounds are listed in
Table 1.1 (Heathcote and Hibbert, 1978; Weast and Astle, 1987). The close
relationship between these aflatoxins was shown by the similarities in their UV and IR
spectra as shown in Appendix B and C respectively.
Table 1.1 Chemical and physical properties of aflatoxin compounds
AFB1 AFB2 AFG1 AFG2
Molecular C17H12O6 C17H14O6 C17H12O7 C17H14O7

formula
Molecular weight 312 314 328 330
Crystals Pale yellow Pale yellow Colorless Colorless
Melting point 268.9 286.9 244.6 237.40

(°C)
Fluorescence Blue Blue Green Green

under UV light
Solubility Soluble in water and polar organic solvent. Normal solvents

are: methanol, water: acetonitrile (9:1), trifluoroacetic acid,
methanol: 0.1N HCl (4:1), DMSO and acetone
Other properties Odorless, colorless and tasteless in solution form.

Incompatible with strong acids, strong oxidising agents and
strong bases. Soluble in water, DMSO, 95% acetone or
ethanol for 24 hours under ambient temperature
11
Many researches have studied the stability of aflatoxins. For example, AFB1
was not found in fruit samples after being irradiated with 5.0 Kgy or more of gamma-
irradiation as reported by Aziz and Moussa (2002). Gonzalez et al. (1998) have studied
the effect of electrolysis, ultra-violet irradiation and temperature on the decomposition
of AFB1 and AFG1. UV irradiation caused an intense effect on aflatoxins where after
60 min of radiation, AFG1 suffers practically with no more decomposition. However,
when the aflatoxin solutions were placed in a 90° C bath for 3 min, a decrease of 20%
from total amount of both AFB1 and AFG1 was obtained. A greater extent of
decomposition (50%) was found for treatment at 100° C during longer time. Levi
(1980) reported that experimental roasting under conditions simulating those of the
typical roasting operation (20 min at 200 ± 5° C) destroyed about 80% of AFB1 added
to green coffee. It was also found that AFB2 decomposed to a larger extent than AFG1
indicating a lower stability against prolonged heat treatment. During food
fermentation which involved other fungi such as Rhizopus oryzae and R. oligosporus,
cyclopentanone moiety of AFB1 was reduced resulting in the formation of aflatoxicol
A as shown in Figure 1.4 which is non-toxic compound. It retains the blue-fluorescing
property of AFB1 under UV light. It has been considered to be one of the most
important B1 metabolites because there is a correlation between the presence of this
metabolite in animal tissues and body fluids with toxicity of AFB1 in different animals
(Lau and Chu, 1983). Aflatoxin solution prepared in water, dimethyl sulphonic acid
(DMSO), 95% acetone or ethanol is stable for 24 hours under ambient conditions.
AFM1 is relatively stable during pesteuring, sterilisation, preparation and storage of
various dairy products (Gurbay et al., 2004).
O
O
OH
O
O
+ R.oryzae O
+ R. oligosporus
O OCH 3 O OCH3
O O
(a) (b)
Figure 1.4 Transformation of toxic (a) AFB1 to non-toxic (b) aflatoxicol A
12
1.2.3 Health Aspect of Aflatoxins
Aflatoxins have received greater attention than any other mycotoxins. There are
potent toxin and were considered as human carcinogen by The International Agency for
Research on Cancer (IARC) as reported in World Health Organisation (WHO)’s
monograph (1987). These mycotoxins are known to cause diseases in man and animals
called aflatoxicosis (Eaton and Groopman, 1994). Human exposure to aflatoxins is
principally through ingestion of contaminated foods (Versantroort et al., 2005).
Inhalation of the toxins may also occur occasionally due to the occupational exposure.
After intake of contaminated feedstuffs aflatoxins cause some undesirable effect in
animals, which can range from vomiting, weight loss and acute necrosis to various
types of carcinoma, leading in many cases to death (Pestka and Bondy, 1990 and
Bingham et al., 2004). Even at low concentration, aflatoxins diminish the immune
function of animals against infection. Epidemiological studies have shown a
correlation between liver cancer and the prevalence of aflatoxins in the food supply.
In views of occurrence and toxicity, AFB1 is extremely carcinogenic while

others are considered as highly carcinogenic as reported by Carlson (2000). It is
immunosuppressive and a potent liver toxin; less than 20 µg of this compound is lethal
to duckling (Hussein and Brasel, 2001). AFB1 is biochemically binding to DNA,
inhibit DNA, RNA, and protein syntheses, and effects DNA polymerase activity as
reported by Egner et al. (2003). Previous research results have demonstrated the
covalent binding of highly reactive metabolite of AFB1 to the N-7 atom of guanine
residues, resulting in major DNA adducts (Moule, 1984; Johnson et al., 1997; Egner et
al., 2003). Major DNA adducts of AFB1 are shown in Figure 1.5.
The four main aflatoxins display decreasing potency in the order AFB1 > AFG1
> AFB2 > AFG2 as reported by Betina (1984). This order of toxicity indicates that the
double bond in terminal furan of AFB1 structure is a critical point for determining the
degree of biological activity of this group of mycotoxins (Hall and Wild, 1994). It
appears that the aflatoxins themselves are not carcinogenic but rather some of their
13
O O O O
O O
OH
OH
O O
O O
O O N OCH3
N OCH3
NH NH CHO
N N NH2
NH2 N NH2
(a) (b)
Figure 1.5 Major DNA adducts of AFB1; (a) 8,9- Dihydro-8-(N7-guanyl)-

9-hydroxy-aflatoxin B1 (AFB1-Gua) and (b) 8,9-Dihydro-8-(N5- Formyl-2’.5’.6 ’-
triamino-4 ’-oxo-N5-pyrimidyl)-9-hydroxy- Aflatoxin B1 (AFB1-triamino-Py)
metabolites (de Vries, 1996). For example, metabolite transformation of AFB1 by

cytochrome P-450 enzyme produces aflatoxin Q1 (AFQ1), AFM1, AFB1-epoxide and
aflatoxin P1 (AFP1) as shown in Figure 1.6. For hamiacetals, AFB2a and AFG2a,
however, are relatively non-toxic despite the close similarity of their structure to those
of AFB1 and AFG1 even at the highest dosages (1200 µg for AFB2a and 1600 µg for
AFG2a).
AFM1 is the main metabolic derivatives of aflatoxins in several animal species

It is found in the cow milk due which had consumed feed which contaminated with
AFB1 as reported by Chang et al. (1983), Yousef and Marth (1985), Van Egmond
(1989), Tuinstra (1990) and Lopez et al. (2002). The relative amount of AFM1
excreted is related to the amount of AFB1 in the feed, and about 0.1% of AFB1
ingested is excreted into milk as AFM1 (Miller, 1987). There was a linear relationship
between the amount of AFM1 in milk and AFB1 in feeds consumed by animals as
reported by Dragacci (1995). AFM1 is produced by hydroxylation of AFB1 in the liver
14
O O
O O
OCH3
B 1 -e p o x id e
O O
O O
OCH3
A flato x in B 1
O O O O O O
O O O
OH
OH
O O O O O O
M1 P1 QI
OCH3 OH OCH3
Figure 1.6 Metabolic pathways of AFB1 by cytochrom P-450 enzymes; B1-

epoxide = AFB1 epoxide, M1 = aflatoxin M1, P1 = aflatoxin P1 and Q1 = aflatoxin
Q1
15
of lactating animals, including humans. It is also known as milk toxin which is much
less carcinogenic and mutagenic than AFB1. It has been classified by the International
Agency For Research Cancer (IARC) as a Group 2 carcinogen (IARC, 1993). It can
generally be found in milk and milk products such as dry milk, whey, butter, cheese,
yogurt and ice cream. AFM2 is the analogous metabolic derivatives of AFB2.
Aflatoxins have been considered as one of the most dangerous contaminant in

food and feed. The contaminated food will pose a potential health risk to human such as
aflatoxicosis and cancer (Jeffrey and Williams, 2005). Aflatoxins consumption by
livestock and poultry results in a disease called aflatoxicosis which clinical sign for
animals include gastro intestinal dysfunction, reduced reproductivity, reduced feed
utilisation, anemia and jaundice. Humans are exposed to aflatoxins by ingestion,
inhalation and dermal exposure as reported by Etzel (2002). LD50 which is the amount
of a materials, given all at once, causes the death of 50% (one half) of a group of test
animal (www.aacohs.ca/oshanswers) for most animal and human for AFB1 is between
0.5 to 10.0 mg kg-1 body weight (Smith and Moss, 1985; Salleh, 1998). Clinical
features were characterised by jaundice, vomiting, and anorexia and followed by
ascites, which appeared rapidly within a period of 2-3 weeks.
Besides causing health problems to humans, aflatoxin also can cause adverse
economic effect in which it lowers yield of food production and fiber crops and
becoming a major constraint of profitability for food crop producer countries, an
example of which is given in Rachaputi et al. (2001). It has been estimated that
mycotoxin contamination may affect as much as 25% of the world’s food crop each
year (Lopez, 1999) resulting in significant economic loss for these countries.
Aflatoxins also inflict losses to livestock and poultry producers from aflatoxin-
contaminated feeds including death and the more subtle effects of immune system
suppression, reduced growth rates, and losses in feed efficiency.
Due to the above reason, aflatoxin levels in animal feed and various human food
products is now monitored and tightly regulated by the most countries. In Malaysia,
16
the action level for total aflatoxins is 15ppb (Malaysian Food Act, 1983). The
European Commission has set limits for the maximum levels of total aflatoxins and
AFB1 allowed in groundnuts, nuts, dried fruit and their products. For foods ready for
retail sale, these limits are 4 ppb for total aflatoxins and 2 ppb for AFB1, and for foods
that are to be processed further the limits stand at 15 ppb for total aflatoxins and 8 ppb
of AFB1 (European Commission Regulation, 2001). The Food and Drug
Administration (FDA) in USA has established an action level of 0.5 ppb of mycotoxin,
AFM1 in milk for humans and 20 ppb for other aflatoxins in food other than milk
(www.ansci.cornel.edu/toxiagents).
In Brazil, the limits allowed for food destined for human consumption are 20
µg/kg of total aflatoxin for corn in grain, flours, peanut and by-products, and 0.5 µg/l of
AFM1 in fluid milk (Sassahara et al., 2005). Australia has established a minimum level
of 15 ppb for aflatoxin in raw peanut and peanut product (Mackson et al., 1999). In
Germany, regulatory levels of total aflatoxins are 4 ppb and 2 ppb for AFB1. For
human dietary products, such as infant nutriment, there are stronger legal limits, 0.05
ppb for AFB1 and the total aflatoxins (Reif and Metzger, 1995). The Dutch Food Act
regulates that food and beverages may contain no more than 5 µg of AFB1 per kg
(Scholten and Spanjer, 1996).
Regulatory level set by Hong Kong government is 20 ppb for peanuts and
peanut products and 15 ppb for all other foods (Risk Assessment Studies report, 2001).
Because of all these reasons, systematic approaches to sampling, sample preparation
and selecting appropriate and accurate method of analysis of aflatoxin are absolutely
necessary to determine aflatoxins at the part-of-billion (ppb) level as reported by Park
and Rua (1991).
1.2.4 Analytical Methods for the Determination of Aflatoxins
Monitoring the presence of aflatoxin in various samples especially in food is

not only important for consumer protection, but also for producers of raw products
17
prior to cost intensive processing or transport. Several methods for aflatoxins

determination in various samples have been developed and reported in the literature.
Method based on thin-layer chromatography (TLC) (Lafont and Siriwardana, 1981;
AOAC, 1984) and high performance liquid chromatography (HPLC) with ultraviolet
absorption, fluorescence, mass spectrometry or amperometry detection, have been
reported (Kok et al., 1986; Taguchi et al., 1995; Ali et al., 2005; Manetta et al.,
2005).
TLC and HPLC techniques are well proven and widely accepted; however,
both techniques have their own disadvantages. These methods require well equipped
laboratories, trained personnel, harmful solvents and time consuming. Moreover,
instrumentations used are expensive (Badea et al., 2004). The main disadvantage
associated with TLC is the lack of precision where a possible measurement error of ±
30 – 50 % is indicated when standard and unknown aflatoxins spot are matched, and
± 15 -25 % when the unknown is interpolated between two standards (Takahashi,
1977b). In this technique, the greater effect was caused by sample matrix which is
usually very complicated (Lin et al., 1998). Poor repeatability is associated with the
sample application, development and plate interpretation steps. HPLC technique is
often viewed as laborious and time intensive, needs complex gradient mobile phase,
large quantity of organic solvent, requiring a significant investment in equipments,
materials and maintenance (Pena et al., 2002).
Pre-column derivatisation technique could improve the sensitivity of the

measurement however, it requires chemical manipulations which are time consuming,
involves aggressive reagents such as bromine, TFA and iodine and also it is difficult
to automate. Other disadvantages of this procedure include the requirement to
prepare iodine solution daily, the necessity for two pumps, dilution of the eluent
stream, the need to thermostat the reactor coil and insufficient day-to-day
reproducibility (Kok et al., 1986). The method using TFA as derivatising agent has
poor reproducibility and is difficult to automate (Reif and Metzger, 1995).
18
Other methods such as enzyme linked immunosorbant assay (ELISA) (Chu et

al., 1987; Lee et al., 1990; Pesavento et al., 1997 and Aycicek et al., 2005),
radioimmunoassay (RIA) (Stroka et al., 2000) and immunoaffinity clean up (Garner,
1993 and Niedwetzki et al., 1994) have also been developed for the detection of these
compounds. The simplicity, sensitivity and rapid detection of aflatoxin by ELISA
has made it possible to monitor several samples simultaneously but ELISA and other
immunochemical methods require highly specific polyclonal or monoclonal sera for
specific and sensitive detection of antigen. The production of specific antibodies for
aflatoxins has allowed the development of this technique based on direct or indirect
competition. This method, however, presents some drawbacks such as long
incubation time, washing and mixing steps (Badea et al., 2004) and is labor intensive
(Carlson et al., 2000). It also requires highly specific polyclonal or monoclonal sera
which is expensive (Rastogi et al., 2001). Blesa et al. (2003) reported that the ELISA
method is a good screening method for investigation of aflatoxins. RIA is very
sensitive but has several disadvantages. The radioisotopes are health hazards,
disposable difficulties and may have short half life (Trucksess et al., 1991).
Cole et al., (1992) have used micellar electrokinetic capillary chromatography

(MECC) technique for rapid separation of aflatoxins. Pena et al., (2002) proposed
capillary electrophoresis (CE) for determination of aflatoxins. They found that the
limit of detection (LOD) of this technique was 0.02 to 0.06 ppb with analysis time of
50 minutes. Due to long duration time and involving use of many reagents such as
benzene and acetonitrile for preparation of aflatoxin stock solution and sodium
dodecyl sulphate (SDS), sodium dihydrogenphosphate, sodium borate and γ-
cyclodextrin for preparing the buffer solution in performing the analysis, this
technique may not be suitable for routine analysis of aflatoxins. A summary of
techniques used for the determination of aflatoxins compounds in various samples
together with its detection limit is shown in Table 1.2.
19
Table 1.2 Summary of analysis methods used for the determination of aflatoxins in
various samples
No Method Aflatoxin / samples Detection Reference

limit (ppb)
1 HPLC-FD B1, B2, G1 and G2 in spices 0.06 Garner et al.

1993)
2 HPLC-FD B1, B2, G1 and G2 in spices 0.5 Akiyama et al.

(2001)
3 HPLC-FD B1, B2, G1 and G2 in peanut 5.0 Beebe (1978)

butter
4 HPLC-FD B1, B2, G1 and G2 in peanut 0.5 Duhart et al.

butter (1988)
5 HPLC-FD B1, B2, G1 and G2 in peanut Not stated Patey et al.

butter (1991)
6 HPLC-FD B1, B2, G1 and G2 in B1, G1, G2 Kussak et al.

airborne dust = 3.1 (1995a)
B2 = 1.8
7 HPLC-FD B1, B2, G1 and G2 in corn, Not stated Trucksess et al.

peanuts and peanut butter (1991)
8 HPLC-FD B1, B2, G1 and G2 in 0.05 Reif and

medicinal herbs and plant Metzger (1995)
extract
9 HPLC-FD B1, B2, G1 and G2 in 0.08 Brera et al.

airborne dust and human sera (2002)
20
Table 1.2 Continued

limit (ppb)
10 HPLC-FD B1, B2, G1 and G2 in 2.0 Chemistry

groundnut, peanut and peanut Department,
butter MOSTI (1993)
11 HPLC-FD B1, B2, G1 and G2 in Not stated Holcomb et al.

standard samples (2001)
12 HPLC-FD B1, B2, G1 and G2 in wine 0.02 Takahashi

(1977a)
13 HPLC-FD B1, B2, G1 and G2 in corn, Not stated Wilson and

almonds, peanuts, milo,rice, Romer (1991)
pistachio, walnuts, corn and
cottonseed
14 HPLC-FD M1 in milk and non fat dry 0.014 Chang and

milk Lawrence
(1997)
15 HPLC-FD B1, B2, G1 and G2 in beer B1, G1= Scot and

0.019, Lawrence
B2, G2 = (1997)
0.015
16 HPLC-FD B1, B2, G1 and G2 in poultry 1.0 Cespedes and

and pig feeds and feedstuffs Diaz (1997)
17 HPLC-FD M1 in milk 0.05 Yousef and

Marth (1985)
18 HPLC-FD M1 in whole milk 0.014 Fremy and

Chang (2002)
21
Table 1.2 Continued

limit (ppb)
19 HPLC-FD B1, B2, G1 and G2 in pistachio B1 = 0.8, Chemistry

kernels and shells B2 = 0.29 Department,
G1 = 0.9 MOSTI (1993)
G2 = 1.1
20 HPLC-FD B1, B2, G1 and G2 in urine 0.0068 Kussak et al.

(1995b)
21 HPLC-FD M1 in milk 10 Gurbey et al.

(2004)
22 HPLC-FD M1 in urine and milk 0.0025 Simon et al.

(1998)
23 HPLC-FD B1, B2, G1 and G2 in sesame Not stated Nilufer and

butter and tahini Boyacio (2002)
24 HPLC-FD B1 and ochratoxin A in bee B1= 0.2 Garcia-

pollen Villanove et al.
(2004)
25 HPLC-FD B1, B2, G1 and G2 in 1.0 Jefferey et al.

and UVD contaminated cocoa beans (1982)
26 HPLC-FD B1 and B2 in wine and fruit 0.02 Takahashi

and UVD juices (1997b)
27 HPLC- B1, B2, G1 and G2 in corn B1, G1 = Fremy and

UVD 1.0, Chang (2002)
B2, G2 =
0.25
22
Table 1.2 Continued

limit (ppb)
28 HPLC- B1in standard sample Not stated Rastogi et al.

UVD (2001)
29 HPLC- B1and B2 in cottonseed 5.0 Pons and Franz

UVD (1977)
30 HPLC- B1 in egg 1.0 Trucksess et al.

UVD (1977)
31 HPLC- B1 and M1 in corn B1 = 3.0 -5.0 Stubblefield and

UVD M1 = not Shotwell (1977)
stated
32 HPLC- B1, B2, G1 and G2 in soy 1.0 Wet et al.

UVD sauces and soybean paste (1980)
33 HPLC-AD B1, B2, G1 and G2 in 7.0 Gonzalez et al.

standard sample (1998)
34 HPTLC B1, B2, G1 and G2 in corn, B1 = 0.2 Kamimura et al.

buckwheat, peanuts and B2, G1, G2 = (1985)
cheese 0.1
35 HPTLC- B1, B2, G1 and G2 in B1 = 3.7, B2 Nawaz et al.

SPE palm kernel = 2.5, G1 = (1992)
3.0, G2 = 1.3
36 TLC B1and M1in artificial B1= 0.03, Stabblefield et

contaminated beef livers M1 = 0.1 al. (1982)
23
Table 1.2 Continued

limit (ppb)
37 TLC B1in milk and milk powder 0.05 Bijl and

Peteghem (1985)
38 TLC Total aflatoxin in wheat Not stated Shotwell et al.

and soybean (1977b)
39 TLC B1, B2, G1 and G2 in B1, G1 = 2.0- Gimeno and

apples, pears, apple juice 2.8, Martins (1983)
and pear jams B2, G2 = 2.0
40 TLC B1, B2, G1 and G2 in corn Corn = 2.0 Bicking et al.

and peanut meal Peanut meal = (1983)
3.0
41 TLC M1 in milk and condensed, Milk = 0.1 Fukayama et al.

evaporated and non-fat Others = 0.2 (1980)
powdered milk
42 TLC B1, B2, G1 and G2 in Not stated Trucksess and

ginger root and oleoresin Stoloff (1980)
43 TLC B1in black olives 3000-7000 Tutour et al.

(1984)
44 TLC B1, B2, G1 and G2 in Not stated Saleemullah et

cereal and nuts al. (1980)
45 TLC-FDs M1in milk 0.005 Gauch et al.

(1982)
24
Table 1.2 Continued

limit (ppb)
46 OPLC-FD B1, B2, G1 and G2 in Not stated Papp et al.

maize, fish, wheat, peanuts, (2002)
rice and sunflower seeds
47 LC-MS B1, B2, G1 anf G2 in figs 1.0 Vahl and

and peanuts Jargensen
(1998)
48 ELISA M1 in milk, yogurt, 0.01 – 0.05 Fremy and Chu.

cheddar and Brie (1984)
49 ELISA M1 in infant milk products Not stated Rastogi et al.

and liquid milk (1977)
50 ELISA M1in curd and cheese Not stated Grigoriadou et

al. (2005)
51 ELISA M1 in cheese Not stated Sarimehtoglu et

al. (1980)
52 ELISA B1, B2, G1 and G2 in Not stated Nilufar and

sesame butter and tahini Boyacio (2000)
53 ELISA ST B1in corn 5.0 Beaver et al.

(1991)
54 MECC B1, B2, G1 and G2 in 0.05 – 0.09 Cole et al.

standard samples (1992)
25
Table 1.2 Continued

limit (ppb)
55 MECC FS B1, B2, G1 and G2 in feed 0.02 – 0.06 Pena et al.

samples (2002)
56 IAC Total aflatoxins and B1 in Total = 1.0 Leszezynska et

nuts B1 = 0.2 al. (1998)
57 IAC B1 in mixed feeds 2.0 Shannon et al.

(1984)
58 FI-IA-AD M1 in raw milk 0.011 Badea et al.

(1977)
59 GC-FID B1, B2, G1 and G2 in Not stated Goto et al.

culture medium (2005)
60 ECS with M1 in skimmed milk 761 Androu et al.

BLMs (1997)
61 DPP B1 in rice, milk, corn and 25 Smyth et al.

peeletised rabbit feed (1979)
62 SIIA M1in an artificial 0.2 Garden and

contaminated food Strachen (2001)
63 MS B1, B2, G1 and G2 in 10 Plattner et al.

contaminated corns (1984)
26
Table 1.2 Continued

limit (ppb)
64 IAFB B1in standard sample 0.1 Carlson et al.

(2002)
65 IACLC B1 and total aflatoxin in Not stated Stroka et al.

peanut butter, pistachio (2000)
paste, fig paste and paprika
powder
66 IACLC B1, B2, G1 and G2 in B1, B2, G1, Chan et al.

maize and peanut butter G2 = 2.0 (1984)
67 ICA B1 in rice, corn and wheat 2.5 Xiulan et al.

(2006)
68 TLC B1, B2, G1 and G2 in Not stated Erdogan (2004)

peppers
69 HPLC-FD B1, B2, G1 and G2 in Not stated Ali et al. (2005)

traditional herbal
medicines
70 LC-MS M1 in bovine milk 0.02 – 0.15 Sorensen and

Elbaek (2005)
71 LC-MS M1in sidestream cigarette 3.75 Edinboro and

smoke Karnes (2005)
72 TLC M1in milk and milk 0.0125 Bakirci (2001)

products
27
Table 1.2 Continued

limit (ppb)
73 ELISA B1and M1 in food and dairy Not stated Aycicek et al.

products (2005)
74 TLC M1 in Iranian feta cheese 0.015 Kamkar et al.

(2005)
75 Ridascreen M1 in pasteurised milk Not stated Alborzi et al.

Test (2005)
76 Ridascreen M1 in raw milk 0.245 Sassahara et al.

Test (2005)
77 TLC B1in melon seeds 2.0 Bankole et al.

(2005b)
78 ECS-ELISA B1in barley 0.02 – 0.03 Ammida et al.

(2004)
79 HPLC-FD B1, B2, G1 and G2 in bee Not stated Gonzalez et al.

pollen (2005)
80 HPLC-FD M1in milk and cheese Milk = 0.001, Manetta et al.

Cheese = (2005)
0.005
81 LC-MS B1, B2, G1 and G2 in B1, B2, G1, Blesa et al.

peanuts G2 = 0.125 – (2001)
2.50
82 ELISA-SPE M1 in milk 0.025 Micheli et al.

(2005)
28
Table 1.2 Continued
Notes:
AD: Amperometric detector

IAC: Immunoaffinity chromatography
IACLC: Immunoaffinity column liquid chromatogtaphy
IAFB: Immunoaffinity fluorometer biosensor
BLMs: Bilayer lipid membranes
DPP: Differential pulse polarography
ECS: Electrochemical sensing
ECS-ELISA: Electrochemical sensing based on indirect ELISA
ELISA: Enzyme linked immunosorbant assay
ELISA ST: Enzyme linked immunosorbant assay for screening
ELISA-SPE: Enzyme linked immuno sorbant assay combined with screen
printed electrode
FD: Fluorescence detector
FI-IA-AD: Flow-injection immunoassay with amperometric detector
GC-FID: Gas chromatography with flame ionization detecter
HPLC: High pressure liquid chromatography
HPTLC: High pressure thin liquid chromatography
HPTLC-SPE: High pressure thin liquid chromatography with solid phase
extraction
ICA: Immnuochromatographic assay
MECC: Micellar electrokinetic’s capillary chromatography
MS: Mass spectrometry
OPLC: Over pressured liquid chromatography
SIIA: Sequential injection immunoassay test
TLC: Thin layer chromatography
TLC-FDs: Thin layer chromatography with fluorescence densitometer
UVD: Ultraviolet detector
1.2.5 Electrochemical Properties of Aflatoxins
Polarographic determination of aflatoxin has been studied by Smyth et al.

(1979) using DPP technique to determine AFB1, AFB2, AFG1 and AFG2 in food
samples using Britton-Robinson buffer solution as the supporting electrolyte. They
found that all aflatoxins exhibited similar polarographic behaviour over a pH range 4 –
29
11. They also obtained that the best-defined waves for analytical purposes were in
Britton-Robinson buffer at pH 9. Slight differences in the potential of reduction of
aflatoxins have been observed owing to their slight differences in structure. They found
that the half wave potentials of reduction of the various aflatoxins were AFB1 = -1.26
V, AFB2 = -1.27 V, AFG1 = -1.21 V and AFG2 = -1.23 V (all versus SCE). The limit
of detection for AFB1 in pure solutions was about 2 x 10 -8 M (25 ppb).
Gonzalez et al. (1998) have studied the cyclic voltammetry of AFG1 in

methanol: water (1:1, v/v) solution on different electrodes such as glassy carbon,
platinum and gold electrodes. They found that AFG1 gave high peak current at a
potential of about +1.2V using glassy carbon electrode compared to other types of
working electrode. The electro activity of the studied aflatoxins increased in the order
AFG2 < AFB1 < AFB2 < AFG1. This order reflects the ability of the aflatoxins
molecules to undergo electrochemical oxidation on glassy carbon electrode.
Duhart et al. (1988) have determined all types of aflatoxin using HPLC
technique with electrochemical detection. They have used differential-pulse mode at
the dropping mercury electrode (DME) with 1 s drop time for detection system, large
drop size, current scale of 0.5 µA and modulation amplitude of 100 mV. They have
found that using mobile phase which consisted of 62.7% BRB at pH 7, 17.9% methanol
and 19.4% acetonitrile, the peak potentials for all aflatoxins were slightly different
which were AFB1 = -1.37 V, AFB2 = -1.36 V, AFG1 = -1.30 V and at -1.30 V for
AFG2. Due to the fairly close together of peak potentials, a potential of -1.28 V has
been selected for detection purposes of these aflatoxins together with HPLC technique.
They found that using this technique average percentage recoveries for peanut butter
samples (n = 4) which have been spiked with a mixture of the four aflatoxins were
AFB1 (76%), AFB2 (77%), AFG1 (87%) and AFG2 (81%). This results show that a
major advantage of this technique was it did not require a derivatisation step as is
common for fluorescent detection.
30
1.3 Voltammetric Techniques
1.3.1 Voltammetric Techniques in General
Voltammetry is an electrochemical method in which current is measured as a

function of the applied potential. It is a branch of electrochemistry in which the
electrode potential, or the faradaic current or both are changed with time. Normally,
there is an interrelationship between all three of these variables (Bond et al., 1989).
The principle of this technique is a measurement of the diffusion controlled current
flowing in an electrolysis cell in which one electrode is polarisable (Fifield and Kealey,
2000). In this technique a time dependent potential is applied to an electrochemical
cell, and the current flowing through the cell is measured as a function of that potential.
A plot of current which is directly proportional to the concentration of an electroactive
species as a function of applied potential is called a voltammogram. The
voltammogram provides quantitative and qualitative information about the species
involved in the oxidation or reduction reaction or both at the working electrode.
Polarography is the earliest voltammetric technique which was developed by

Jaroslav Heyrovsky (1890-1967) in the early 1920s, for which he was awarded the
Nobel Prize in Chemistry in 1959. It was the first major electro analytical technique
(Barek et al., 2001a). Since then many different forms of voltammetry have been
developed such as direct current polarography (DCP), normal polarography (NP),
differential pulse polarography (DPP), square-wave polarography (SWP), alternate
current polarography (ACP), cyclic voltammetry (CV), stripping voltammetry (SV),
adsorptive stripping voltammetry (AdSV) and adsorptive catalytic stripping
voltammetry (AdCSV) techniques. Working electrodes and limit of detection (LOD)
for these modern voltammetric techniques are shown in Table 1.3 (Barek et al., 2001a).
The advantage of this technique include high sensitivity where quantitative and
qualitative determination of metals, inorganic and organic compounds at trace levels,
10-4 – 10-8 M (Fifield and Kealey, 2000), selectivity towards electro active species
31
(Barek et al., 2001b), a wide linear range, portable and low-cost instrumentation,
speciation capability and a wide range of electrode that allow assays of many types of
samples such as environmental samples (Zhang et al., 2002; Ghoneim et al., 2000;
Buffle et al., 2005), pharmaceutical samples (Yaacob, 1993; Yardimer et al., 2001;
Abdine et al., 2002; Hilali et al., 2003 and Carapuca et al., 2005 ), food samples
(Ximenes et al., 2000; Volkoiv et al., 2001); Karadjova et al., 2000; Sabry and Wahbi,
1999 and Sanna et al., 2000), dye samples (Mohd Yusoff et al., 1998 and Gooding et
al., 1997) and forensic samples (Liu et al., 1980; Wasiak et al., 1996; Pourhaghi-Azar
and Dastangoo, 2000; Woolever et al., 2001).
Various advances during the past few years have pushed the detectability of
voltammetric techniques from the submicromolar level for pulse voltammetric
techniques to the subpicomolar level by using an adsorptive catalytic stripping
voltammetry (Czae and Wang, 1999). The comparison of using polarographic and other
analytical techniques in different application fields is depicted in Table 1.4 (Barek et al.,
2001a).
1.3.2 Voltammetric Measurement
1.3.2.1 Instrumentation
Voltammetry technique makes use of a three-electrode system such as working

electrode (WE), reference electrode (RE) and auxiliary electrode (AE). The whole
system consist of a voltammetric cell with a various volume capacity, magnetic stirrer
and gas line for purging and blanketing the electrolyte solution.
32
Table 1.3 Working electrodes and LOD for modern polarographic and
voltametric techniques
Technique Working LOD

electrode
TAST DME ~ 10 -6 M
Normal pulse polarography (NPP) DME ~ 10 -7 M

Normal pulse voltammetry (NPV) HMDE ~ 10 -7 M
Stair case voltammetry (SCV) HMDE ~ 10 -7 M
Differential pulse polarography (DPP) DME ~ 10 -7 M

Differential pulse voltammetry (DPV) HMDE ~ 10 -8 M
Square wave polarography (SWP) DME ~ 10 -8 M

Square wave voltammetry (SWV) HMDE ~ 10 -8 M
Alternate current polarography (ACP) DME ~ 10 -7 M

Alternate current voltammetry (ACV) HMDE ~ 10 -8 M
Anodic stripping voltammetry (ASV) HMDE ~ 10 -10 M

Cathodic stripping voltammetry (CSV) MFE ~ 10 -9 M
Potentiometric stripping analysis (PSA) MFE ~ 10 -12 M

33
Table 1.4 The application range of various analytical techniques and their
concentration limits when compared with the requirements in different fields of
chemical analysis.
Field of chemical analysis Concentration range
Environmental monitoring 10-12 M to 10 -4 M
Toxicology 10-11 M to 10 -2 M
Pharmacological studies 10-10 M to 10 -4 M
Food control 10-8 M to 10-4 M
Forensic 10-7 M to 10-3 M
Drug assay 10-5 M to 10 -2 M
Analytical techniques Application range
Adsorptive stripping voltammetry 10-12 M to 10-7 M
Anodic / cathodic stripping voltammetry 10-11 M to 10-6 M
Differential pulse voltammetry 10-8 M to 10-3 M
Differential pulse polarography 10-7 M to 10-3 M
Tast polarography 10-6 M to 10-3 M
d.c.polarography 10-5 M to 10-3 M
spectrophotometry 10-6 M to 10-3 M
HPLC with voltammetric detection 10-7 M to 10-3 M
HPLC with fluorescence detection 10-9 M to 10-3 M

34
Table 1.4 Continued
Analytical techniques Application range
Spectrofluorometry 10-9 M to 10-3 M
Atomic absorption spectrometry 10-8 M to 10-3 M
Atomic fluorescence spectrometry 10-9 M to 10-3 M
Radioimmunoanalysis 10-13 M to 10 -3 M
Neutron activation analysis 10-9 M to 10-3 M
x-ray fluorescence analysis 10-7 M to 10-3 M
mass spectrometry 10-9 M to 10-3 M
A typical arrangement for a voltammetric electrochemical cell is shown in Figure 1.7

(Fifield and Haines, 2000).
N2
Inlet
RE WE AE
Cell
cover
Solution
Level
Stirrer
Figure 1.7 A typical arrangement for a voltammetric electrochemical cell

(RE: reference electrode, WE: working electrode, AE: auxiliary electrode)
35
The arrangement of the electrodes within the cell is important. The RE is

placed close to the WE and the WE is located between the RE and the AE. Using the
three-electrode-cell concept, a potentiostat monitors the voltage over the WE and AE
which is automatically adjusted to give the correct applied potential. This is obtained
by continuously measuring the potential between the WE and the RE, by comparing it
to the set voltage and by adjusting the applied voltage accordingly if necessary. The
cell material depends on application that it is usually a small glass beaker with a close
fitting lid, which includes ports for electrodes and a nitrogen gas purge line for
removing dissolved oxygen and an optional stir bar.
The WE is the electrode where the redox reaction of electroactive species takes
place and where the charge transfer occurs. It is potentiostatically controlled and can
minimise errors from cell resistance. It is made of several different materials including
mercury, platinum, gold, silver, carbon, chemically modified and screen printed
electrode. The performance of voltammetry is strongly influenced by the WE. The
ideal characteristics of this electrode are a wide potential range, low resistance,
reproducible surface and be able to provide a high signal-to-noise response. The WE
must be made of a material that will not react with the solvent or any component of the
solution over as wide a potential range as possible. The potential window of such
electrodes depends on the electrode material and the composition of the electrolyte as
summarised in Table 1.5 below (Wang, 2000). Majority of electrochemical methods
use HMDE and MFE (Economou and Fielden, 1995) for use in the cathodic potential
area, whereas solid electrode such as gold, platinum, glassy carbon, carbon paste are
used for examining anodic processes.
Mercury has been used for the WE in earlier voltammetry techniques,

including polarography. Since mercury is a liquid, the WE often consists of a drop
suspended from the end of a capillary tube. It has several advantages including its high
over potential for the reduction of hydronium ion to hydrogen gas. This allows for the
application of potential as negative as -1.0 V versus SCE in acidic solution, and -2.0V
versus SCE in basic solution.
36
Table 1.5 List of different type of working electrodes and its potential windows.
ELECTRODE ELECTROLYTE POTENTIAL WINDOWS
1 M H2SO4 +0.3 to -0.1V

Hg
1 M KCl 0 to -1.9V
1 M NaOH -0.1 to -2.0V
0.1 M Et4NH4 OH -0.1 to -2.3V
1 M H2SO4 +1.0 to -0.5V

Pt
1 M NaOH +0.5 to -1.0V
1 M HClO4 +1.5 to -1.0V

C
0.1M KCl +1.0 to -1.3V
Other advantages of using mercury as the working electrode include the ability
of metals to dissolve in the mercury, resulting in the formation of an amalgam. The
greatest advantages of this electrode is that new drops or new thin mercury films can be
readily formed, and this cleaning process removes problems that could be caused by
contamination as a result of previous analysis. In contrast, this is not the generally case
for electrodes made from other materials, with the possible exception of carbon
electrodes, where the electrode cleaning is made of cutting off a thin layer of the
previous electrode surface. Another advantage is the possibility to achieve a state of
pseudostationary for linear sweep voltammetry (LSV) using higher scan rate.
Miniaturised and compressible mercury electrode offer new possibilities in
voltammetry especially for determination of biologically active species and surfactant.
One limitation of mercury electrode is that it is easily oxidised at + 0.3 V and cannot be
used at potential more positive than + 0.4 V versus the SCE, depending on the
composition of the solution (Dahmen, 1986 and Fifield and Haines, 2000). At this
potential, mercury dissolves to give an anodic polarographic wave due to formation of
mercury (I) (Skoog et al., 1996).
37
There are three main types of mercury electrode used in voltammetry

techniques including hanging mercury drop electrode (HMDE), dropping mercury
drop electrode (DME) and static mercury drop electrode (SMDE). In the HMDE, a
drop of the desired size is formed and hanged at the end of a narrow capillary tube. In
the DME, mercury drops form at the end of the capillary tube as a result of gravity.
The drop continuously grows and has a finite lifetime of several seconds. At the end
of its lifetime the mercury drop is dislodged, either manually or by the gravity, and
replaced by a new drop. In the SMDE, it uses a solenoid-driven plunger to control
the flow of mercury. It can be used as either HMDE or DME. A single activation of
solenoid momentarily lifts the plunger, allowing enough mercury to flow through the
capillary to form a single drop. To obtain a dropping mercury electrode the solenoid
is activated repeatedly. The diagram of HMDE is shown in Figure 1.8 (Metrohm,
2005).
Figure 1.8 A diagram of the HMDE

38
Other type of mercury electrode is the controlled growth mercury drop

electrode (CGME). In this electrode, a fast response valve controls the drop growth.
The cross sectional view of the CGME is shown in Figure 1.9 (BAS, 1993).
Figure 1.9 A diagram of the CGME
The capillary has a stainless stell tube embedded in the top end of the
capillary. Mercury flow through the capillary is controlled by a fast response valve.
The valve is rubber plug at the end of a shaft which when displaced slightly up will
allow mercury to flow. Since the contact between filament of mercury in the capillary
and the reservoir is a stainless steel tube, the electrode has low resistance. The total
resistance from the contact point to the mercury is about 7 ohm. The valve seal is
controlled by the valve seal adjustment knob. The opening of this valve is controlled
by a computer-generated pulse sequence, which leads to a stepped increase in the
drop size. Changing the number of pulse and/or the pulse width, so a wide range of
drop sizes is available can therefore vary the drop size. Varying the time between the
39
pulses can control the rate of growth of the mercury drop. Therefore, a slowly
growing mercury drop suitable for stripping experiments can be generated. An
important advantage of the CGME compared to HMDE is no contamination of the
capillary due to back diffusion (Dean, 1995).
In addition to the mercury drop electrode, mercury may be deposited onto the
surface of a solid electrode to produce mercury film electrode (MFE). The MFE is
based on an electrochemically deposited mercury film on conventional substrate
electrode such as a solid carbon, platinum, or gold electrode. The solid electrode is
placed in solution of mercury ion (Hg2+) and held at a potential at which the reduction
of Hg2+ to Hg is favorable, forming a thin mercury film. It displays the properties of a
mercury electrode, having various electro analytical advantageous such as a high
hydrogen evolution over potential and simple electrochemistry of many metals and
other species of analytical interest. For example, Castro et al. (2004) have used thin-
film coated on a glassy carbon electrode (GCE) for quantitative determination of
polycyclic aromatic hydrogen (PAH). In this experiment, they plated the mercury over
5 min at a cell voltage of -0.9 V. Economou and Fielden (1995) have developed a
square wave adsorptive stripping voltammetric technique on the MFE for study of
riboflavin. In this work, riboflavin was absorbed on the MFE at a potential of 0.0 V
(vs. Ag/AgCl) in pH 12.0 of electrolyte solution.
However, the increased risks associated with the use, manipulation and disposal
of metallic mercury or mercury salt, have led to general trend for more environmental-
friendly analytical methods such as using bismuth film electrode (BFE) instead of
mercury (Kefala et al., 2003; Economou, 2005; Lin et al., 2005a and Lin et al., 2005b ).
The BFE consists of a thin bismuth film deposited on a carbon paste substrate that has
shown to offer comparable performance to the MFE. Morfobos et al. (2004) have
studied square wave adsorptive stripping voltammetry (SWAdSV) on a rotating-disc
BFE for simultaneous determination of nickel (II) and cobalt (II). Hocevar et al. (2005)
have developed a novel bismuth-modified carbon paste (Bi-CPE) for a convenient and
40
reliable electrochemical sensor for trace heavy metals detection in conjunction with
stripping electroanalysis.
In another study, using pencil-lead BFE as the working electrode, Demetriades

et al. (2004) have determined trace metals by anodic stripping voltammetry (ASV).
They revealed that pencil-lead BFE were successfully applied to the determination of
plumbum and zinc in tap water with results in satisfactory agreement with atomic
absorption spectrometry (AAS). Zahir and Abd Ghani (1997) have developed a pencil
2B graphite paste electrode which was fabricated with polymerized 4-vinylpyridine for
glucose monitoring.
Other solid or metal electrode commonly used as WE are carbon, platinum

(Salavagione et al., 2004; Santos and Machado, 2004; Aslanoglu and Ayne, 2004 ),
gold (Hamilton and Ellis,1980; Parham and Zargar, 2001; Parlim and Zarger, 2003;
Moressi et al., 2004; Munoz et al., 2005;), graphite (Orinakova et al., 2004; Pezzatini et
al., 2004; Jin and Lin, 2005), diamond (Sonthalia et al., 2004) and silver (Iwamoto et
al., 1984). Solid electrodes based on carbon are currently in widespread use in
voltammetric technique, primarily because of their broad potential window, low
background current, rich surface chemistry, low cost, chemical inertness, and suitability
for various sensing and detection application (Wang, 2000). It includes glassy carbon
electrode (GCE), carbon paste electrode (CPE), chemically modified electrode (CME)
and screen-printed electrode (SPE).
For the GCE, the usual electrode construction is a rod of glassy carbon, sealed
into an inert electrode body, a disc of electrode material is exposed to the solution. It is
the most commonly used carbon electrode in electro analytical application (Ozkan and
Uslu, 2002; Ibrahim et al., 2003; Erk, 2004). The cleaning of this electrode is
important, to maintain a reactive and reproducible surface. Pre-treated
electrochemically GCE have increased oxygen functionalities that contribute to more
rapid electron transfer. Wang et al. (1997) have used in adsorptive potentiometric
stripping analysis of tamoxifen, a nonsteriodal anti-estrogen used widely for the
41
treatment of hormone-dependent breast cancer. The electrode was anodised at + 1.7 V

for 1 min in the electrolyte containing tomixifen. Using cyclic voltammetric technique,
they found that a large definite anodic peak corresponding to the oxidation of the
adsorbed drug at GCE at + 0.985 V. Other workers using GCE as the WE were Wang
et al. (2004), Shi and Shiu (2004) and Ji et al. (2004).
The CPE, a mixture of carbon powder and a pasting medium at certain ratio,
were the result of an attempt to produce electrode similar to the dropping mercury
electrode (Kizek et al., 2005). They are particularly useful for anodic studies, modified
electrode and also for stripping analysis. CMEs are electrodes which have been
deliberately treated with some reagents, having desirable properties, so as to take on the
properties of the reagents (Arrigan, 1994; Kutner et al., 1998). A few examples of
these applications were reported by Abbaspour and Moosavi, (2002), Abbas and
Mostafa (2003); Ciucu et al. (2003); Ferancova et al. (2000) and Padano and Rivas
(2000).
SPE are increasingly being used for inexpensive, reproducible and sensitive
disposable electrochemical sensors for determination of trace levels of pollutant and
toxic compounds in environmental and biological fluids sample (Wring et al., 1991).
A disposable sensor has several advantages, such as preventing contamination between
samples, constant sensitivity and high reproducibility of different printed sensors (Kim
et al., 2002). The most useful materials for printing electrochemical sensors could be
carbon-based inks because they have a very low firing temperature (20-120° C) and can
be printed on plastic substrate. Carbon can also be directly mixed with different
compounds, such as mediator and enzymes. A few examples of the experiments which
used SPE as the WE were reported by Kim et al. (2002), for detection of phenols using
α-cyclodextrin modified screen printed graphite electrodes. Ohfuji et al. (2004) have
constructed a glucose sensor based on a SPE and a novel mediator pyocyanin from
Pseudomonas aeruginosa. Carpini et al. (2004) have studied oligonucleotide-modified
screen printed gold electrodes for enzyme-amplified sensing of nucleic acid. Lupu et
al. (2004) have developed SPE for the detection of marker analytes during wine
42
making. In this work they have developed biosensors for malic acid and glucose with a
limit of detection of 10-5 M and 10 -6 M for malic acid and glucose respectively. The
sensors were applied in the analysis of different samples of wine.
Besides using macro-electrode, voltammetric technique also utilizes

microelectrode (Lafleur et al. 1990) with the size of electrode radius much smaller than
the diffusion-layer thickness, typically between 7 to 10 µM (Hutton et al., 2005 and
Buffle and Tercier-Waeber, 2005) as the WE. It is constructed from the same materials
as the macro-electrode but with a smaller diameter to enhance mass transport of analyte
to the electrode surface due to smaller electrode than the diffusion layer. Hence,
increasing signal-to noise ratio and measurement can be made in highly resistive media
due to decrease of the ohmic drop that results when the electrode size reduced
(Andrieux et al., 1990). The microelectrode with diameter as small as 2 µm, allow
voltammetric measurements to be made on even smaller sample (Harvey, 2002). Aoki
(1990) reported that ultra microelectrodes influence electrode kinetics more specifically
than conventional electrode because of lateral diffusion promotes mass transport. Since
it minimise uncompensated resistance, they are useful for determination of kinetic
parameters. Almeida et al. (1998) have done voltammetric studies of the oxidation of
the anti-oxidant drug dipyridamole (DIP) in acetonitrile and ethanol using ultra
microelectrode (UME). In this work they studied cyclic voltammetric technique of the
drug with the UME which was 12.7 µm diameters. They found that with that electrode,
the diffusion current of DIP is proportional to the electrode radii for low scan rates in
cyclic voltammetry.
The second electrode used in the voltammetric system is auxiliary electrode

(AE). The AE is made of an inert conducting material typically a platinum electrode
wire (Wang, 2000). It provides a surface for a redox reaction to balance the process that
occured at the surface of the WE. It does not need special care, such as polishing. In
order to support the current generated at the WE, the surface area of the AE must be
equal to or larger than of the WE. The function of the AE is to complete the circuit,
allowing charge flow through the cell (Fifield and Haines, 2000). In an electro
43
analytical experiment, there is no need to place the AE in a separate compartment since

the diffusion of product that was produced by a redox reaction at the surface of the AE
does not significantly interfere the redox reaction at the WE.
The third electrode used in the voltammetric technique is reference electrode

(RE). This RE provides a stable potential so that any change in cell potential is
attributed to the working electrode. The major requirement for the RE is that the
potential does not change during the recording voltammetric curve at different applied
voltage (Heyrovsky and Zuman, 1968). The common RE are standard hydrogen
electrode (SHE), calomel electrode (SCE) and silver/silver electrode (Ag/AgCl). The
SHE is the reference electrode used to establish standard-state potential for other half
reaction. It consists of a platinum electrode immersed in a solution in which the
hydrogen ion activity is 1.00 and in which hydrogen gas is bubbled at a pressure of 1
atm. The standard-state potential for the reaction;
2H+ (aq) + 2e- → H2 (g) (1.0)
is 0.00 V for all temperatures. It is rarely used because it is difficult to prepare and
inconvenient to use.
The second reference electrode is the Standard Calomel Electrode (SCE)

which is based on the redox couple between mercury chloride (Hg2Cl2) and Hg as
below;
Hg2Cl2(s) + 2e 2Hg(l) + 2Cl-(aq) (1.1)
The potential of this electrode is determined by the concentration of chloride ion. It is

constructed using an aqueous solution saturated with potassium chloride (KCl) which
has a potential of + 0.2444 V at 25 0 C. It consists of an inner tube packed with a paste
of Hg, Hg2Cl2 and saturated KCl. A small hole connects the two tubes, an asbestos fiber
serves as a salt bridge to the solution in which the SCE is immersed.
44
Other type of reference electrode is the Ag/AgCl electrode which is the most
common RE since it can be used at higher temperature. This electrode is based on the
redox couple between silver chloride ( AgCl ) and silver ( Ag ) as illustrated below;
AgCl(s) + 2e Ag(s) + Cl- (1.2)
The potential of this electrode is determined by the concentration of Cl-. For saturated
KCl the potential is + 0.197 V whereas for 3.5 M KCl the potential electrode is + 0.205
V at 25° C (Harvey, 2000).
1.3.2.2 Solvent and Supporting Electrolyte
Electrochemical measurements are commonly carried out in a medium which

consists of solvent containing a supporting electrolyte. Sometimes in most cases,
supporting electrolyte has to be added to the dissolved sample in an attempt to achieve
the following (Heyrovsky and Zuman 1968);
a) To make solution conductive

b) To control the pH value so that organic substances are reduced in a
given potential range and inorganic substances are not hydrolysed
c) To ensure the formation of such complexes that give well
developed and well separated waves
d) To shift the hydrogen evaluation towards more negative potentials
and to eliminate catalytic effects on hydrogen evolution
e) To suppress unwanted maxima by addition of surface-active
substances to the supporting electrolyte.
The choice of the solvent is primarily by the solubility of the analyte, its redox
activity and also by solvent properties such as electrical conductivity, electrochemical
activity and chemical reactivity. The solvent should not react with the analyte and
45
should not undergo electrochemical reaction over a wide potential range. In aqueous
solution the cathodic potential is limited by the reduction of hydrogen ions:
2 H+ (aq) + 2 e- → H2 (g) (1.3)
resulting hydrogen evolution current. The more acidic the solution the more positive
is the potential of this current due to the reaction expressed by;
E = E0H2/H+ - 0.059 pH (1.4)
The composition of the electrolyte may affect the selectivity of voltammetric

measurements. The ideal electrolyte should give well-separated and well-shaped peaks
for all the analytes sought, so that they can be determined simultaneously. For example
Kontoyannis et al. (1999) used tris buffered saline (TBS) at pH 7.4 as the supporting
electrolyte for simultaneous determination of diazepam and liposome using DPP
technique. Inam and Somer (1998) have determined selenium (Se) and lead (Pb)
simultaneously in whole blood sample by the same technique using 0.1 M HCl as the
supporting electrolyte. They observed that there were three peaks at -0.33 V, -0.54 V
and -0.41 V which belonged to an intermetallic compound (PbSe), Se and Pb
respectively. Barbiera et al. (1997) have developed anodic stripping voltammetric
technique for simultaneous determination of trace amounts of zinc, lead and copper in
rum without pre-treatment and in the absence of supporting electrolyte. They observed
that there were three peaks at -0.92 V, -0.42 V and 0.05 V which belong to Zn, Pb and
Cu respectively.
Because of the sensitivity of the voltammetric method, certain impurities in

supporting electrolyte can affect the accuracy of the procedures. It is thus necessary to
prepare the supporting electrolyte from highly purified reagents and should not easily
oxidised and reduced. To obtain acceptable ionic strength of supporting electrolyte,
certain concentration should be prepared which is usually about 0.1 M. This level is a
compromise between high conductivity and minimum contamination (Wang, 2000).
46
The low ionic strength which is 0.01 M of supporting electrolyte (HClO4 – NaClO4)
was very effective for the adsorptive accumulation of analyte on the electrode as found
by Berzas et al. (2000) when they developed adsorptive stripping square wave
technique for determination of sildenafil citrate (Viagra) in pharmaceutical tablet.
Dissolved oxygen must be removed from supporting electrolyte first since the
reduction of dissolved oxygen will cause two cathodic peaks at -0.05 V and -0.9 V
(versus SCE) as reported by Fraga et al. (1998) and Reinke and Simon (2002). With
increasing pH, the waves due to reduction of oxygen are shifted to more negative
potential. The oxygen reduction generates a large background current, greater than that
of the trace analyte, and dissolved oxygen therefore tends to interfere with
voltammetric analysis (Colombo and van den Berg, 1998). The common method for the
removal of dissolved oxygen is by purging with an inert gas such as nitrogen or argon
where longer time may be required for large sample volume or for trace measurements.
To prevent oxygen from reentering, the cell should be blanketed with the gas while the
voltammogram is being recorded. However, this conventional procedure is time
consuming and not suitable for flow analysis. Due to this reason, Colombo and van den
Berg (1998) have introduced in-line deoxygenating for flow analysis with voltammetric
detection. They have used an apparatus which is based on the permeation of oxygen
through semi-permeable silicone tubing into an oxygen free chamber and enables the
determination of trace metals by flow analysis with voltammetric determination.
1.3.2.3 Current in Voltammetry
When an analyte is oxidised at the working electrode, a current passes electrons

through the external electric circuitry to the auxiliary electrode, where reduction of the
solvent or other components of the solution matrix occurs. Reducing an analyte at the
working electrode requires a source of electrons, generating a current that flows from
the auxiliary electrode to the cathode. In either case, a current resulting from redox
reaction at the working electrode and auxiliary electrode is called a faradaic current. A
current due to the analyte’s reduction is called a cathodic current. Anodic currents are
47
due to oxidation reaction. The magnitude of the faradaic current is determined by the
rate ofthe resulting oxidation or reduction reaction at the electrode surface. Two factors
contribute to the rate of the electrochemical reaction which are the rate at which the
reactants and products are transported to and from the electrode, and the rate at which
electron pass between the electrode and the reactants and products in solution.
There are three modes of mass transport that influence the rate at which reactant
and products are transported to and from the electrode surface which are diffusion,
migration and convection. Difussion from a region of high concentration to region of
low concentration occurs whenever the concentration of an ion or molecule at the
surface of electrode is different from that in bulk solution. When the potential applied
to the WE is sufficient to reduce or oxidise the analyte at the electrode surface, a
concentration gradient is established. The volume of solution in which the
concentration gradient exists is called the diffusion layer. Without other modes of mass
transport, the width of the diffusion layer increases with time as the concentration of
reactants near electrode surface decreases. The contribution of diffusion to the rate of
mass transport is time-dependent.
Convection occurs when a mechanical means is used to carry reactants toward

the electrode and to remove products from the electrode. Ther most common means of
convection is to stirr the solution using a stir bar. Other methods include rotating the
electrode and incorporating the electrode into a flow cell.
Migration occurs when charged particles in solution are attracted or repelled

from an electrode that has a positive or negative surface charge. When the electrode is
positively charged, negatively charged particles move toward the electrode, while the
positive charged particles move toward the bulk solution. Unlike diffusion and
convection, migration only affects the mass transport of charged particles.
The rate of mass transport is one factor influencing the current in voltammetry
experiment. When electron transfer kinetics are fast, the redox reaction is equilibrium,
48
and the concentrations of reactants and products at the electrode are those psecified by
Nersnt Equation. Such systems are considered electrochemically reversible. In other
system, when electron transfer kinetics are sufficiently slow, the concentration of
reactants and products at the electrode surface, and thus the current, differ from that
predicted by the Nersnt euqation. In this case the system is electrochemically
irreversible.
Other currents that may exist in an electrochemical cell those are unrelated to
any redox reaction are nonfaradaic and residual currents. The nonfaradaic current must
be accounted for if the faradaic component of the measured current is to be determined.
This current occurs whenever the electrode’s potential is changed. Another type of non-
faradaic current is charging current which occur in electrochemical cell due to the
electrical double layer’s formation. Residual current is a small current that inevitably
flows through electrochemical cell even in the absence of analyte (Harvey, 2000).
1.3.2.4 Quantitative and Qualitative Aspects of Voltammetry
Quantitative information is obtained by relating current to the concentration of

analyte in the bulk solution and qualitative information is obtained from the
voltammogram by extracting the standard-state potential for redox reaction. The
concentration of the electroactive species can be quantitatively determined by the
measurement of limiting current which is linear function of the concentration of electro
active species in bulk solution.
Half- potential serves as a characteristic of a particular species which undergoes

reduction or oxidation process at the electrode surface in a given supporting electrolyte,
and it is independent of the concentration of that species. Its function in the qualitative
determination is the same as retention time in chromatographic technique.
49
1.3.3 Type of Voltammetric Techniques
1.3.3.1 Polarography
Polarography is a subclass of voltammetry in which the WE is the DME. This

technique has been widely used for the determination of many important reducible
species since the DME has special properties particularly its renewable surface and
wide cathodic potential range. In this technique, it takes place in an unstirred solution
where a limiting current is diffusion limiting current (Harvey, 2000). Each new
mercury drop grows in a solution whose composition is identical to that of the initial
bulk solution. The relationship between the concentration of analyte, CA, and limiting
current ( Id ) is given by Ilikovic equation ( Ewing, 1997; Heyrosky, 1968; Ilkovic,
1934 and Dahmen,1986).
1 2 1
I d = 708nD m t C A
2 3 6
(1.5)
where:
n = number of electrons transferred in the redox reaction
D = analyte’s diffusion coefficient ( cm2 sec-1)
m = flow rate of mercury drop (g sec-1)
t = drop time (sec)
CA = concentration of depolariser (mol l-1)
The above equation represents the current at the end of the drop life. The average
current (Iave) over the drop life is obtained by integrating the current over this time
period:
1 2 1
I ave = 607nD 2 m 3 t 6 C A (1.6)
From the above equation, there is a linear relationship between diffusion current and
concentration of electroactive species. It also indicates that the limiting diffusion
50
current is a function of the diffusion coefficient which depends on the size and shape
of the diffusion particle. As compared to another technique such as cyclic
voltammetry technique, in this technique, the peak current is directly proportional to
concentration and increases with the square root of the scan rate as given by the
Randles-Sevcik equation (Equation 1.7) for a reversible system (Wang, 2000).
Ip = (2.69 x 105) n 3/2 ACD1/2υ1/2 (1.7)

where;
n = number of electron
A = electrode area (cm2)
C = concentration (mol cm-3)
D = diffusion coefficient (cm2 s-1)
υ = scan rate (V s -1)
There are several types of polarographic techniques as was mentioned earlier.

Polarography is used extensively in the analysis of metal ion, inorganic anions and
organic compounds containing easily reducible or oxidisable functional group. A list
of electroreducible and electrooxidisable organic functional groups is shown in Table
1.6 (Dean, 1995).
Table 1.6 Electroreducible and electrooxidisable organic functional groups
Electroreducible Aromatic carboxylic acid, azomethines, azoxy compounds

organic functional conjugated alkene, conjugated aromatic, conjugated carboxylic
groups acid conjugated halide, conjugated ketone, diazo compounds,
dienes, conjugated double bond, nitroso compounds,
organometallics, disulfide, heterocycles, hydroquinones,
acetylene, acyl sulfide aldehyde, hydroxylamines, imines,
ketones, nitrates, nitriles nitro compounds, oximes, peroxides,
quinones, sulfones, sulfonium salts and thiocyanates.
Electrooxidisable Alcohols, aliphatic halides, amines, aromatic amines, aromatic

organic functional ides, carboxylic acids, ethers, heterocyclic amines, heterocyclic
groups aromatics, olefins, organometallic and phenols.
51
1.3.3.2 Cyclic Voltammetry
Cyclic voltammetry (CV) is a potential-controlled reversal electrochemical

experiment. A cyclic potential sweep is imposed on an electrode and the current
response is observed (Gosser, 1993). CV is an extension of linear sweep
voltammetry (LSV) in that the direction of the potential scan is reversed at the end of
the first scan (the first switching potential) and the potential range is scanned again in
the reverse direction. The experiment can be stopped at the final potential, or the
potential can be scanned past this potential to the second switching potential, where
the direction of the potential scan is again reversed. The potential can be cycled
between the two switching potentials for several cycles before the experiment is
ended at the final potential. CV is the most widely used technique for acquiring
qualitative information about electrochemical reactions.
Analysis of the current response can give considerable information about the
thermodynamics of redox processes, the kinetics of heterogeneous electron-transfer
reaction and the coupled chemical reactions or adsorption processes. It is often the first
electrochemical experiment performed in an electrochemical study especially for any
new analyte since it offers a rapid location of redox potentials of the electro active
species and convenient evaluation of the effect of media upon the redox process. In the
CV technique, the applied potential sweep backwards and forwards between two limits,
the starting potential and the switching potentials. In this technique, the potential of the
WE is increased linearly in an unstirred solution. The resulting plot of current versus
potential is called a cyclic voltammogram. Figure 1.10a to 1.10c show the cyclic
voltammograms for reversible, irreversible and quasireversible reactions. For a typical
reduction and oxidation process in reversible reaction (as in Figure 1.10a), during the
forward sweep the oxidised form is reduced, while on the reverse sweep the reduced
form near the electrode is reoxidised. Chemical reaction coupled to the electrode
reaction can drastically affect the shape of the CV response. In the case of irreversible
reaction, no reverse peak is observed (Figure 1.10b).
52
(a)
(b)
(c)
Figure 1.10 Cyclic voltammograms of (a) reversible, (b) irriverisble and (c)
quasireversible reactions at mercury electrode (O = oxidised form and R = reduced
form)
53
The cyclic voltammogram shows characteristics of an analyte by several

important parameters such as peak currents and peak potentials that can be used in the
analysis of the cyclic voltammetric response either the reaction is reversible,
irreversible or quasi-reversible as listed in Table 1.7. Cyclic voltammogram will
guide the analyst to decide the potential range for the oxidation or reduction of the
analyte and that it can be a very useful diagnostic tool.
Table 1.7 The characteristics of different type of electrochemical reaction
Type of reaction Characteristics
Reversible Cathodic and anodic peak potential are separated by 59/n mV.
The position of peak voltage do not alter as a function of voltage

scan rate
The ratio of the peak current is equal to one
The peak currents are proportional to square root of the scan rate
The anodic and cathodic peaks are independent of the scan rate
Disappearance of a reverse peak

Irreversible
The shift of the peak potential with scan rate ( 20 – 100 mV/s)
Peak current is lower than that obtained by reversible reaction.
Quasi-reversible Larger separation of cathodic and anodic peak potential

(> 57/ n mV) compared to those of reversible system.
54
1.3.3.3 Stripping Voltammetry
Stripping technique is one of the most important and sensitive electrochemical

technique for measuring trace metals and organic samples. The term stripping is
applied to a group of procedures involving preconcentration of the determinant onto
the working electrode, prior to its direct or indirect determination by means of a
potential sweep (Wang, 1985). The preconcentration (or accumulation) step can be
adsorptive, cathodic or anodic. Its remarkable sensitivity is attributed to the addition
of an effective preconcentration step with advanced measurement procedures that
generate an extremely favorable signal-to-noise ratio as reported by Blanc et al.
(2000). Brainina et al. (2000) list advantages of this technique such as high
sensitivity, low detection limit, wide spectrum of test materials and analytes, both of
organic and inorganic origin, insignificant effect of matrix in certain instances,
compatibility with other methods, relative simplicity and low cost of equipment and
finally, it can be automatic on-line and portable options. This technique is able to
measure many analytes simultaneously such as reported by Ghoneim et al. (2000).
They have determined up to eleven metals in water sample simultaneously using this
technique. Stripping voltammetry technique utilises a few steps as follows;
a) Deposition step:
In this step, analyte will be preconcentrated on the WE within a certain time

while solution is stirred. The deposition potential imposed on the WE is chosen
according to the species to be determined and is maintained for a deposition period
depending on their concentration. The choice of deposition potential can provide some
selectivity in the measurement (Sun et al., 2005). Deposition time must be controlled
since the longer the deposition time, the larger the amount of analye available at the
electrode during stripping. During deposition step, the solution is stirred to facilitate
transportation of ions of interest to the surface of WE.
55
b) Rest step:
In this step, it allows formation of a uniform concentration of the ions of interest

on the mercury. As the forced convection is stopped at the end of the deposition period,
the deposition current drops almost to zero and a uniform concentration is established
very rapidly. It also insures that the subsequent stripping step is performed in a
quiescent solution.
c) Stripping step:
This step consists of scanning the potential anodically for anodic stripping and
cathodically for cathodic stripping. When the potential reaches the standard potential
of a certain ion of interest–metal ion complex, the particular ion of interest is reoxidised
or reduced back into solution and a current is flowing. The resultant voltammogram
recorded during this step provide the analytical information of the ions of interest. The
stripping step after preconcentration gives the possibility for selective determination of
different substances assuming that they also have different peak potential as reported by
Kubiak et al. (2001). The potential-time sequence in stripping analysis is shown in
Figure 1.11 which shows all three steps that were mentioned earlier.
Deposition Stripping
Stirred Quiescent
Applied
Potential Deposition potential
Final potential
Time
Figure 1.11 The potential-time sequence in stripping analysis

56
Most stripping measurements require the addition of appropriate supporting

electrolyte and removal of dissolved oxygen. The former is needed to decrease the
resistance of the solution and to ensure that the metal ions of interest are transported
toward the electrode by diffusion and not by electrical migration. Contamination of the
sample by impurities in the reagents used for preparing the supporting electrolyte is a
serious problem. Dissolved oxygen severely hampers the quantitation and must be
removed.
The main types of interference in stripping analysis are overlapping stripping

signals, the adsorption of organic surfactants on the electrode surface, the presence of
dissolved oxygen and the formation of intermetallic compounds by metals such as
copper and zinc co-deposited in the mercury electrode. Overlapping signals cause
problems in the simultaneous determination of analytes with similar redox potential
such as lead and tin. Intermetallic-compounds formation and surfactant cause a
depression of the stripping response and also shifting the signal location. Stripping
voltammetry is composed of three related techniques that are anodic, cathodic and
adsorptive stripping voltammetry.
1.3.3.3a Anodic Stripping Voltammetry (ASV)
ASV is mainly used in the determination of metal ions that can be reduced and
then re-oxidised at a mercury electrode. Voltammetric measurements of numerous
metal ions in various types of samples have been reported (Arancibia et al., (2004) and
Shams et al., (2004)). The term ASV is applied to the technique in which metal ions
are accumulated by reduction at an HMDE held at a suitable negative potential. The
deposition potential is usually 0.3 to 0.5 V more negative than a standard potential for
reduced metal ion to be determined. ASV consists of two steps. The first step is a
controlled potential electrolysis in which the working electrode is held at a cathodic
potential sufficient to deposit the metal ion (M n+) on the electrode to form an amalgam,
M(Hg). This step is called accumulation step which is represented by an equation;
57
Mn+ + ne- + Hg → M(Hg) (1.8)
The solution is stirred during this process to increase the rate of deposition. Near the
end of the deposition time, stirring is stopped to eliminate convection as a mode of
mass transport. The duration of the deposition step is selected according to the
concentration level of the metals ion.
In the second step, the potential is scanned anodically toward more positive
potential. When the potential of the WE is sufficiently positive the analyte is stripped
from the electrode, returning to solution as its oxidised form. This step is called
stripping which is represented by an equation
M(Hg) → Mn+ + ne- (1.9)
The current during the stripping step is monitored as a function of potential giving rise
to a peak-shaped voltammogram. The peak current is proportional to the analyte’s
concentration.
1.3.3.3b Cathodic Stripping Voltammetry (CSV)
CSV is used to determine a wide range of organic compounds, and also

inorganic compounds that form insoluble salts with the electrode material. It has been
found to be widely applicable to many problems of clinical and pharmaceutical interest
(Wang, 1988). Voltammetric measurements of numerous electro active species of
biological significance such as drugs (Ghoneim et al., 2003; Arranz et al., 1999;
Rodriguez et al., 2004; Ghoneim and Beltagi, 2003 and Cabanillas et al., 2003 ) and
toxic substances ( Hourch et al., 2003; Safavi et al., 2004 ) have been reported.
The term CSV was used originally for the indirect trace determination of
organic compounds as mercury salt, involving anodic oxidation of mercury and
subsequently cathodic reduction of mercury. This technique is similar to the previous
58
technique with two exceptions. First, the deposition step involves the oxidation of the
mercury electrode from Hg to Hg2+, which then reacts with the analyte to form an
insoluble salt at the surface of the electrode. For example, when chloride ion (Cl-) is the
analyte, the deposition step is
2Hg(l) + 2Cl- (aq) → Hg 2Cl2 (s) + 2e- (1.10)
Secondly, stripping is accomplished by scanning cathodically toward a more negative

potential, reducing Hg2+ back to Hg and returning the analyte to the solution.
Hg2Cl2(s) + 2e- → 2Hg(l) + 2Cl-(aq) (1.11)
1.3.3.3c Adsorptive Stripping Voltammetry (AdSV)
The term AdSV seems to have been first used by Lam et al. in 1983. It is a
powerful analytical technique for the determination of nmol levels of a wide range of
organic compounds (Smyth and Smyth, 1978; Smyth and Vos, 1992). In technical
report for International Union Of Pure And Applied Chemistry under Analytical
Chemistry Division Commission On Electro analytical Chemistry (Fogg and Wang
1999), suggested that AdSV technique is applied to stripping voltammetric technique in
which accumulation is effected by adsorption of mainly organic determinants and that
can be justified less in case of the adsorption of metal complexes in determining metal
ions. The AdSV term should not be applied when there is a change of oxidation state
of the metal ion during the accumulation for example in the accumulation of copper (I)
complexes or salts or in other cases where an organic compounds is being accumulated,
and determined indirectly, as a metal salts or complex such as mercury salts or nickel
complexes.
In this technique the deposition step occurs without electrolysis process.

Instead, the analyte adsorbs on to the electrode’s surface. During deposition, the
electrode is maintained at a potential that enhances adsorption. When deposition is
59
sufficient the potential is scanned in an anodic or cathodic direction depending on

whether we wish to oxidise or reduce the analyte. In recent years, AdSV which
improves sensitivity and selectivity, have promoted the development of many
electrochemical methods for ultra-trace measurement of a variety of organic (Ghoneim
et al., 2003; Ghoniem and Taufik, 2004; Farias et al., 2003 and Hourch et al., 2003)
and inorganic species (Ensafi et al., 2004; Zimmerman et al., 2001) and Jurado-
Gonzalez et al. (2003). Barek et al. (2001a) reported that AdSV on HMDE is much
more sensitive with a typical LOD between 10-9 and 10 -10 M.
1.3.3.4 Pulse voltammetry
This technique uses pulse waveform in recording its voltammogram which

offers enhanced sensitivity and resolution. The advantage of pulse techniques is that the
waveform is designed so as to discriminate against non-faradic current hence, increase
sensitivity. The enhanced resolution is particularly useful when several electroactive
species are being analysed simultaneously (Fifield and Haines, 2000). In this
technique, current sampling takes place at the end of the pulse and utilise the different
time dependence of faradic (if) and charging current (ic), as shown in Figure 1.12.
Faradaic
Current, i f
Sampling time
+ Changing
Current, i c
Current
0 Pulse time
Time
Figure 1.12 Schematic drawing showing the if and ic versus pulse time course
60
This technique, is aimed at lowering the detection limits of voltammetric measurement

down to 10-8 M in its differential pulse mode. Increasing the ratio between the faradic
and non-faradic current permit convenient quantitation down to the 10-8 M
concentration level (Wang, 2000). Differential pulse and square wave techniques are
the most commonly used pulse technique. Differential pulse polarograms or
voltammograms are peak shaped because current difference is measured. The
following section gives an overview of three important waveforms in pulse technique.
1.3.3.4a Differential Pulse Voltammetry (DPV)
In DPV, fixed magnitude pulse (10 to 100 mV) superimposed on a linear

potential ramp are applied to the working electrode at a time just before the end of the
drop as shown in Figure 1.13. The current is sampled twice, just before the pulse
application and again late in the pulse life normally after 40 ms, when the charging
current has decayed. Subtraction of the first current sampled from the second provides
a stepped peak-shape derivative voltammogram. The resulting differential pulse
voltammogram consists of current peaks, the height of which is directly proportional to
the concentration of corresponding analyte. The differential-pulse operation results in a
very effective correction of the charging background current.
Pulse Width
Step E Sample Period
E Pulse
Amplitude
Sample Period
Pulse Period
Outlet
Time
t
Figure 1.13 The schematic diagram of steps in DPV by superimposing a periodic
pulse on a linear scan
61
1.3.3.4b Square Wave Voltammetry (SWV)
SWV is a large-amplitude differential technique in which a waveform

composed of a symmetrical square wave, superimposed on a base staircase potential, is
applied to the working electrode. The current is sampled twice during each square-
wave cycle, once at the end of forward pulse and another at the end of the reverse pulse.
Since the square-wave modulation amplitude is very large, the reverse pulses cause the
reverse reaction of the product of the forward pulse. The difference between the two
measurements is plotted versus the base staircase potential as shown in Figure 1.14.
The resulting peak current is proportional to the concentration of the analyte. Excellent
sensitivity accrues from the fact that the net current is larger than either the forward or
reverse components. Coupled with the effective discrimination against the charging
current, very low detection limits can be attained.
ESW
∆E
2
τ
Applied
Potential
Accumulation Time
Time
Figure 1.14 Waveform for square-wave voltammetry
The advantage of SWV is that a response can be found at a high effective scan
rate, thus reducing the scan time. Because of its high scan rate, it provides a great
economy of time (Arranz et al., 1999 and Ghoneim and Tawfik., 2004). There are
62
several reports on application of the SWV technique for determination of several

samples as listed in Table 1.8 which involves deoxygenation step prior analysis.
However, in certain case beside it offers the additional advantage of high speed; it can
increase analytical sensitivity and relative insensitivity to the presence of dissolved
oxygen as reported by Economou et al. (2002).
Table 1.8 Application of SWV technique
No Sample Supporting electrolyte Reference
1 Ketorolac in human serum Acetate buffer, pH 5.0 Radi et al. (2001)
2 Imidacloprid in river water BRB, pH 7.2 Guiberteau et al.

(2001)
3 Cocaine and its metabolite Phosphate buffer, pH 8.5 Pavlova et al.

(2004)
4 Amlodipine besylate in BRB, pH 11.0 Gazy (2004)

tablets and biological fluids
5 EDTA species in water Diluted HCl, pH 2.8 and Zhao et al. (2003)
0.05 M NaCl
6 RDX in soil 0.1 M acetate buffer, pH Ly et al. (2002)

4.5
7 Levofloxacin in human 0.05 M acetate buffer, pH Radi and El-Sherif

urine 5.0 (2002)
8 Sertraline in commercial 0.1 M borate, pH 8.2 Nouws et al.

products (2005a)
63
Table 1.8 continued
No Sample Supporting electrolyte Reference
9 Cadmium in human hair 0.01 M TEA, pH 11.0 Arancibia et al.

(2004)
10 Copper, stannous, 0.1 M dibasic ammonium Locatelli (2005)

antimony, thallium, and citrate, pH 6.3
plumbum in food and
environmental matrices
11 Imatinib (Gleevec) and its 0.012 M HClO4, pH 2.0 Rodriguez et al.

metabolite in urine (2005)
12 Famotidine in urine 0.02 M MOPS buffer, pH Skrzypek et al.

6.7 (2005)
13 Haloperidol in bulk form, BRB, pH 9.0 El-Desoky et al.

pharmaceutical formulation (2005)
and biological fluids
14 Copper, cadmium and zinc Acetic acid, pH 7.34 El-Maali et al.

complexes with (2004)
cephalosporin antibiotic
15 Triprolidine in 0.04 M BRB, pH 11.0 Zayed and Habib

pharmaceutical tablets (2005)
16 Metoclopramide in tablet 0.4 M HCl-sodium Farghaly et al.

and urine acetate, pH 6.2 and 0.2 M (2005)
KCl
17 Cefoperazone in bacterial BRB, pH 4.4 Billova et al.

culture, milk and urine (2005)
64
Table 1.8 continued
Notes:
BRB: Britton-Robinson buffer

EDTA: Ethylene diamine tetraacetic acid
HClO4: Perchloric acid
MOPS: 3-(N-morpholino)propanesulphonic
RDX: Hexahydro-1,3,5-trinitro-1,3,5-triazine
TEA: Triethylammonium
1.4 Objective and Scope of Study
1.4.1 Objective of Study
The development of methods for the determination of aflatoxins has been

constantly in demand due to the fact that aflatoxins are a major concern as the toxic
contaminants of foodstuffs and animal feed, and have been recognised as a potential
threat to human health since the early 1960s resulting in frequent economic losses.
The widespread occurrence of aflatoxins producing fungi in our environment and the
reported natural occurrence of toxin in a number of agricultural commodities has led
the investigator to develop a new method for aflatoxins analysis.
In order to maintain an effective control of aflatoxins in food and foodstuffs

proper analytical procedures must be applied. Different analytical methods such as
thin layer, liquid chromatography or enzyme immunoassay test which were
mentioned in Chapter I, have been developed for aflatoxins determination. However
all these proposed techniques have their disadvantages. For HPLC technique, the
method requires well equipped laboratories, trained personnel, harmful solvents as
well as time consuming, and costly in buying and maintenance.
65
For immunological methods such as ELISA technique, it has a few

disadvantages such as long incubation time, washing and mixing steps and also labour
intensive. This method requires highly specific polyclonal or monoclonal sera which
is costly. For radioimmunoassay (RIA) technique, it uses radioisotope which raises
concerns in radiation safety in dealing with and also in disposing radioactive waste. For
micellar electrokinetic capillary chromatography (MECC) technique, it needs preparing
a lot of reagents for the buffering system and takes considerable time to complete the
analysis.
With regards to voltammetric analysis, no stripping voltammetric study of

aflatoxins is reported until now. This study has been proposed in order to develop a
new alternative technique for determination of aflatoxins. This technique is the most
promising method for determination of aflatoxins due to its main advantages compared
to competing analytical techniques such as excellent sensitivity, reasonable speed,
minimal sample pre-treatment, satisfactory selectivity, wide applicability, ability to
undertake speciation analysis and low cost of instrumentation and maintenance as
reported by Economou et al. (2002) and Radi (2003). Stripping analysis has been
proven to be a powerful technique for the determination of both organic and inorganic
electroactive species (Bond, 1980). Differential pulse cathodic stripping voltammetry
satisfies the requirement of an efficient and senstitive technique for the determination
of aflatoxin compounds because it has been shown to be a powerful technique in
determination of other organic compounds in various sample origins down to ppb level
as reported by Zima et al. (2001), Sun and Jiao (2002), Yardimer and Ozaltin (2004)
and Nouws et al. (2005b).
In this research, the voltammetric behaviour of these compounds would be

studied in great detail and the stripping voltammetry especially differential pulse
stripping voltammetry (DPSV) and square wave stripping voltammetry (SWSV)
techniques could provide accurate and sensitive methods for aflatoxins determination
especially in food sample such as groundnuts, would be investigated.
66
The groundnut is selected for the sample to be analysed in this study since
among foods and foodstuff, peanut and peanuts products are widely utilized as health
food (www.copdockmill.co.uk/aflatoxin). It seem to be significantly contaminated with
aflatoxins. It contains high content of carbohydrate which provides a substrate that is
particularly suitable for toxin production (Neal, 1987). This will affect the quality and
safety of humans life. Futhermore, Aspergillus moulds which produces aflatoxins grow
easy on groundnut.
AFB1, AFB2, AFG1 and AFG2 were selected as the subjects in this study due
to the regulatory requirement for their determination in imported raw grounnuts as
imposed by the Malaysian government to assure that this commodity is free from any
toxin contamination before being supplied for the domestic market. AFM1 and AFM2
are not involved in this study since currently no such regulation imposed by the
Malaysian government even though a regulatory standard has already been set which is
not more 0.05 ppb AFM1 and AFM2 should be present in milk and milk products. For
the time being, the analysis on AFM1 and AFM2 are not being carried out in Malaysia.
The other reason, globally, AFB1, AFB2, AFG1 and AFG2 are more concerned with
their contamination in many food samples compared to AFM1 and AFM2 which
contaminate milk and milk products only.
The objectives of this study are:
a) To investigate the electrochemical behaviour of aflatoxins B1 (AFB1), AFB2,

AFG1 and AFG2 on the mercury electrode in appropriate supporting electrolyte.
b) To establish optimum conditions for the determination of those aflatoxins by

the method of differential pulse cathodic stripping voltammetry (DPCSV) and
square wave stripping voltammetry (SWV) techniques with a HMDE as the
working electrode using Britton-Robinson buffer (BRB) solution as the
67
c) To develop an accurate, sensitive, fast and simple method for the determination
of all studied aflatoxins in food samples such as ground nut and comparing the
results with the established method such as HPLC.
1.4.2 Scope of Study
The studies are as follows:
a) Studies on the voltammetric behaviour of aflatoxin compounds using cyclic

voltammetry (CV) technique as an introductory step. Using this technique, the
effect of increasing concentration of aflatoxins, scan rate and repetitive scanning
on the peak height (Ip) and peak potential (Ep) of each aflatoxin will be
investigated.
b) Studies on the differential pulse cathodic and anodic stripping voltammetriy

(DPCSV) of all aflatoxins. Parameters optimisation include pH of supporting
electrolyte, accumulation potential (Eacc), accumulation time (tacc), scan rate (υ),
initial potential (Ei), final potential (Ef) and pulse amplitude.
c) Using optimised analytical parameters and experimental conditions, the effect of

increasing concentration of aflatoxins to the Ip of the compounds will be studied.
Regression equation, R2 value, linearity range, limit of detection (LOD), limit of
quantification (LOQ), accuracy and reliability of the method will be obtained.
Ruggedness and robustness tests also will be studied for the proposed technique.
d) The proposed technique will be further investigated in terms of interference

where each aflatoxin will be reacted with increasing amounts of metals ion such
as zinc, aluminum, nickel, lead and copper, and with organic compounds such as
ascorbic acid, L-cysteine and β-cyclodextrin.
68
e) The optimised parameters for DPCSV technique will be applied for square wave
stripping voltammetry (SWSV) including optimisation steps such as the Eacc,
tacc, pulse amplitude, scan rate, voltage step and frequency. The last 3
parameters are interrelated to each other in the SWSV technique.
f) Both DPCSV and SWSV techniques that were successfully developed will be
applied in the determination of aflatoxins content in real samples such as
groundnut. The recovery studies also will be carried out for the accuracy test of
the developed method. The results will be compared with that obtained by
accepted technique such HPLC. For the HPLC analysis, the final solutions from
the extraction and clean-up procedures of groundnut in chloroform were sent to
the Chemistry Department, Penang Branch, Ministry of Science, Technology
and Innovation (MOSTI).
g) The stability of aflatoxins will be determined for aflatoxin stock and standard
solutions according to the following procedures
i) Aflatoxin stock solutions prepared in benzene: acetonitrile (98:2)

will be studied using ultra-violet-visible spectrophotometer. In
this analysis, each aflatoxin stock solution will be monitored
every month (from 0 to 12 months) and the concentrations of
aflatoxins will be calculated from the measured absorbance.
ii) Aflatoxin standard solutions in BRB which was kept in the

freezer at -4.0 0 C will be measured using voltammetric technique
monthly up to 6 months
iii) Aflatoxin standard solutions in BRB which have been added into
the voltammetric cell and exposed to ambient temperature will be
onitored every hour (from 0 to 8 hours) by measuring their peak
height and peak potential
69
iv) Aflatoxin standard solution in BRB which was added into the
voltammetric cell containing BRB at different pH (6.0, 7.0, 9.0
and 11.0) and exposed to ambient temperature will be monitored
every hour (from 0 to 3 hours) by measuring their peak height
and peak potential.
70
CHAPTER II
RESEARCH METHODOLOGY
2.1 Apparatus, Materials and Reagents
2.1.1 Apparatus
A BAS CV-50W voltammetric analyser in connection with a Control Growth

Mercury Electrode (CGME) stand equipped with a three-electrode system and a 20 ml
capacity BAS MR-1208 cell as shown in Figure 2.0 were used for all the voltammetric
determinations. The working electrode was a Hanging Mercury Drop Electrode
(HMDE). As a reference and counter-electrode, a silver-silver chloride (Ag / AgCl)
and a platinum wire were used, respectively. All potentials are quoted relative to this
reference electrode. BAS CV-50W was connected to a computer for data processing.
For robustness test which is required in validating procedure for developed technique,
VA 757 Computrace Metrohm Voltammetric analyzer as shown in Figure 2.1 was used.
A pH meter Cyber-scan model equipped with a glass electrode combined with an
Ag/AgCl reference electrode, was employed for all pH measurements. UV-VIS
spectrophotometer (UV-2501/PC Shimadzu) was used for measurement of absorbance
of all 10 ppm aflatoxin stock solutions and 1 ppm aflatoxin standard in BRB solution.
71
(a) (b)
Figure 2.0 BAS CGME stand (a) which is connected to CV-50W voltammetric
analyser and interface with computer (b) for data processing
Figure 2.1 VA757 Computrace Metrohm voltammetric analyzer with 663 VA

Stand (consists of Multi Mode Electrode (MME))
72
2.1.2 Materials
2.1.2.1 Aflatoxin Stock and Standard Solutions
Aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin

G2 ( AFG2) (SIGMA) are supplied by Chemopharm with a quantity of 1 mg per bottle
each. The batch numbers of these aflatoxins are listed in Table 2.0.
Table 2.0 List of aflatoxins and their batch numbers used in this experiment.
Aflatoxins Batch Number
AFB1 112K4049
AFB2 120K4014
AFG1 11K4003
AFG2 082K4091
Stock solutions of these aflatoxins with concentrations of 10 ppm each were

prepared by dissolving the total amount of aflatoxin with 2.0 ml acetonitrile. The
solution was transfered into 100 ml volumetric flask. Benzene was added into the flask
until 100 ml mark. The flasks were covered with aluminium foil and kept in
refrigerator with a temperature of 16° C. The absorbances of these stock solutions were
measured using ultraviolet-visible (UV) spectrometer and the concentrations of
aflatoxins were calculated. This measurement of these aflatoxins stock solutions were
carried out monthly per a year while kept under cool condition for stability study.
73
For preparation of aflatoxins standard solution with concentration of 1 ppm, 1

ml of the stock solution was pipetted and placed into an amber bottle. Nitrogen was
bubbled into the solution to remove all the organic solvent until dryness followed with
an addition of 10 ml BRB at pH of 9.0. The solution was well mixed and kept in
freezer at temperature of -4 0 C. This standard solution was analysed by voltammetric
technique very month for 6 months to observe the stability of the aflatoxins in buffered
solution. To obtain the concentration of 0.1 µM of AFB1, AFB2, AFG1 and AFG2
each in 10 ml supporting electrolyte, an amount of 312, 315, 328 and 330 µl was
injected into the cell respectively.
2.1.2.2 Real Samples
Real samples such as groundnut were purchased from a few domestic shops
which are located in Taman Universiti, Skudai, Johor. These samples were labeled as
S01 to S15.
2.1.3 Reagents
The chemicals used were all of analytical reagent quality grade and the
solutions were prepared with de-ionised water obtained from a Barnstead Ultra
Nanopure water system. 1% of sodium hypochlorite was used to clean all glasswares
used.
2.1.3.1 Britton-Robinson Buffer (BRB), 0.04 M
BRB with a buffering range from 2.0 to 12 was prepared by dissolving 2.47 g of
boric acid (Fluka) in a 1liter flask containing 2.3 ml of glacial acetic acid (MERCK)
and 2.70 ml orthophosphoric acid (Ashland Chemical) and then further diluting to the 1
liter mark with deionised water. The pH of the buffer solution was adjusted to the
74
required value by addition of 1.0 M sodium hydroxide (MERCK) or 1.0 M

hydrochloric acid solutions (MERCK).
2.1.3.2 Carbonate Buffer, 0.04 M
Carbonate buffer with a buffering range from pH 9.0 to 12 was prepared by

mixing two different solutions which were firstly prepared. The first solution, 0.04 M
sodium bicarbonate was prepared by dissolving 3.36 g sodium bicarbonate (MERCK) in
a 1 liter of de-ionised water. Similarly, for the second solution, 0.04 M sodium
carbonate was prepared by dissolving 4.24 g sodium carbonate (MERCK) in 1 liter of
deionised water. The pH of the buffer was adjusted to the required value by mixing the
two solutions.
2.1.3.3 Phosphate Buffer, 0.04 M
Phosphate buffer was prepared by mixing two different solutions which were
firstly prepared. The first solution, 0.04 M potassium hydrogen phosphate was prepared
by dissolving 5.44 g potassium hydrogen phosphate (BDH) in a 1 liter of deionised
water. Similarly, for the second solution, 0.04 M sodium hydrogen phosphate was
prepared by dissolving 5.68 g sodium hydrogen phosphate (BDH) in 1 liter of deionised
water. The pH of the buffer was adjusted to the required value by mixing the two
solutions.
2.1.3.4 Ascorbic Acid Solution, 1.0 mM
This solution was prepared by dissolving 0.0176 g of ascorbic acid (GCE) in

100 ml of de-ionised water.
75
2.1.3.5 β-cyclodextrin (β-CD) Solution, 1.0 mM
This solution was prepared by dissolving 0.1135 g of C42H70O35 (Fluka) in 100

ml of de-ionised water.
2.1.3.6 L-cysteine, 1.0 x 10-5 M
This solution was prepared by dissolving 0.012 g of cysteine (SIGMA) in 100

ml of de-ionised water. 1.0 ml of this solution was diluted to 100 ml with de-ionised
water.
2.1.3.7 2,4-dihydrofuran, 0.15 M
This solution was prepared by diluting 1.105 ml of 13.57 M of 2,4 dihydrofuran

(SIGMA) into 100 ml with de-ionised water.
2.1.3.8 Coumarin, 3.0 x 10 -2 M
This solution was prepared by dissolving 0.21 g of coumarin (SIGMA) in 50 ml

methanol.
2.1.3.9 Poly-L-lysine (PLL), 10 ppm
This solution was prepared by dissolving 0.001 g of poly-l-lysine (SIGMA) in

100 ml de-ionised water.
2.1.3.10 Standard Aluminium (III) Solution, 1.0 mM
The standard aluminium (III) solution was prepared by dissolving 0.5212 g of

Al(NO 3)3.9H2O (MERCK) in 100 ml de-ionised water.
76
2.1.3.11 Standard Plumbum (II) Solution, 1.0 mM
The standard plumbum (II) solution was prepared by dissolving 0.0530 g

Pb(NO3)2 (EMORY) in 100 ml deionised water.
2.1.3.12 Standard Zinc (II) Solution, 1.0 mM
The standard zinc (II) solution was prepared by dissolving 0.121g ZnSO4.5H2O
(BDH) in 100 ml de-ionised water.
2.1.3.13 Standard Copper (II) Solution, 1.0 mM
The standard copper (II) solution was prepared by dissolving 0.0980 g.

CuSO4.7H2O (GCE) in 100 ml de-ionised water.
2.1.3.14 Standard Nickel (II) Solution, 1.0 mM
The standard nickel (II) solution was prepared by dissolving 0.0963 g

NiCl2.6H2O (MERCK) in 100 ml deionised water.
2.1.3.15 Methanol: 0.1 N HCl Solution, 98 %
This solution was prepared by mixing 98 ml of methanol and 2 ml 0.1 N HCl.
2.1.3.16 Zinc Sulphate Solution, 15%
This solution was prepared by dissolving 15 g of zinc sulphate (MERCK) in 100

ml de-ionised water.
77
2.2 Analytical Technique
2.2.1 General Procedure for Voltammetry Analysis
The general procedure used to obtain stripping voltammograms was according

to the following procedures. A 10 ml aliquot of buffer solution was placed in a
voltammographic cell and the required aflatoxin standard solution was added using a
micropipette. The stirrer was switched on at 300 rpm and the solution was purged with
nitrogen gas for 10 minute. After forming a new HMDE, accumulation was effected for
the required time at the appropriate potential while stirring the solution. A medium
mercury drop size was used. At the end of the accumulation time the stirring was
switched off and after 10 s had elapsed to allow the solution to become quiescent, the
negative going potential was initiated. When further volume of aflatoxin were added to
the cell, the solution was redeoxygenated with nitrogen gas for another 2 minute before
carrying out further voltammetric determination following the mentioned general
procedures.
2.2.2 Cyclic Voltammetry (Anodic and Cathodic Directions)
Cyclic voltammetry of all aflatoxins were carried out by anodic and cathodic
cyclic techniques. The initial parameters for cathodic cyclic voltammetry are as
follows; initial potential (Ei) = 0 V, high potential, (EH) = 0 V, low potential (EL) = -
1.50 V, scan rate (υ)= 200 mV/s, quiet time = 10 s and sensitivity = 1 µA/V. For anodic
cyclic voltammetry, all previous parameters were maintained except for Ei = -1.50 V.
The concentration of aflatoxins used for these experiments were fixed at 1.3 µM.
2.2.2.1 Standard Addition of Sample
5 ppm (1.59 x 10-5 M) AFB2 standard solution was prepared by taking 5.0 ml
aflatoxin stock solution, degassed until dryness and redissolved in BRB pH 9.0. For
cyclic voltammetry experiment, general procedure as previouly mentioned was
78
followed. A 818 µl of 1.59 x 10-5 M AFB2 standard solution was then added into
voltammetric cell containing 10 ml of supporting electrolyte which resulted in
concentration of aflatoxin of 1.3 µM in the cell. The solution was purged with nitrogen
for 120 s before being scanned. Further series of scans were carried out with increasing
concentration of the aflatoxins from 2.0 to 3.4 µM. Subsequently a purge of 120 s was
employed between the cyclic voltammetry. A graph of peak current against aflatoxin
concentration was plotted and linearity of the curve was calculated. This method was
repeated for other aflatoxins.
2.2.2.2 Repetitive Cyclic Voltammetry
Using the aflatoxins with concentration of 1.3 µM, repetitive cyclic voltammetry
for anodic and cathodic direction were run using the same parameters as mentioned
before with the υ of 200 mV/s and 10 cyclic number. Any change in the Ip and Ep
height of all aflatoxins due to the increasing number of the cycle were observed.
2.2.2.3 Effect of Scan Rate (υ)
The whole procedure for cyclic voltammetry was repeated for all aflatoxins with
different υ from 20 mV/s to 500 mV/s while other parameters kept constant. The
applied υ were 20, 40, 60, 80, 100, 200, 300, 400 and 500 mV/s. Any change in the Ip
and Ep of all aflatoxins due to the changing of the υ were observed. Graphs of log Ip
against log υ, Ep versus log υ and Ip versus υ were plotted.
2.2.3 Differential Pulse Cathodic Stripping Voltammetric Determination of

AFB2
A 315 µl of 1 ppm (0.318 x 1-0-5 M) AFB2 sample solution in BRB at pH 9.0

was added by means of micropipette into the cell which contained 10 ml of the BRB
with the same pH. The resulting concentration of sample in the voltammetric cell is 0.1
µM (31.4 ppb). The solution was deoxygenated with nitrogen free oxygen for 120 s
79
before carrying out further voltammetry. Three further series of sample additions were
employed with a purge of 120 s were performed between each cathodic stripping cycle.
2.2.3.1 Effect of pH
The following general procedure was carried out. A 1.26 ml of 5 ppm (1.59 x
-5
10 M) AFB2 standard solution was added by means of micropipette into the cell which
contained 8.74 ml of BRB at pH 9.0. The resulting concentration of sample in
voltammetric cell is 2.0 µM (628 ppb). The solution was deoxygenated with nitrogen
for 120 s before carrying out further voltammetry. The whole procedure was repeated
for series of buffer from pH 4.0 to 13.0 and a graph of Ip vs. pH was then plotted.
2.2.3.2 Method Optimisation for the Determination of AFB2
The optimisation steps for determination of AFB2 were carried out using two
different concentrations of AFB2 which were 2.0 uM and 0.02 µM for high and low
concentrations respectively. The initial parameters used for this optimisation steps are;
Ei = 0 mV, Ef = -1500 mV, Eacc = 0 mV, t acc = 0, υ = 20 mV/s, pulse implitude = 100
mV and quite time = 10 s. General procedure for voltammetric analysis was employed.
The Ip and Ep values were observed. The same procedure was repeated with the same
parameters except for tacc was 30 s. For the optimisation steps, the influence of each
parameter on the Ip and Ep of AFB2 was studied. In each experiment, one of the
parameters was varied while others were kept constant.
2.2.3.2a Effect of Scan Rate (υ)
After the best condition of pH was chosen, the effect of υ was carried out. The υ
were varied from 20 to 100 mV/s while maintaining other parameters constant.
Subsequently a purge of nitrogen gas for 120 s was employed between cathodic
stripping cycles. A graph of Ip against υ was then plotted and the best υ was then
selected.
80
2.2.3.2b Effect of Accumulation Potential (E acc)
The general procedure was followed with the best condition of pH and υ set to
the required value. Effect of Eacc was carried out with other parameters kept unchanged
as previously mentioned. A graph of Ip against Eacc was then plotted.
2.2.3.2c Effect of Accumulation Time (tacc)
Using the best condition for Eacc, tacc and pH of BRB solution, general procedure
was followed where a series of scans with increasing tacc were carried out. A graph of Ip
against tacc was then plotted.
2.2.3.2d Effect of Initial Potential (Ei)
The effect of Ei to the Ip using the chosen previous optimised parameters was
carried out where a series of scans with different Ei while Ef kept constant were
performed. The effect of Ei was carried out with variation of 0 V to -1.10 V and
increasing towards more negative potential until it reached the required maximum Ip. A
graph of Ip against Ei was then plotted.
2.2.3.2e Effect of Pulse Amplitude
The best conditions of other parameter were chosen and the pulse amplitude was
optimised as the final step in this optimisation procedure. The effect of pulse amplitude
was carried out with variation of 30 to 100 mV. A graph of Ip against pulse amplitude
was then plotted.
2.2.3.3 Method Validation
Validation of the method was examined via evaluation of linearity, limit of

detection (LOD), limit of quantification (LOQ), accuracy, repeatability, reproducibility,
81
recovery, robustness and ruggedness. Multiple scanning of blank before addition of

AFB2 standard solution was performed. LOD was calculated by standard addition of
low concentration of aflatoxins until a response was obtained that is significantly
different from the blank sample (Barek et al., 2001a). The intra-assay and inter-assay
precision for AFB2 was calculated from repeated analysis of certain concentration of
AFB2 during one working day and on different days respectively. Recovery study for
the accuracy of the method were performed by adding an amount of AFB2 into the
eluate of real samples and after extraction and clean-up procedures before being
analysed for AFB2 and percentage recovery of AFB2 was calculated. The robustness,
as a measure of procedure’s capability to remain unaffected by small variations of some
important procedure conditions, was examined by analysis of 0.1 µM AFB2 (31.4 ppb)
under the successive variation of pH of the supporting electrolyte (pH 8.5 – 9.5), Eacc
(from - 0.59 to - 0.61 V) and tacc (75 – 85 s). The ruggedness of the measurement was
examined by applying the proposed procedure to assay of aflatoxin using two different
models of voltammetry instruments that were BAS and Metrohm voltammetry
analysers.
For other aflatoxins (AFB1, AFG1 and AFG2) the same procedures were
followed as mentioned for AFB2 including method validation as well. For all cases,
each calibration curve was constructed and the same statistical parameters were
evaluated.
2.2.3.4 Interference Studies
2.2.3.4a Effect of Cu (II), Ni (II), Al (III), Pb (II) and Zn (II)
The effect of all the above metals on the measurement of 0.1 µM of aflatoxin in
BRB pH 9 was carried out by adding a series of concentration of all metal standard
solutions into the aflatoxin solution in the voltammetric cell started from 0.1 to 1.0 µM
concentration. The graphs of Ip of respective aflatoxin against the concentration of all
standard solutions were plotted.
82
2.2.3.4b Effect of Ascorbic Acid, β-CD and L-cysteine
The effect of ascorbic acid, β-CD and L-cysteine on the measurement of 0.1µM
of aflatoxin in BRB pH 9 were carried by adding a series of concentration of ascorbic
acid, β-CD and L-cysteine solutions into the aflatoxin solution in the voltammetric cell
from 0.1 to 1.0 µM concentration of each compound. The graphs of Ip of respective
aflatoxin against the concentration of added acorbic acid, β-CD and L-cysteine were
plotted.
2.2.3.5 Modified Mercury Electrode with Poly-L-Lysine (PLL)
10 ml of BRB solution at pH 9.0 was pipetted into voltammetric cell followed

with 10 µl of PLL and 0.1 µM aflatoxins. The solution was purged for 10 minutes
before general procedure for voltammetric determination was applied. Ip of 0.1µM
AFB2 was measured. The procedure was repeated by using different pH of BRB.
2.2.4 Square Wave Cathodic Stripping Voltammetry (SWSV)
All optimised parameters which were obtained using DPCSV were applied using
SWSV for determination of 0.1 µM of each aflatoxin.
2.2.4.1 SWSV Parameters Optimisation
All SWSV parameters such as pulse amplitude, voltage step and frequency were
optimised to obtain maximum Ip and defined peak for AFB2. For this optimisation
procedure, 0.1 µM AFB2 was used.
2.2.4.2 SWSV Determination of All Aflatoxins
Other aflatoxins with concentration of 0.1 µM have been measured using SWSV
technique with optimised parameters. The results of Ip and Ep of all aflatoxins were
83
compared with those obtained by DPCSV technique. The effect of concentration on Ip

and Ep of aflatoxins has been observed. The plot of Ip versus concentration of
respective aflatoxin was constructed and all statistical parameters were evaluated.
2.2.5 Stability Studies of Aflatoxins
These studies were carried out by a series of experiments according to the

procedures elaborated below;
2.2.5.1 Stability of 10 ppm Aflatoxins
Stability of 10 ppm aflatoxin stock solutions prepared in benzene: acetonitrile

(98%) was monitored using UV-VIS spectrophotometer using 98% of benzene:
acetonitrile as a blank. 1 ml of sample was scanned three times starting from λ of 500
nm to 280 nm. This measurement was done every month from first day of preparation
for 12 months period time. The concentration of each aflatoxin were calculated using
the formula as stated in Appendix D.
2.2.5.2 Stability of 1 ppm Aflatoxins
Stability of 1ppm aflatoxin standard solutions prepared in BRB pH 9.0 was

performed using DPCSV method. In this experiment, 0.1 µM of each aflatoxin was
scanned every month from first to 6th month. The graph of Ip current against months of
keeping period was plotted.
2.2.5.3 Stability of 0.1 µM Aflatoxins Exposed to Ambient Temperature
Stability study of 0.1 µM aflatoxins in BRB pH 9.0 which was exposed to the
ambient temperature in voltammetric cell was performed using DPCSV method. In this
experiment, 0.1 µM of each aflatoxin was scanned every hour from 0 to 8th hour
84
exposure time. The graph of Ip against exposure time was plotted. Nitrogen gas was
purged for 10 min before performing each scan.
2.2.5.4 Stability of 0.1 µM Aflatoxins in Different pH of BRB
Stability of 0.1µM aflatoxins in different pH of BRB (6 to 11.0) were observed.

For each pH value, the Ip of aflatoxin was measured for 3 hours. The graph of Ip against
exposure time for each pH of BRB was plotted.
2.2.6 Application to Food Sample
Fifteen batches of real samples (groundnut) were purchased and labeled with
S01 to S15 were used in this study. The samples were subjected to the extraction and
clean-up procedure prior voltammetric analysis using DPCSV and SWSV techniques.
A few techniques which were named as Technique 1, 2 and 3 were studied for
extraction and clean-up of real samples.
2.2.6.1 Technique 1
50 g of groundnut samples were weighed into 250 ml blender, then 100 ml of

acetonitrile-water (9:1) solution were added and shaken for 1 hour. The extraction
solution was filtered through a pre-pleated filter paper to remove solid material and
extract was collected and kept for analysis (Pena et al., 2002).
2.2.6.2 Technique 2
Groundnut samples were first ground in a household blender at high speed for
3 minutes. For extraction, 10 ml of methanol-water (80:20) was added to 5g of sample,
followed by 5 ml hexane. The suspension was hand shaken for 3 minutes and then
passed through Whatman No. 4 filter paper. The aqueous layer was diluted 1 in 10 for
the assays to avoid any interference from methanol, hence the consequence aflatoxin’s
85
concentration in the extracted solution was 1/20 th that in groundnut samples. The
extraction took approximately 15 minutes to perform (Garden et al., 2001)
2.2.6.3 Technique 3
This technique is adapted by The Chemistry Department, Penang Branch,

Ministry of Science, Technology and Innovation (MOSTI), Pulau Pinang (2003) as
shown in Appendix E. 1.0 ml of final solution in chloroform was taken and put into an
amber bottle followed with degassing and re-dissolved in 1.0 ml BRB at pH 9.0 before
spiking into voltammetric cell for analysis.
2.2.6.4 Blank Measurement
For blank measurement, the procedure of extraction and clean-up steps were
followed as previously mentioned in Appendix E without addition of real sample. The
blank consisted of methanol, 0.1 N HCl, 15% ZnSO4 and chloroform. 1.0 ml of final
solution was collected from extraction procedure in chloroform, degassed and re-
dissolved in 1.0 ml of BRB at pH 9.0.
2.2.6.5 Recovery Studies
Recovery studies for aflatoxins in groundnut samples were performed using

DPCSV and SWSV techniques. For this study, a certain volume of 1.0 ppm of
aflatoxins as listed in Table 3.3 were injected into 20.0 ml of eluate from blended real
sample (Ammida et al., 2004). The sample with injected aflatoxins was then mixed
with 20.0 ml zinc sulphate, 15%. The total amount of added aflatoxins in the cell
obtained by respective injected volume is shown in Table 2.1. The solution was well
mixed for 30 s and subjected to extraction procedure as previously mentioned. Sample
solution was prepared by degassed of 1 ml of chloroform until dryness followed with
dissolution in BRB at pH 9.0.
86
Table 2.1 Injected volume of aflatoxins into eluate of groundnut and the final
concentrations obtained in voltammetric cell
Injected volume of 1 ppm Final concentration in

aflatoxins (ml) voltammetric cell (ppb)
1.6 3.0
5.1 9.0
9.3 15.0
2.2.6.6 Voltammetric Analysis
Voltammetric analysis of blank, recovery and real samples were carried out
using DPCSV and SWSV techniques. For each sample, 200 µl of the solution was
spiked into 10 ml supporting electrolyte followed with general voltammetric
determination procedure. Voltammograms of each samples were recorded and any peak
appeared was observed. For determination of aflatoxin in real sample, standard addition
method was applied by spiking 10 ppb of aflatoxin standard followed with general
procedure for voltammetric analysis. The amount of aflatoxin in groundnut sample was
calculated according to the formula as stated below;
Aflatoxin (ppb) = P’ / P x C x 12.5 (2.0)

where;
P’ = Ip of sample (nA)
P = Ip of aflatoxin standard (after substract from P’) (nA)
C = Concentration of aflatoxin spiked in the cell (ppb)
12.5 = Factor value after the sample weight, volume of chloroform used
in the extraction and preparation of injection sample have been
considered
87
The detailed information on how this formula was formulated are shown in Appendix F
The results were compared with that obtained by HPLC technique. The HPLC
system used was a Waters model comprising WATERS 600 controller, WATERS 717
auto sampler, WATERS temperature control module and MILLENIUM
chromatography manager, equipped with a model WATERS 420-AC fluorescence
detector filled with standard optical filters with 338 nm bandpass excitation filter and
425 nm longpass emission filter, gain; 16 and maximum span. For chromatographic
separation, a Nova-Pak C-18 steel column (150 x 3.9 mm, 4um particle size) was
employed. The separation was carried out at room temperature using, as the mobile
phase of 70% water, 20 % methanol and 10 % acetonitrile with a flow rate of 1.6 ml /
min at column temperature of 30° C. The injection volume was 30 µl with 12 min for
running time.
88
CHAPTER III
RESULTS AND DISCUSSION
3.1 Cyclic Voltammetric Studies of Aflatoxins
Cyclic voltammetric (CV) experiment is normally carried out for the initial
electroanalytical studies of any compound to obtain information on electroanalytical
properties of the compounds before being investigated in depth using other
electroanalytical techniques is carried out (Wang, 2000). In this work, all aflatoxins
(AFB1, AFB2, AFG1 and AFG2) have been initially analysed by this technique. The
main objective of this experiment was to obtain the electroanalytical properties of
aflatoxin at different scan rates in various pH of BRB solution. Any current peak that
appeared from the voltammogram was evaluated to confirm the type of reaction of
aflatoxin on the mercury electrode. The repetitive cyclic voltammetry were also
performed at certain CV parameters such as scan rate of 200 mV/s in BRB solution with
pH of 9.0. This experiment was carried out to observe the process of accumulation of
aflatoxins on mercury electrode with longer accumulation time. Various pH medium of
BRB have been used in these experiments to observe any involvement of hydronium
ion in the electrode processs of aflatoxins on the mercury electrode (Kamal et al.,
1996). The peak potentials of these compounds which were measured throughout this
work were against an Ag/AgCl as the reference electrode.
89
3.1.1 Cathodic and Anodic Cyclic Voltammetric of Aflatoxins
The parameters set up for cathodic cyclic voltammetric measurement were

initial potential (Ei ) = 0 mV, high potential (EH ) = 0 mV, low potential (EL) = -1500
mV, scan rate (ν) = 200 mV/s and sensititvity = 1 µA/V. A BRB solution with pH 9.0
was initially used as the supporting electrolyte following the previous experiment
carried out by Smyth et al. (1979) using DPP technique. The result that were obtained
showed that the BRB solution with pH 9.0 was the best medium for reduction of
aflatoxins on the mercury electrode. CV experiments results also revealed that the BRB
pH 9.0 is the optimum pH for this reduction to take place. Figure 3.0 shows the Ip of
0.6 µM AFB1 obtained by cathodic cyclic voltammetry in different pH of BRB. The Ep
of AFB1 was shifted to the more negative direction with increasing pH as shown in
Figure 3.1 indicating that proton was involved in the reduction of AFB1 (Sun et al.,
2005).
30
25
20
Ip (nA)
15
10
5
0
5 6 7 8 9 10 11 12 13
pH of BRB
Figure 3.0 Cathodic peak current of 0.6 µM AFB1 in various pH of BRB obtained
by cathodic cyclic voltammetry. Ei = 0, Elow = -1.5 V, Ehigh = 0 and scan rate = 200
mV/s.
90
From the Figure 3.0, the curve rises from pH 6.0 to 9.0 then goes down from pH
9.0 to 12.0. According to Smyth et al. (1979), for pH 6.0 to 8.0, the reduction peak of
AFB1 was caused by the reduction of double bond in benzene ring which conjugates to
the keto group and followed by the protonation of molecule that preceded the reduction
until it achieved maximum reduction at pH 9.0. The possible mechanism for this
reaction is shown in Figure 3.2.
1.34
1.33
1.32
Ep (-m V)
1.31
1.3
1.29
1.28
1.27
5 6 7 8 9 10 11 12 13
pH of BRB
Figure 3.1 Shifting of Ep of AFB1 with incresing pH of BRB. Parameter conditions

O O
O O
O
O
H H
2e-
2H+
OCH3
OCH3
Figure 3.2 Mechanism of reduction of AFB1 in BRB at pH 6.0 to 8.0

91
For pH 10 to 12.0, due to decreasing number of hydrogen ions in the supporting

electrolyte that is involved in the reduction process, anion radical was dimerised and
produced a reduction peak. Their mechanism is shown in Figure 3.3.
O O
O O
O
O
2e -
2 2
O CH 3
OC H3
2 H 2O
D im er + 2 OH-
Figure 3.3 Mechanism of reduction of AFB1 in BRB at pH 9.0 to 11.0
Using BRB at pH 9.0 as the optimum medium for the supporting electrolyte,
cathodic cyclic voltammograms of 1.3 µM of all aflatoxins were obtained where all
aflatoxins produced a sharp and well defined cathodic wave around -1.315 V for both
AFB1 and AFB2, -1.245 V for AFG1 and -1.250 V for AFG2 with a peak current of 42
nA, 30 nA, 47 nA and 39 nA for AFB1, AFB2, AFG1 and AFG2 respectively. The peak
was located not far from the peak of hydrogen over potential which was at -1.450 V.
For all aflatoxins, no oxidation wave appeared in the anodic branch, which indicates
that the aflatoxins reductions are irreversible. Cyclic voltammograms of all aflatoxins
are shown in Figures 3.4 to 3.7.
The effect of initial potential (Ei)) on cathodic peak of AFB1 was investigated.
Varying Ei caused no extra peak to appear for both cathodic and anodic waves which
92
show that the Ei did not produce any significant change on the cathodic peak of AFB1.
The Ip were almost the same for Ei at 0 to -0.8 V which was around 22 nA.
Figure 3.4 Cathodic cyclic voltammogram for 1.3 µM AFB1, obtained at

scan rate of 200 mV/s, Ei = 0, Elow = -1.5 V and Ehigh = 0 in BRB solution at pH
9.0.
Figure 3.5 Cathodic cyclic voltammogram for 1.3 µM AFB2 in BRB solution at pH
9.0. All parameter conditions are the same as in Figure 3.4.
93
Figure 3.6 Cathodic cyclic voltammogram for 1.3 µM AFG1 in BRB solution at pH
Figure 3.7 Cathodic cyclic voltammogram for 1.3 µM AFG2 in BRB solution at pH
For Ei of more than -0.80 V, the Ip decreased to 17 nA as shown in Figure 3.8.

This is because the presence of Zn+2 in BRB solution facilitates the migration and
adsorption of aflatoxin towards the working electrode. This is proven by the study of
94
Zn +2 using CV technique with various Ei and the results are shown in Figure 3.9. There
is no significant change on Ep of AFB1 (-1.30 V) with changing Ei .
30
25
20
I p (nA)
15
10
5
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Ei (-V)
Figure 3.8 Effect of Ei to the Ip of 0.6 µM AFB1 in BRB at pH 9.0 obtained by
cathodic cyclic voltammetry. Parameter conditions are the same as in Figure 4.4.
20
15
Ip (nA)
10
5
0
0 0.2 0.4 0.6 0.8 1 1.2
Ei (-V)
Figure 3.9 Effect of Ei to the Ip of 0.1 µM Zn2+ in BRB at pH 9.0 obtained by
cathodic cyclic voltammetry. Parameter conditions are the same as in Figure 3.4.
Anodic cyclic voltammetry measurement was carried out using the same
parameters with the exception of Ei = -1500 mV (versus Ag/AgCl). The anodic cyclic
voltammograms confirmed that the reduction reaction of all aflatoxins are irreversible
95
whereby only cathodic peak appeared but with a slightly higher current of 46.76 nA at -
1.318 V for AFB1, 35 nA at -1.311 V for AFB2, 52 nA at 1.243 V for AFG1 and 50 nA
at -1.259 V for AFG2. This may be due to longer time of anodic scanning itself, which
gave a longer time for accumulation of aflatoxins on the mercury electrode surface
compared to the cathodic measurement. For example, anodic cyclic voltammogram of
AFB2 is shown in Figure 3.10.
Figure 3.10 Anodic cyclic voltammogram of 1.3 µM AFB2 obtained at scan rate of
200 mV/s, Ei = -1.5 V, Elow = -1.5 V and Ehigh = 0 in BRB solution at pH 9.0.
Standard additions of each aflatoxin shows that the peak current at their
respective potential increases with increasing concentrations. The voltammograms of
AFB2 (Figure 3.11) shows that the peak current at -1.315 V increases with increasing
AFB2 concentration. This dependence is represented in Figure 3.12. The
corresponding equation for this dependence is given below;
Ip (nA) = 21.65 x (µM) + 6.605 (R2 = 0.9857, n = 4) (3.0)
No extra peak was observed with this standard addition either at the anodic
direction or cathodic direction. This result reveals that the appeared peak was due to the
respective aflatoxin and does not from other electroactive compounds.
96
Figure 3.11 Effect of increasing AFB2 concentration on the peak height of cathodic
cyclic voltammetric curve in BRB at pH 9.0; (a) 1.30 µM, (b) 2.0 µ M (c) 2.70 µM and
(d) 3.40 µM. Parameter conditions are the same as in Figure 3.4
100
80
Ip (nA)
60
40 y = 21.65x + 6.605
20 R2 = 0.9857
0
1 1.5 2 2.5 3 3.5 4
[AFB2] / uM
Figure 3.12 Peak height of reduction peak of AFB2 with increasing concentration of
AFB2. Experimental conditions are the same as in Figure 3.4.
97
For other aflatoxins, the dependence of current peak to their concentrations in

BRB at pH 9.0 is listed in Table 3.0. The regression equations of this relationship show
that the peak heights of aflatoxins are correlate well with the concentration of
aflatoxins. Cathodic cyclic voltammograms of other aflatoxins for increasing
concentrations are shown in Appendix G. The plots of Ip versus concentrations for
other aflatoxins are shown in Appendix H.
Table 3.0 The dependence of current peaks of aflatoxins to their concentrations

obtained by cathodic cyclic measurement in BRB at pH 9.0
Aflatoxin Regression equation for increasing R2

concentration from 1.30 to 3.40 µM (n=4)
AFB1 y = 22.451x + 10.989 0.9899
AFB2 y = 21.650x + 6.605 0.9807
AFG1 y = 24.756x + 13.072 0.9812
AFG2 y = 33.55x + 1.435 0.9914
Repetitive cathodic stripping voltammetry studies for all aflatoxins were also
carried out in order to observe whether any adsorption phenomenon took place at the
surface of mercury electrode as reported by Ghoneim et al. (2003a). The results show
that the reduction peak of all aflatoxins increases with the number of cycles, which may
be attributed to adsorption of aflatoxins at the mercury electrode surface (Farias et al.,
2003). No other cathodic peak was observed for all cases which show that no other
species was produced by the reduction of aflatoxins. The peak potentials of all
98
aflatoxins were shifted to less negative direction with increasing number of cycles. For
example, repetitive cathodic cyclic voltammograms of AFB2 is shown in Figure 3.13.
Figures 3.14 and 3.15 show the increasing of peak current of AFB2 cathodic peak and
shifting of its peak potential to less negative direction with increasing number of cycle
respectively. For other aflatoxins, their repetitive cyclic voltammogram are shown in
Appendix I.
Figure 3.13 Repetitive cathodic cyclic voltammograms of 1.3 µM AFB2 in BRB

solution at pH 9.0. Experimental conditions are the same as in Figure 3.4.
60
50
40
Ip (nA)
30
20
10
0
1 2 3 4 5
No of cycle
Figure 3.14 Increasing Ip of 1.3 µM AFB2 cathodic peaks obtained from repetitive
cyclic voltammetry. Experimental conditions are the same as in Figure 3.4.
99
1316
1312
Ep (-mV)
1308
1304
1300
0 1 2 3 4 5 6
No of cycle
Figure 3.15 Peak potential of 1.3 µM AFB2 with increasing number of cycle
obtained by repetitive cyclic voltammetry.
The adsorption of aflatoxins on the electrode surface is expected to be due to the

aflatoxins chemical structures that consist of many functional groups such as ketones,
ester and ether. The presence of these functional groups increases the polarity and
adsorption (Volke and Liska, 1994) of the compound and may be attributed to the
adsorption process taking place on the electrode surface. Furthermore, due to the
presence of the phenyl ring, aflatoxins have surface activity and are readily adsorbed
onto mercury surface (El-Hefnawey et al., 2004). However, a further increase in the
repeatitive cycle tends to slow down the increment of the Ip due to the formation of a
double layer at the mercury electrode (Fifield and Kealey, 2000).
The effect of increasing scan rate, υ (from 20 to 500 mV/s) to the Ep and Ip of
aflatoxins cathodic peaks were observed under the same experimental conditions.
Linear relationship was observed between the log Ip versus log υ for all aflatoxins with
slope values of 0.5346, 0.5097, 0.5623 and 0.5439 for AFB1, AFB2, AFG1 and AFG2
respectively. The slope value of more than 0.5 indicates that the diffusion current
100
produced by the reduction of all aflatoxins on the mercury electrode are influenced by
adsorption in the electrochemical process at the electrode surface (Gosser, 1994 and
Yaridimer and Ozaltin, 2001). For example, a plot of log Ip versus log υ for AFB2 is
shown in Figure 3.16.
1.9
1.7
1.5
1.3
Lo g Ip
1.1
0.9 y = 0.5097x + 0.4115
0.7 R2 = 0.995
0.5
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8
log scan rate
Figure 3.16 Plot of log Ip versus log υ for 1.3 µM AFB2 in BRB solution at pH 9.0.
Experimental conditions are the same as in Figure 3.4.
Plotting Ep versus log υ for scan rates in the range of 20 to 500 mV/s for all
aflatoxins, linear graphs were obtained according to the equation:
a) Ep (-mV) = 61.415 log υ + 1171.6 (R2 = 0.9987, n = 9) for AFB1 (3.1)

b) Ep (-mV) = 40.065 log υ + 1223.2 (R2 = 0.9936, n = 9) for AFB2 (3.2)
c) Ep (-mV) = 48.484 log υ + 1134.8 (R2 = 0.9978, n = 9) for AFG1 (3.3)
d) Ep (-mV) = 49.297 log υ + 1146.0 (R2 = 0.9984), n= 9) for AFG2 (3.4)
The Ep linearly shifted to the more negative direction which confirms the
irreversibility of the reduction processes on the mercury electrode (de Betono et al,
1996 and Arranz et al. 1999). Figure 3.17 shows a plot of Ep of AFB2 versus log υ in
the range of 20 to 500 mV/s. For other aflatoxins, these plots are shown in Appendix J.
101
1340
1330
1320
Ep (-mV)
1310
1300
1290 y = 40.065x + 1223.3
1280 R2 = 0.9936
1270
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8
log scan rate
Figure 3.17 Plot of Ep versus log scan rate for 1.3 µM AFB2 in BRB solution at pH
9.0
A linear plot was obtained for the effect of scan rate on Ip of aflatoxins with 2
linear regions. Taking AFB2 for an example, in the first region which is for the faster
scan rate (100 to 500 mV/s) the equation was linear according to the equation Ip =
0.0759x + 24.566 (R2 = 0.9917, n = 5) as shown in Figure 3.18. At the second region
where the scan rate was slow (20 to 80 mV/s), the linear equation is Ip = 0.2639x + 6.16
(R = 0.9979, n = 5) which was also linear. Due to the slope of slow scan rate region
that is higher than the fast scan rate region, it can be concluded that the reduction of
aflatoxins on the mercury electrode prefers a slow reaction. The plots for other
aflatoxins are illustrated in Appendix K.
As far as the chemical structures of AFB1, AFB2, AFG1 and AFG2 are
concerned, the main difference of AFB1 and AFG1 with AFB2 and AFG2 is the
absence of double bond in the terminal furan ring of AFB2 and AFG2 compared to
AFB1 and AFG1. From the result, it suggests that this double bond does not play a
major role in the reduction process at the mercury electrode since all of them gave
single cathodic peak at very close potentials. However, the double bond in the benzene
102
ring which conjugates with the keto group has the potential to be reduced on the
mercury electrode and produce cathodic peak.
70
60
50
40
Ip (nA)
30
20
10
0
0 50 100 150 200 250 300 350 400 450 500 550
Scan rate (mv/s)
Figure 3.18 Plot of Ip versus υ for 1.3 µM AFB2 in BRB solution at pH 9.0.
From overall results of CV studies, all aflatoxins reduced at mercury electrode

gave a single cathodic peak with Ip at about 40 ± 5.0 nA for the concentration of 1.3 µM
with Ep between -1.250 V and -1.315 V. All the aflatoxins were adsorbed onto the
mercury electrode. Due to these properties, an adsorptive cathodic stripping
voltammetric technique was developed to obtain more sensitive method for the
determination of trace levels of aflatoxins.
3.2 Differential Pulse Cathodic Stripping Voltammetry (DPCSV) of AFB2
In this experiment, AFB2 was used to represent the aflatoxins since all of them
have the same electrochemical behaviour as those found in previous experiments.
Since the results from CV studies showed that all aflatoxins underwent reduction
103
process only, AFB2 was analysed by DPCSV technique using these parameters; Ei = 0,
Ef = –1.50 V, Eacc = 0 mV, no tacc at υ of 50 mV/s.
In this study, AFB2 standard solution was redissolved in BRB at pH 9.0 after the
organic solvent had been completely removed to avoid any effect on the diffusion rate
of electroactive species and the electrical double layer thickness which may increase the
current resistance and further increase the noise level (Kotoucek et al. 1997). BRB
solution at pH 9.0 was used as the supporting electrolyte because from the previous
experiment by CV technique, the BRB solution at pH 9.0 was the best solution for the
determination of aflatoxin. The initial result in Figure 3.19 shows that a single peak
with a peak height (Ip) about 5.0 nA was observed at a peak potential (Ep) of -1.256 V
for 1.0 uM AFB2. The other peak which appears at Ep of -0.92 V is the Zn peak from
the contamination of BRB solution. The single peak at -1.256 V was due to the AFB2
reduction peak since no extra peak was observed as previously confirmed by the CV
technique.
Figure 3.19 DPCS voltammograms of 1.0 µM AFB2 (Peak I) in BRB at pH 9.0 (a) at
tacc = 0 (b) and 30 s (c). Other parameter conditions; Ei = 0, Ef = -1.50 V, Eacc = 0 V, υ =
50 mV/s and pulse amplitude = 100 mV. Peak II is the Zn peak.
104
The effect of tacc on the Ip of AFB2 peak was also examined. When tacc was set
to 30 s, the Ip of AFB2 increased to 22 nA and at the same time, no increase for the Ip of
the Zn peak (Figure 3.19). This result suggests that AFB2 has been actively adsorbed on
the mercury electrode surface. This phenomenon is similar to the previous result found
by the CV study.
3.2.1 Optimisation of Conditions for the Stripping Analysis
3.2.1.1 Effect of pH and Type of Supporting Electrolyte
The adsorptive cathodic stripping voltammetric response for 2.0 µM AFB2 was
examined in BRB solution over the pH range from 3.0 to 13.0 using cathodic stripping
voltammetry, under the operational conditions: Ei = 0 V, Ef = -1.5 V, Eacc = 0 V, tacc =
30 s, υ = 50 mV/s and pulse amplitude = 100 mV. The results are presented in Figure
3.20.
Figure 3.20 DPCS voltammograms for 2.0 µM AFB2 in BRB (Peak I) at different
pH values: (a) 6.0, (b) 7.0 (c) 8.0 (d) 9.0 (e) 11.0 and (f) 13.0. Eacc = 0 V, tacc = 30 s,
υ= 50 mV/s and pulse amplitude = 100 mV. Peak II is the Zn peak.
105
Since only a single cathodic peak for AFB2 was obtained, it can be concluded
that the AFB2 molecule exhibits one reduction peak over the studied pH. It was found
that for pH less than 4.0, no peak was observed. At pH 4.0 and less, the peak of AFB2
was probably overlapped by hydrogen over potential which appeared at less negative
potential (Abu Zuhri et al, 2000). In acidic conditions, the concentration of hydrogen
ion is higher and more readily reduced at the mercury electrode producing a peak at
potential value around -1.20 V.
The single and characteristic AFB2 peak was initially observed at pH 5.0 with Ip
of 11.0 nA and Ep located at -1.119 V. The increase in the pH gradually increased the Ip
until it reached its maximum value at pH 9.0 (45 nA at Ep of -1.192 V). When there is
further increase of pH beyond pH 9.0, the Ip decreased until it disappeared at pH 13.0 as
shown in Figure 3.21. This result is not contradictory with that obtained by Smyth et al.
(1979) when they studied the effect of pH of BRB on the peak height of AFB1 using the
differential pulse polarographic (DPP) technique. They also found that BRB at pH 9.0
gave the highest Ip of AFB1.
60
50
.
40
Ip (nA)
30
20
10
0
4 6 8 10 12
pH
Figure 3.21 Dependence of the Ip for AFB2 on the pH of 0.04 M BRB solution.
AFB2 concentration: 2.0 µM, Ei = 0, Ef = -1.5V, Eacc = 0, tacc = 30 sec, υ = 50 mV/s and
106
The sharp decrease in Ip at pH 10.0 to 12.0 and no current peak obtained for pH
13.0 indicates that there is deficiency in the amount of hydrogen for reduction process
to take place, hence the peak height decreased. At pH 13.0 no peak was observed
which indicates that at this pH, the chemical structure of AFB2 may be totally damaged
or another reaction has taken place under alkaline medium. This finding was confirmed
by measuring AFB2 in BRB at pH 9.0 and followed with the lower pH such as 4.0 and
subsequently increased to pH 9.0. The measurements were then carried out in the
higher pH such as 10.0, 11.0, 12.0 and 13.0 and then back to 9.0 without changing the
solution. The result of this experiment is shown in Figure 3.22.
70 70
60 60
50 50
40 40
I p (n A )
I p (n A )
30 30
20 20
10 10
0 0
9 6 4 6 9 9 10 11 12 13 12 10 9
pH of BRB pH of BRB
(a) (b)
Figure 3.22 Ip of 2.0 µM AFB2 obtained in BRB (a) pH from 9.0 decreases to 4.0
and reincrease to 9.0 (b) pH from 9.0 increase to 13.0 and redecrease to 9.0.
Results show that at pH 4.0, no peak was observed but while the pH was
increased to 6.0 and pH 9.0, the peak appeared which indicated that the structure of
AFB2 was not damaged by the strong acidic condition. When pH was adjusted to pH
13.0, the peak dissapeared and no peak was observed even when the pH was re-adjusted
to 9.0 which showed that at pH 13.0, the structure of AFB2 was totally damaged or
transformed to nonelectroactive compound in the strong basic media.
107
The above finding was further confirmed by measuring the absorbance of the
AFB2 using UV-VIS spectrophotometer. The results from UV-VIS technique are
shown in Figures 3.23a, 3.23b and 3.23c for AFB2 in BRb pH 6.0, 9.0 and 13.0
respectively. The results show that no absorbance was obtained at wavelength (λ) of
365 nm for AFB2 in BRB at pH 13.0 while for AFB2 in BRB at pH 6.0 and 9.0 the
absorbance were 0.104 and 0.102 respectively. This was due to the fact that in BRB pH
13.0 the functional group (ketone), one of the UV chromophore which conjugates to
double bond in benzene ring is damaged due to the opening of this ring by the high
basic condition as reported by Janssen et al. (1997).
(a) (b)
(c)
Figure 3.23 UV-VIS spectrums of 1 ppm AFB2 in BRB at pH (a) 6.0, (b) 9.0 and (c)
13.0.
108
Figure 3.24 shows the opening of the lactone ring when AFB2 reacts with high
alkaline solution. When no keto group conjugated with the double bond, it is difficult
for the carbon-carbon double bond in the benzene to be reduced (Smyth and Smyth,
1978) which produce no reduction peak.
O O
O
O HO
O O
OCH3 OCH3
O O
Figure 3.24 Opening of lactone ring by strong alkali caused no peak to be observed
for AFB2 in BRB at pH 13.0.
This results shows that the reduction of AFB2 is totally dependent on the
supporting electrolyte which suggests that the reduction of AFB2 involves the reaction
of AFB2 with hydrogen ions originating from the supporting electrolyte (Abu Zuhri,
2000, Herdenandez et al., 1977 and Rodriguez et al. 2005).
The peak response was also examined in the presence of different buffers such
as phosphate and acetate buffers at the pH 9.0. BRB was selected as the most suitable
supporting electrolyte since it gave the highest Ip and good repeteability, as shown in
Table 3.1. It was found that the presence of peak of AFB2 reduction is strongly
influenced by pH and the buffer constituent.
The supporting electrolyte has two roles to play. The concentration of the
supporting electrolyte regulates the electrical resistance of the cell and it also controls
the migration of ions between the working and secondary electrode (Riley and
Tomlinson, 1987).
109
Table 3.1 Effect of buffer constituents on the Ip of 2.0 µM AFB2 at pH 9.0.

Experimental conditions are the same as in Figure 3.21
Buffer solution Ip (nA) Ep (V) % RSD (n =5)
Britton Robinson 50.08 -1.192 0.51
Carbonate 27.82 -1.130 1.19
Phosphate 43.74 -1.140 1.35
The effect of different concentrations of BRB at pH 9.0 as the supporting

electrolyte has been carried out and the results are shown in Figure 3.25 which shows
that BRB with 0.04 M concentration gave the highest Ip of AFB2 compared with other
studied concentrations. In 0.08 M, the Ip decreases due to the excess of supporting
electrolyte concentration causing the ionic migration of the aflatoxin to be reduced
(Riley and Tomlinson, 1987).
60
50
40
Ip (nA)
30
20
10
0
0.02 M 0.04 M 0.08 M
[BRB]
Figure 3.25 Ip of 2.0 µM AFB2 in different concentration of BRB at pH 9.0.

110
The effect of different pH and concentrations of BRB has been studied and the
results are shown in Figure 3.26. The figure shows that BRB with concentration of 0.04
M at pH 9.0 is the most suitable solution for the voltammetric determination of AFB2.
This can be explained that for 0.04 M at pH 9.0, the migration and adsorption of AFB2
at the working electrode was maximum as compared to other conditions.
Voltammograms of AFB2 in different concentrations of BRB at pH 9.0 are shown in
Figure 3.27
60
50
40 pH = 6.0
Ip (nA)
30 pH = 7.0
20 pH = 9.0
10
0
0.02 M 0.04 M 0.08 M
[BRB]
Figure 3.26 Ip of 2.0 µM AFB2 in different pH and concentrations of BRB
Figure 3.27 DPCS voltammograms of 2.0 µM AFB2 (peak I) in (a) 0.04 M, (b) 0.08
M and (c) 0.02 M BRB at pH 9.0 as the blank. Ef = -1.5 V, Eacc = 0 v, tacc = 30 s, υ = 50
mV/s and pulse amplitude = 100 mV. Peak II is the Zn peak
111
As determined in previous CV and DPCSV experiments, AFB2 were reduced at the

mercury electrode. Based on the chemical structure of aflatoxin, there are a few sites
that may undergo this process. There are double bonds in the terminal furan rings for
AFB1 and AFG1 and a double bond in the benzene ring which is conjugated with the
ketone group for all aflatoxins. Further investigations have been carried out to confirm
which site is actually involved in this reduction reaction at the mercury electrode by
voltammetric study of separate basic compounds that form aflatoxins which are 2,3
dihydrofuran and coumarin.
Another compound is tetrahydrofuran but it does not have any double bond in
their chemical structure which may not be reducable at the electrode, hence no detailed
investigation has been carried out. Their chemical structures are shown in Figures 3.28a
to 3.28c. Other possibility of the chemical structure that form aflatoxin compound was
not studied due to the unavailability of the compound.
O O
O O
(a) (b) (c)

Figure 3.28 Chemical structures of (a) 2,3 dihydrofuran (b) tetrahydrofuran and (c)
coumarin
Voltammograms of 0.02 to 0.2 µM of 2,3 dihyrofuran in BRB pH 9.0 are shown

in Figure 3.29. There is a small peak observed at -1.21 V which has not significantly
increased when the concentration was increased. The result indicates that 2,3
dihydrofuran is not reduced at the mercury electrode which indicates that the reduction
of AFB1 and AFG1 is not due to the reduction of double bond in their terminal furan
rings.
112
Figure 3.29 Voltammograms of 2,3 dihydrofuran (peak I) for concentrations from (b)
0.02 to 0.2 µM in (a) BRB at pH 9.0. Peak II; Zn peak.
The second compound that may undergo reduction at the mercury electrode is
coumarin (Figure 3.27c). Increasing the concentration of coumarin yields a linear
relationship of Ip corresponding to an equation of y = 25.877x – 35.5 (R2 = 0.9953).
A plot of Ip versus concentration of coumarin is shown in Figure 3.30.
500
400
300
Ip (nA)
200
y = 25.877x - 35.5
100 R2 = 0.9953
0
0 5 10 15 20
[Coumarin] x 10-5M
Figure 3.30 Dependence of Ip of coumarin to its concentrations

113
The voltammograms of coumarin with increasing concentration are represented

in Figure 3.31which shows that it was reduced at the mercury electrode producing sharp
and characteristic peak at Ep of -1.49 V. The result of voltammetric study of coumarin
shows that it is solely involved in the reduction of all studied aflatoxins.
As electrochemical behaviour of the aflatoxins closely parallels that of

coumarin, it is likely that the electroactivity of these compounds is associated with
coumarin moiety in their molecules structure. The main difference in behaviour lies in
the fact that the reduction of the aflatoxins occurs at a potential of about 300 – 400 mV
more positive than that for coumarin. This can be explained by the increased
conjugation caused by ketone group in the neighbouring cyclopentanone (AFB1 and
AFB2) or δ-lactone (AFG1 and AFG2) rings. Based on this reason, aflatoxins are
easier to reduce compared to coumarin and their Ep are located at more positive than
that for coumarin.
Figure 3.31 Voltammograms of coumarin at concentration of (b) 34 µM, (c) 68 µM,

(d) 102 µM, (e) 136 µM and (f) 170 µM in (a) BRB at pH 9.0
114
Based on all previous results, it can be concluded that a double bond in the
benzene ring which conjugates with the ketone group undergoes a reduction reaction
that produces a single cathodic peak for all studied aflatoxins. This finding is also in
agreement with a few reports that have been previously published for compounds that
have a double bond in the benzene ring which has conjugated with the ketone group
reduced at the mercury electrode. For example, cortisone and testosterone were reduced
and peaks were obtained for the reduction of the C = C double bond at -1.77 V and -
1.84 V (versus SCE) respectively in 0.1 M tri-ethyl ammonium per chlorate (TEAP)–
50 % methanol as reported by Smyth and Smyth (1978). Both compounds are 3-keto
steroids with un-saturation between C4 and C5 as shown in Figure 3.32a and 3.32b.
OH
O CH3
HO
O CH3
HO
O
(a) (b)
Figure 3.32 Chemical structures of (a) cortisone and (b) testosterone.
Smyth and Smyth (1978) also reported that cardiovascular drugs such as digoxin
and digitoxin (Figure 3.33) also undergo reduction reaction which occurs at C=C bond
in the five membered ring structure containing the conjugated carbonyl group. Using
DPP technique, digoxin and digitoxin gave peak at -2.285 V and -2.325 V (versus SCE)
respectively in propan-2-ol – 0.01 M tetrabutylammonium bromide (TBAB).
115
O O
OH
CH 3 CH3
O O
CH 3 CH3
OH OH
(C 18H 31O 9 )O H
(C18H31O9)O H
(a) (b)
Figure 3.33 Chemical structures of (a) digoxin and (b) digitoxin.
Other examples for the compounds that undergo reduction reaction which occurs
at C=C bond in their chemical structures are listed in Table 3.2. Other compounds that
have the same chemical structure and reduced at the mercury electrode produce
cathodic peak are enoxacin (Zhang et al. 1996), ofloxacin (Rizk et al. 1998) and
Alizarin Red S (ARS) (Sun and Jiao, 2002). Based on the previous reports, taking into
account that the molecular structure of AFB2 is similar in case where the carbonyl
group conjugates to the double bond in the benzene ring, it is assumed that AFB2
undergoes reduction reaction at the mercury electrode whereby C=C bond in its
chemical structure is reduced at the mercury electrode.
The Ep of AFB2 is also pH dependent and shifted towards a more negative

direction in increasing pH of the BRB solution which indicates the involvement of
protons in the AFB2 electrode reaction (Ghoneim et al., 2003a and Rodriguez et al.,
2004). A plot of Ep versus pH of supporting electrolyte is illustrated in Figure 4.34.
Two ranges were observed in the Ep – pH plot (Range I for pH from 5 to 8 and Range II
for pH from 9 to 12 as in Figure 4.33) with an intersection point at pH 8.7 which was
thought to correspond to the pKa value of the adsorbed AFB2 (Navalion et al. 2002).
This result again suggests that the deprotonated form of the AFB2 is the electroactive
species adsorbed in the HMDE.
116
Table 3.2 Compounds reduced at the mercury electrode
Compound Chemical structure Ep (V) Supporting Reference

electrolyte
O NHCH2N(CH2CH3)2
Imidazoacridinone -1.41 Phosphate Cakala et

buffer at al., (1999)
N
pH 7.4
N
O O
Warfarin -1.40 BRB at Ghoneim
H
pH 5.0 and Tawfik,
(2004)
ONa CH2COCH3
HO
CCH
Lavonogestrel -1.44 BRB at Ghoneim et

pH 3.0 al., (2004a)
O
C2H5
Nalidixic acid N N
-1.30 BRB at Ibrahim et
H3C
pH 5.0 al., (2002)
COOH
O
117
1250
Range II
E p (-m V ) 1200
Range I
1150
1100
4 6 8 10 12 14
pH
Figure 3.34 Effect of pH of BRB solution on the Ep for AFB2. AFB2 concentration:
2.0 µM, Ei = 0, Ef = -1.5 V, Eacc = 0, tacc = 30s, υ = 50 mV/s.
The relation between Ep and pH of the medium over the range, 5.0 to 8.0 is expressed
by the following equation:
Ep (-mV) = 21.4 pH + 1015.9 (R2 = 0.9777, n = 4) (3.5)
The Ep remains constant for pH 10.0 to pH 12.0 which suggests that within this range of
pH, the reaction is independent of the number of protons involved in the reduction
process.
3.2.1.2 Optimisation of Instrumental Conditions
The voltammetric determination of analyte at trace level normally involves very

small current responses. For that reason it is important to optimise all the parameters
which may have an influence on the measured current. From the previous experiment,
BRB solution at pH 9.0 was chosen as the best supporting electrolyte, thus this solution
has been used throughout this optimisation procedure using differential pulse cathodic
118
stripping voltammetry (DPCSV) technique. The effect of Ei, Ef, Eacc, tacc, υ and pulse
amplitude to the Ip and Ep and of AFB2 peak have been studied. This optimisation
procedure used two different concentrations of AFB2 which are 2.0 µM and 0.06 µM
for high and low concentrations respectively. The former has been initially used
followed with the latter as the final optimisation step. The initial parameters used for
this optimisation were; Ei = 0 mV, Ef = -1500 mV, Eacc= 0 mV, tacc = 15 s, υ = 20 mV/s,
pulse amplitude = 100 mV and quiescent time = 10 sec. Using all the stated parameters,
the Ip obtained for 2.0 µM of AFB2 was 29 nA and the Ep was -1.240 V.
3.2.1.2a Effect of Scan Rate (υ)
The dependence of Ip and Ep of AFB2 on υ has been studied. The υ was varied from 20
to 100 mV/s while other parameters were unchanged. Figure 3.35a shows a maximun
response (36 nA) obtained at scan rate of 40 mV/s.
50 1320
40
Ip (n A )
30 1290
Ep (-m V)
20
10 1260
0
0 20 40 60 80 100 120 1230
0 30 60 90 120
Scan rate (mV/s)
Scan rate (mV/s)
(a) (b)
Figure 3.35 Effect of various υ to the (a) Ip and (b) Ep of 2.0 µM AFB2 peak in
BRB pH 9.0. Ei = 0, Ef = -1.50 V, Eacc = 0, tacc = 15 s and pulse amplitude = 100 mV.
Ip decreases slowly for the increasing υ from 50 to 80 mV/s and sharply drops for 100
mV/s. The increasing υ has shifted the Ep of AFB2 towards a more negative direction
according to the equation;
119
E (-mV) = 0.6x (mV/s) + 1230.4 (R2 = 0.9896, n = 5) (3.6)
as shown in Figure 3.35b. From this study, the υ of 40 mV/s was used for further
optimisation step.
3.2.1.2b Effect of Accumulation time (tacc)
The effect of tacc to the Ip and Ep of AFB2 peak have been studied over the range
of 15 to 60 s as shown in Figure 3.36. It was found that, for the first 40 s, the Ip
increases with tacc. This is due to the larger amount of AFB2 which has accumulated at
the electrode surface with longer tacc. Since the amount of AFB2 is proportional to the
tacc, the resulting peak currents yield a straight line when plotted against the tacc within
the stated accumulation range as shown in Figure 3.36a. This relationship follows the
equation;
Ip (nA) = 0.4895x (x 10-6 M) + 26.132 (R2 = 0.9727, n = 5) (3.7)
60 1280
1270
E p (-m V )
40
Ip (n A )
1260
1250
20
1240
0 1230
0 20 40 60 0 20 40 60
Accumulation time (s) Accumulation time (s)
(a) (b)
Figure 3.36 Effect of tacc on (a) Ip and (b) Ep of 2.0 µM AFB2 in BRB at pH 9.0. Ei =
0, Ef = - 1.50 V, Eacc = 0 mV, υ = 40 mV/s and pulse amplitude = 100 mv.
However, when the tacc is longer than 40s, the Ip slowly decreases which is due to the
complete coverage of the electrode surface (Wang, 1985). This complete coverage
120
phenomenon can be confirmed by increasing drop size of HMDE which resulting

increased of the peak height.
3.2.1.2c Effect of Accumulation Potential (E acc)
The influence of Eacc to the Ip and Ep of AFB2 reduction peak has been
observed. An Eacc means the potential at which the AFB2 is deposited at the mercury
electrode surface. The optimised Eacc refers to the potential that is the most effective for
the deposition of the AFB2 at the mercury electrode surface while AFB2 solution
remains unstirred which produces the largest Ip and minimum side reaction. (Wang,
1985). The result shown in Figure 3.37 shows that over a range of 0 to -1200 mV, a
highest Ip was found at Eacc of -800 mV which indicates that at this potential, strong
adsorption of AFB2 takes place on the electrode surface. The Ip and Ep of AFB2 at this
optimum Eacc are 50 nA and -1.248 V respectively.
60 1258
50 1256
40
E p (-m V )
1254
Ip (nA )
30 1252
20 1250
10 1248
0 1246
0 400 800 1200 0 200 400 600 800 1000 1200 1400
Eacc (-mV) Eacc (-mV)
(a) (b)
Figure 3.37 The relationship between (a) Ip and (b) Ep with Eacc for 2.0 µM AFB2
in BRB at pH 9.0. Ei = 0, Ef = -1.50 V, tacc = 15 s, υ = 40 mV/s and pulse amplitude =
100 mV.
A linear response was obtained for the Eacc over a range of 0 to -0.80 V ( R2 = 0.9769, n
= 5). From Figure 3.37b, for Eacc at more negative than -0.80 V with the same Ei (Ei =
0), Ep of AFB2 were constant which indicates that after the maximum Eacc was achieved
and the accumulation process of AFB2 at electrode surface was not effective, there are
121
no more changes in Ep. At the same time, Ip decreased until it totally disappeared at Eacc
of -1.5 V. The result does not contradict with the suggestion made by Harvey (2000)
that the Eacc was usually several tenths of a volt (0.3 to 0.5 V) before the Ep of
electroactive species being investigated. Since the highest Ip with no extra peak was
obtained at an Eacc of -0.80 V, this potential was chosen as the optimum Eacc.
3.2.1.2d Effect of Initial Potential (Ei)
Further optimisation was performed by observing the effects of Ei to the Ip and

Ep of AFB2. The Ei used in this study were 0, 200, 400, 600, 800 and 1000 mV while Ef
was kept constant at -1500 mV. The result is shown in Figure 3.38a. The result indicates
that when the Ei was changed to more negative values, the Ip slowly decreased until the
Ei was -800mv. However at -1000 mV the Ip sharply increased and gave a maximum
response (53 nA). In other words, when the Ei was changed through this manner, the
window of scanning potential was smaller and hence, the time for analysis became
shorter. For Ei which was more negative than -1000 mV, the scan was not performed
due to the very short range of scanning potential. Also because of the instrumental
limitations, this experiment cannot be continued unless other instrumental parameters
such as pulse amplitude and υ are changed.
70 1260
60 1256
50
E p (-m V )
Ip (nA )
40 1252
30 1248
20
1244
10
0 1240
0 300 600 900 1200 0 300 600 900 1200
Ei (-mV) Ei (-mV)
(a) (b)
Figure 3.38 Effect of Ei on (a) Ip and (b) Ep of 2.0 µM AFB2 in BRB at pH 9.0. Ef = -
1.50 V, Eacc = -0.80 V, tacc = 40 s, υ = 40 mV/s and pulse amplitude = 100 mV
122
From Figure 3.38b, Ep remains constant for Ei of 0 to -400 mV. It started to shift
towards a more negative direction when the Ei was -600 mV and remained constant
after this value. This experiment reveals that the best potential window was from -1.0
V to -1.50 V which can save the analysis time and at the same time gave maximum Ip.
3.2.1.2e Effect of Pulse Amplitude
The effect of the pulse amplitude on the Ip shows that the Ip increased and Ep
displaced towards less negative direction when the pulse amplitude increased from the
range of 30 to 100mV as shown in Figure 4.39. The result shows that a maximum value
of Ip (53 nA) was obtained at pulse amplitude of 100 mV. At the higher value of pulse
amplitude, the Ip is slightly increased but peak broadening was observed, so 100 mV is
chosen for optimum pulse amplitude.
80 1300
60 1260
E p (-m V )
Ip (nA )
40 1220
20 1180
0 1140
0 30 60 90 120 150 0 30 60 90 120
Pulse amplitude (mV) Pulse amplitude (mV)
(a) (b)
Figure 3.39 Effect of pulse amplitude on (a) Ip and (b) Ep of 2.0 µM AFB2 in BRB at
pH 9.0. Ei = -1.0 V, Ef = -1.50 V, Eacc = -0.80 V, t acc = 40 s and υ = 40 mV/s.
From this study, the optimum conditions for electroanalytical determination of

2.0 µM AFB2 by DPCSV technique were as follows; Ei = -1.0 V, Ef = - 1.5 V,
Eacc = -0.80 V, tacc = 40 s, υ = 40 mV/s and pulse amplitude = 100 mV. Using this
optimised parameters, the Ip and Ep of 2.0 uM AFB2 were found to be 53 nA and -1.208
V respectively which was able to enhance the peak current to about 83% as compared to
that obtained by unoptimised parameters.
123
From the previous optimisation procedures, the Ip of AFB2 was enhanced by

83% and an attempt was made to get higher Ip by further optimisation steps using the
lower concentration of AFB2. The reason is using high concentration of AFB2 may lead
to the formation of mercury electrode saturation due to the adsorption of AFB2. This
optimisation was carried out using 0.06 µM AFB2 and all previous optimised
parameters were applied.
The optimisation steps followed the same procedure as for 2.0 µM AFB2. The Ip
and Ep initially obtained for 0.06 µM AFB2 were 13.90 nA and -1.232 V respectively.
The effect of Eacc on the Ip and Ep of AFB2 as shown in Figure 3.40 revealed that the Ip
obtained for E acc of -200 mV, -400 mV, -600 mV, -800 mV and -1000mV are not
similar which were about 14 nA compared to 11.37 nA and 12.42 nA for 0 and -1100
mV respectively. No peak was observed for Eacc more negative than -1100 mV.
20
15
ip (nA)
10
5
0
0 400 800 1200 1600
Accumulation potential (-mV)
Figure 3.40 Effect of Eacc on Ip of 0.06 µM AFB2. Ei = - 1.0 V, Ef = -1.50 V, tacc = 40

s, scan rate = 40 mV/s and pulse amplitude = 100 mV.
Using the potential window of Ei = -1.0 V and Ef = -1.4 V, the optimum Eacc
was -0.60 V which gave the Ip of 16.84 nA compared to another potential range (Ei = -
1.0 V and Ef = -1.50 V) which produced only 13.78 nA. The Ep of AFB2 remained
constant for all studied Eacc. The results suggested that using lower concentration, the
optimum Eacc is less negative compared to the higher concentration because for the
lower concentration, the electroactive species could easily completely reduce at the
mercury electrode. The Ep of AFB2 was independent of the Eacc at the low
124
concentrations. From this experiment, Eacc = -0.6 V, Ei = -1.0 V and Ef = -1.40 V were
chosen as the optimum condition for E acc and potential window respectively.
The effect of the tacc to the Ip of AFB2 was also investigated and the result is
shown in Figure 3.41a which shows that by increasing tacc, for the range of 20 to 80 s,
the Ip linearly increased following the equation:
Ip (nA) = 0.4038x (x s) - 0.2847 (R2 = 0.999, n = 5) (3.8)
The maximum Ip of 31.86 nA was obtained at an Eacc of 80 s. When AFB2 was

deposited at the mercury electrode for more than 80 s, the Ip was not significantly
increased (Figure 3.41a) which may be due to the saturation of the mercury electrode
surface and adsorptive equilibrium was achieved (Shams et al. 2004). Due to this
reason, the tacc at 80 s was chosen as the optimum tacc . The Ep of AFB2 was shifted
toward less negative direction with increasing tacc as shown in Figure 3.41b.
50 1250
40 1240
E p (-m V )
1230
Ip (nA )
30
20 1220
10 1210
0 1200
0 30 60 90 120 150 0 30 60 90 120 150
Accumulation time (s) tacc (s)
(a) (b)
Figure 3.41 Effect of tacc on (a) Ip and (b) Ep of 0.06 µM in BRB at pH 9.0. Ei = - 1.0
V, Ef = -1.40 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 100 mV.
The relationship between the υ and Ip of AFB2 cathodic peak was also
investigated, which revealed that when the υ is increased, the Ip also increased as shown
125
in Figure 3.42a. For the υ between 20 to 50 mV/s, the Ip is linearly proportional to the υ
according to the following equation:
Ip (nA) = 0.6481x (x mV/ s) + 5. 8407 (R2 = 0.9999 n = 3) (3.9)
The Ip was not significantly different when the υ was faster than 60 mV/s. Therefore,
50 mV/s was selected as the optimum υ which gave the maximum Ip of AFB2. The Ep
of AFB2 has linearly shifted towards a more negative direction for this range and a
small change for the υ of 60 and 80 mV/s as shown in Figure 3.42b. Due to
instrumentation limitations, for the υ of 50 mV/s, the pulse amplitude was changed to
80 mV instead of 100 mV. Without that, the Ip of AFB2 could not be automatically
calculated by the computer software. The maximum Ip that was obtained from this
optimisation was 38.18 nA at Ep of -1.23 V which was about 200% higher than before
being optimised (13.90 nA at -1.232 V). Figure 3.44 illustrates the voltammograms of
0.06 uM AFB2 obtained under optimised and unoptimised conditions.
50 1225
40 1220
E p (-m V )
I p (n A )
30 1215
20 1210
10 1205
0 1200
0 20 40 60 80 100 0 20 40 60 80 100
Scan rate (mV/s) Scan rate (mV/s)
(a) (b)
Figure 3.42 Effect of υ on (a) Ip and (b) Ep of 0.06 µM AFB2. Ei = - 1.0 V, Ef = -1.40
V, Eacc = -0.60 V and pulse amplitude = 80 mV.
126
Figure 3.43 Voltammogramms of 0.06 µM AFB2 obtained under (a) unoptimised and
(b) optimised parameters in BRB at pH 9.0.
From this optimisation steps, the final optimum parameters of DPCSV for
determination of 0.06 µM AFB2 compound are; Ei = -1.0 V, Ef = -1.40 V, Eacc =
-0.60 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV. Table 3.3 represents all
optimised parameters for both low and high concentrations of AFB2.
Table 3.3 Optimum parameters for 0.06 µM and 2.0 µM AFB2 in BRB at pH 9.0
Parameters 0.06 µM AFB2 2.0 µM AFB2
Ei (V) -1.0 -1.0
Ef (V) -1.4 -1.5
Eacc (V) -0.6 -0.8
tacc (s) 80 40
υ (mV/s) 50 40
Pulse amplitude 80 100

127
For voltammetric study of other aflatoxin except AFG1, the optimum

parameters for lower concentration of AFB2 was used throughout the experiments since
it give more sensitive and shorter analysis time compared to other optimum parameters.
For AFG1, only Ei was changed to -0.95 V to obtain the peak that is located at the
middle of the potential range.
3.2.2 Analysis of Aflatoxins
The optimum parameters determined were then used for determination of 0.1
µM AFB2, AFB1, AFG1 and AFG2. The values of Ip and Ep obtained from these
analysis are listed in Table 3.4. The relative standard deviation (RSD) for five
measurements of each aflatoxins are less than 5% which means that the precision of the
method is good and the obtained analytical information has good reliability as reported
by Aboul-Eneim et al. (2001). Voltammograms of each aflatoxin are shown in Figure
3.44a to 3.44d. Voltammograms of four aflatoxins which were mixed together are
shown in Figure 3.45 which shows that the combination of peak from AFB1 and AFB2
and also from AFG1 and AFG2 produced two shoulders.
Table 3.4 The Ip and Ep of aflatoxins obtained by optimised parameters in BRB at

pH 9.0 using DPCSV technique
Aflatoxin, 0.1 µM Ip (nA), (n=5) RSD (%) Ep (V)
AFB1 57.38 ± 0.43 0.75 -1.22
AFB2 57.49 ± 1.02 1.77 -1.23
AFG1 56.85 ± 1.28 2.25 -1.15
AFG2 56.20 ± 1.16 2.06 -1.17

128
(a) (b)
(c) (d)
Figure 3.44 Voltammograms of 0.1 µM (a) AFB1, (b) AFG1, (c) AFB2 and (d)
AFG2 in BRB at pH 9.0. Ei = -1.0 V (except for AFG1 = -0.95 V), Ef = -1.4 V,
Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.
129
Figure 3.45 Voltammograms of (b) mixed aflatoxins in BRB at pH 9.0 as the blank
(a). Parameters condition; Ei = -0.95 V, Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50
mV/s and pulse amplitude = 80 mV.
A study was carried out to observe a relationship between Ip and concentration

of aflatoxins. For each aflatoxin, the calibration curve was prepared by a series of
standard addition of aflatoxins. Statistical parameters such as linearity range, R2 value,
limit of detection (LOD), limit of quantification (LOQ), accuracy and precision were
evaluated. For ruggedness and robustness tests of the proposed technique, only AFB2
was used.
3.2.2.1 Calibration Curves of Aflatoxins and Validation of the Proposed Method
3.2.2.1a Calibration curve of AFB2
The calibration curve for AFB2 determination was established by applying the
developed procedure. The Ip of AFB2 with increasing concentration is shown in Figure
3.46 which shows that after Ip achieved its maximum value it tends to level off with
further addition of AFB2 standard solution as expected for a process that was limited by
adsorption of analyte. This phenomenon was similarly observed in the previous study
130
carried out by the CV technique. The linear plot of Ip versus the concentration of AFB2
is shown in Figure 3.47. Their voltammograms are shown in Appendix L
250
200
150
Ip (nA)
100
50
0
0 10 20 30 40 50
-8
[AFB2] x 10 M
Figure 3.46 Increasing concentration of AFB2 in BRB at pH 9.0. The parameter

conditions are Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.60 V, tacc = 80 s, υ = 50 mV/s and
200
160
120
Ip (nA)
80
y = 5.5385x + 3.2224
40 2
R = 0.9980
0
0 10 20 30 40
-8
[AFB2] X10 M
Figure 3.47 Linear plot of Ip versus concentration of AFB2 in BRB at pH 9.0. The
parameter conditions are the same as in Figure 4.46
It shows that the range of linearity was found to be from 2.0 to 32.0 x 10-8 M
(6.29 to 100.63 ppb) with a LOD of 0.796 x 10-8 M (2.50 ppb) and LOQ was 2.65 x
10-8 M (8.33 ppb). The R2 value was 0.9980. The LOD was determined by standard
131
addition of lower concentration of analyte until a sample peak is obtained that was
significantly different from the blank signal (Barek et al., 1999).
For the determination of precision of the developed method, the reproducibility

was calculated from 8 independent measurements of 0.10 µM and 0.20 µM of AFB2
solution on the same day, obtaining relative standard deviations (RSD) of 2.83 % and
0.72% respectively. The precision is good since the mean values do not exceeds 15%
of the variance coefficient (CV) (Torreiro et al. 2004). Voltammograms of these
measurements are shown in Appendix M. For the inter-days (within 3 days) accuracy
determinations, the RSD for day 1, day 2 and day 3 obtained for measurement of 0.10
µM are 2.83%, 2.39% and 1.31% respectively and for 0.20 µM were 0.72 %, 1.03 %
and 0.98 %. Voltammograms of these measurements for 0.10 µM AFB2 are shown in
Appendix N. The results of these precision studies for intra-day and inter-day are
shown in Table 3.5.
Table 3.5 Ip (in nA) obtained for intra-day and inter-day precision studies of 0.10
µM and 0.20 µM by the proposed voltammetric procedure (n=8)
Intra-day Inter-day measurement

[AFB2] measurement Ip ± SD (RSD)
Ip ± SD (RSD)
Day 1 Day 2 Day 3
0.1 µM 57.68 ± 1.63 57.68 ± 1.63 56.52 ± 1.35 56.59 ± 0.74

(2.83%) (2.83%) (2.39%) (1.31%)
0.2 µM 114.87 ± 0.83 114.87 ± 0.83 113.85 ± 1.17 112.78 ± 1.11

(0.72%) (0.72%) (1.03 %) (0.98%)
132
The accuracy of the method is defined as the closeness of agreement between

the experiment result and the true value (Chan et al., 2004). It is determined by
calculating the percentage of relative error between the measured mean concentrations
and the added concentrations (Torriero, 2004). In order to determine the accuracy of
the proposed method, a recovery study was carried out by the addition of an amount of
three different concentrations of AFB2 (0.10 µM, 0.15 µM and 0.20 µM) into the
voltammetric cell and measuring the peak currents of respective concentrations. The
actual amount of AFB2 found in the cell were calculated using the obtained regression
equation (y = 5.5385x + 3.2224). The recoveries obtained were 97.29 ± 1.29 %, 97.15 ±
0.79 % and 99.39 ± 0.50 % respectively as shown in Table 3.6. The result shows that
the recovery of AFB2 standard is considered good.
Table 3.6 Mean values for recovery of AFB2 standard solution (n = 3)
No of Amount Ip (nA) Amount Recovery %Recovery ± SD

experiment added found (%) (RSD)
(x 10-8 M) (x 10-8 M)
1 10 56.32 9.59 95.90
55.45 9.44 94.40 96.37 ± 2.24

(2.39 %)
58.04 9.88 98.80
2 15 85.21 14.80 98.67
84.78 14.73 98.25 98.47 ± 0.24

(0.24 %)
85.10 14.78 98.53
3 20 113.85 19.97 99.85
114.20 20.03 100.15 99.90 ± 0.23

(0.23 %)
113.75 19.94 99.70
133
The robustness of the proposed technique was evaluated by examining the

influence of small variation in some of the most important procedural conditions,
including pH (8.5 to 9.5), Eacc (-0.59 V and -0.61 V) and tacc (75 s and 85 s) to the Ip of
AFB2 (Ghoneim et al., 2004b). The results of these studies are shown in Table 3.7.
Using F test with 95 % degree of freedom, the results showed that none of these
variables significantly affected the obtained Ip of AFB2. Thus, the proposed technique
could be considered robust.
Table 3.7 Influence of small variation in some of the assay conditions of the
proposed procedure on its suitability and sensitivity using 0.10 µM AFB2
Variable Condition Ip of AFB2 (nA) Recovery ±SD (%)

± SD, n=5 n=5
pH 9.0 Eacc = -0.60 V 60.62 ± 0.88 103.01 ±1.50

8.5 tacc = 80 s 58.82 ± 0.79 100.33 ± 1.35
9.5 59.30 ± 0.44 101.15 ± 0.75
Eacc -0.60 V pH = 9.0 60.62 ± 0.88 103.01 ± 0.48

-0.59 V tacc = 80 s 58.78 ± 1.02 100.26 ± 1.75
-0.61 V 59.82 ± 0.91 102.04 ± 1.56
tacc 80 s pH = 9.0 60.62 ± 0.88 103.01 ± 0.48

75 s Eacc = -0.60 V 58.78 ± 0.48 100.26 ± 0.82
85 s 60.90 ± 0.76 103.49 ± 1.30
Moreover, the ruggedness of the proposed technique was examined by applying

the procedure to the analysis of 0.1 µM AFB2 using two potentiostat models, BAS CV
134
450 W and VA 757 Metrohm Analysers, under the same optimised experimental
parameters. The results of these studies are listed in Table 3.8. Using the same
statistical test, the results show no significant difference in the Ip of AFB2 obtained by
using both analysers which indicates that the proposed procedure could be considered
rugged. Detailed information on F test for robustness and ruggedness evaluations are
elaborated in Appendix O.
Table 3.8 Results of ruggedness test for proposed method using 0.10 µM AFB2
Voltammetric analyser Ip of AFB2 ± SD Recovery ± SD (%)

(nA) , n = 5 n=5
BAS CV50W 58.08 ± 1.63 99.11 ± 2.79
VA 757 Metrohm 57.98 ± 0.83 98.94 ± 1.42
3.2.2.1b Calibration Curve of AFB1
Using the same approach as carried out for AFB2, the calibration curve for
AFB1 was constructed by plot of Ip of AFB1 versus their concentrations. DPCSV
measurements of increasing concentrations were applied and the result is shown in
Figure 3.48. Figure 3.49 shows the linear curve for AFB1 which has a linear range
between 2.0 to 32.0 x 10-8 M (6.29 to 100.63 ppb) with a R2 value of 0.9989. LOD of
the measurement of AFB1 was 0.5 x 10-8 M (1.56 ppb). LOQ was 1.67 x 10-8 M (5.2
ppb). Voltammograms of AFB1 with successive standard addition for AFB1 are shown
in Appendix P.
135
250
200
150
Ip (nA)
100
50
0
0 10 20 30 40 50
-8
[AFB1] x 10 M
Figure 3.48 Standard addition of AFB1 in BRB at pH 9.0. Ei = -1.0 V, Ef = -1.4 V,

Eacc = -0.60 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.
200
150
I p (nA)
100
y = 5.4363x + 3.7245
50
R2 = 0.9989
0
0 10 20 30 40
[AFB1] x 10-8M
Figure 3.49 Linear plot of Ip versus concentration of AFB1 in BRB at pH 9.0. All
parameter conditions used are the same as stated in Figure 3.48.
136
Repeatability was examined by performing five replicate measurements for 0.10

and 0.20 µM AFB1 both intra-day and inter-day measurements for it precision and the
result is shown in Table 3.9. Relative standard deviation (RSD) of 0.10 µM and 0.20
µM for intra-day measurement were 1.83% and 0.74% respectively have been achieved.
For inter-day measurements, RSD of 0.10 µM of AFB1 for day 1, day 2 and day 3 were
1.83%, 4.37% and 2.86% respectively and for 0.20 µM RSD of Ip were 0.74%, 1.93%
and 0.56%. The results indicate the high precision of the proposed procedure.
The accuracy of the proposed method was evaluated by applying the same
procedure as for AFB2. The results are shown in Table 3.10 which shows that the
accuracy of the proposed method is considered high.
µM and 0.20 µM AFB1 by proposed voltammetric procedure (n=5)

[AFB1] measurement Ip ± SD (RSD)
Ip ± SD (RSD)
Day 1 Day 2 Day 3
0.10 µM 58.64 ± 1.11 58.64 ± 1.11 58.53 ± 1.01 57.49 ± 0.67

(1.89%) (1.89%) (1.72%) (1.13%)
0.20 µM 112.84 ± 0.82 112.84 ± 0.82 112.68 ± 0.81 113.26 ± 0.63

(0.73%) (0.73%) (0.72%) (0.56%)
137
Table 3.10 Mean values for recovery of AFB1 standard solution (n = 3)
No of Amount Ip (nA) Amount Recovery Recovery ± SD (%)

(x 10-8 M) (x 10-8 M)
1 10 58.49 10.07 100.70 99.50 ± 1.20

57.11 9.83 98.30 (0.21 %)
57.80 9.95 99.50
2 15 84.70 14.89 99.27 99.87 ± 0.63

85.24 14.97 99.80 (0.63 %)
85.85 15.08 100.53
3 20 112.26 19.99 99.97 100.15 ± 0.49

113.32 20.06 100.31 (0.26 %)
112.58 19.96 99.80
3.2.2.1c Calibration Curve of AFG1
The validation of this proposed method for quantitative assay of AFG1 was
examined via the evaluation of the same parameters as for AFB1. The effect of
increasing concentration of AFG1 to their Ip up to 45 x 10-8 M (147.6 ppb) is shown in
Figure 3.50. The calibration curve with a linear range of 2.0 to 32.0 x 10-8 M (6.56 to
105 ppb) with a R2 value of 0.9992 was obtained as shown in Figure 3.51. LOD value
for this measurement was 1.0 x 10-8 M (3.28 ppb) and LOQ was 3.33 x 10 -8 M (10.93
ppb) Voltammograms of AFG1 with increasing concentration are shown in Appendix
Q. The results for the precision and accuracy tests are listed Table 3.11 and 3.12
respectively.
138
250
200
150
Ip (nA)
100
50
0
0 10 20 30 40 50
-8
[AFG1] x 10 M
Figure 3.50 Effect of concentration to Ip of AFG1 in BRB at pH 9.0. Ei = -0.95 V,

Ef = -1.4 V, Eacc = -0.60 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.
200
150
Ip (nA)
100
50 y = 5.5899x + 3.708
R2 = 0.9992
0
0 10 20 30 40
-8
[AFG1] x 10 M
Figure 3.51 Linear plot of Ip versus concentration of AFG1 in BRB at pH 9.0.

The parameter conditions are same as in Figure 3.50.
139
µM and 0.20 µM of AFG1 by proposed voltammetric procedure (n=5)

[AFG1] measurement Ip ± SD (RSD)
Ip ± SD (RSD)
Day 1 Day 2 Day 3
0.10 µM 58.11 ± 0.93 58.11 ± 0.93 59.34 ± 0.81 57.56 ± 1.09

(1.60%) (1.60%) (1.37%) (1.89%)
0.20 µM 117.60 ± 1.24 117.60 ± 1.24 118.85 ± 1.12 117.65 ± 1.21

(1.05%) (1.05%) (0.94 %) (1.02%)
Table 3.12 Mean values for recovery of AFG1 standard solution (n = 3)
No of Amount Ip (nA) Amount Recovery Recovery ± SD (%)

(x 10-8 M) (x 10 -8 M)
1 10 58.83 9.97 99.70 99.20 ± 0.95

58.16 9.98 99.80 (0.95 %)
57.16 9.81 98.10
2 15 88.20 14.78 98.53 99.28 ± 0.80

89.62 15.02 100.13 (0.81 %)
88.78 14.88 99.20
3 20 118.16 19.80 99.00 98.80 ± 0.30

117.52 19.69 98.45 (0.30 %)
118.10 19.79 98.95
140
3.2.2.1d Calibration Curve of AFG2
The applicability of the proposed DPCSV technique as an analytical method for

the last type of aflatoxin which is AFG2 was examined by measuring the peak current
of AFG2 as a function of its concentration under optimised operational parameters.
Figure 3.52 shows the effect of increasing concentration of AFG2 to the Ip. A
calibration graph for AFG2 was constructed by standard addition method.
250
200
150
Ip (nA)
100
50
0
0 10 20 30 40 50
[AFG2] / X10-8M
Figure 3.52 Effect of concentration of AFG2 to Ip of AFG2 in BRB at pH 9.0.

Ei = -1.0 V, E f = -1.4 V, Eacc = -0.60 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude =
80 mV.
The correlation between Ip and concentration of AFG2 was linear within the
range 2.0 to 32.0 x 10-8 M (6.60 to 105.61 ppb) as shown in Figure 3.53. The
calibration graph was represented by the equation: ip (µA) = 5.7132x (x 10-8 M) +
1.1682; R2 = 0.9989 and n = 10. The LOD was found to be 0.758 x 10-8 M (2.50 ppb)
and LOQ was 2.53 x 10-8 M (8.33 ppb). The voltammograms of AFG2 with successive
addition of its concentration are shown in Appendix R. The results of precision studies
for 0.10 µM and 0.20 µM of AFG2 which were obtained by proposed method are
represented in Table 3.13.
141
200
150
I p (nA)
100
y = 5.7132x + 1.1682
50
R2 = 0.9989
0
0 10 20 30 40
-8
[AFG2] x 10 M
Figure 3.53 Linear plot of Ip versus concentration of AFG2 in BRB at pH 9.0. All
parameters used are the same as stated in Figure 3.52.
µM and 0.20 µM of AFG2 by proposed voltammetric procedure (n=5).

[AFG1] measurement Ip ± SD (RSD)
Ip ± SD (RSD)
Day 1 Day 2 Day 3
0.10 µM 58.53 ± 1.05 58.53 ± 1.05 59.11 ± 1.19 58.72 ± 1.14

(1.79%) (1.79%) (2.01%) (1.94%)
0.20 µM 115.40 ± 1.69 115.40 ± 1.69 115.20 ± 1.82 115.40 ±1.14

(1.46%) (1.46%) (1.58 %) (0.99%)
For accuracy, it was examined by performing three replicate measurements for

0.1 µM, 0.15 µM and 0.2 µM AFG2 solutions under the same operational parameters.
Percentage recoveries (R%) of 100.12 +/- 0.25 %, 99.66 +/- 0.90 % and 100.03 +/- 0.26
142
% were achieved respectively with a mean value of % RSD of 0.47%, which indicates
the high accuracy of the proposed technique. Table 3.14 shows the results of accuracy
study for AFG2.
Table 3.14 Mean values for recovery of AFG2 standard solution (n = 3)
No of Amount Ip (nA) Amount Recovery Recovery ± SD

experiment added found (%) (%)
(x 10-8 M) (x 10 -8 M) (RSD)
1 10 58.14 9.97 99.97 100.12 ± 0.25

58.20 9.98 99.80 (0.25 %)
59.10 10.04 100.40
2 15 86.20 14.88 99.22 99.66 ± 0.90

87.50 15.10 100.70 (0.90 %)
86.10 14.86 99.06
3 20 115.40 19.99 99.97 100.03 ± 0.26

115.80 20.06 100.31 (0.26 %)
115.20 19.96 99.80
The Ip and Ep of the aflatoxins with concentration of 0.10 µM which were

obtained by BAS C50W were not significantly different compared to those obtained by
VA 757 Metrohm Analyser as shown in Table 3.15, indicating that the proposed
technique can be applied using different types of voltammetry analysers. Figures 3.54a
to 3.54d show the voltammograms of AFB1, AFB2, AFG1 and AFG2 which were
obtained using Metrohm voltammetry analyser. From the validation of the proposed
technique, Table 3.16 shows all statistical parameters for the aflatoxins.
143
Table 3.15 Ip and Ep of 0.10 uM aflatoxins obtained by BAS and Metrohm

voltammetry analysers under optimised operational parameters for DPCSV method.
Voltammetry analysers
BAS CV 50W 757 VA Metrohm
Aflatoxins
Ip (nA) ± SD Ep (V) Ip (nA) ± SD Ep (V)
(RSD) (RSD)
AFB1 58.28 ± 2.79 -1.22 59.88 ± 0.94 -1.23

(4.78%) (1.57%)
AFB2 58.08 ± 1.63 -1.23 57.98 ± 0.83 -1.24

(2.81%) (1.43%)
AFG1 56.85 ± 1.87 -1.15 59.26 ± 0.84 -1.15

(3.29%) (1.42%)
AFG2 56.20 ± 1.34 -1.17 57.56 ± 0.63 -1.18

(2.38%) (1.10%)
3.2.2.2 Determination of Limit of Detection (LOD)
The limit of detection (LOD) is an important quantity in chemical analysis. The

LOD is the smallest concentration or amount that can be detected with reasonable
certainty for a given analytical procedure (Ahmad, 1993; Miller and Miller, 1993). It is
the lowest concentrations that can be distinguished from the noise level as described by
Bressole et al. (1996). Thompson (1998) reported that the detection limit caused
problems for analytical chemists because they are difficult to interpret and the arbitrary
dichotomising of the concentration domain provides misleading viewpoint of the
behaviour of analytical systems.
144
(a) (b)
(c) (d)
Figure 3.54 Voltammograms of 0.10 µM (a) AFB1, (b) AFB2, (c) AFG1 and (d)
AFG2 in BRB at pH 9.0 obtained by 757 VA Metrohm voltammetry analyser. Ei = -1.0
V (except for AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.8 V, tacc = 80 s, υ = 50 mV/s and
In order to estimate the LOD, some alternative methods have been used to
overcome the difficulties such as:
145
Table 3.16 Analytical parameters for calibration curves for AFB1, AFB2, AFG1 and
AFG2 obtained by DPCSV technique using BRB at pH 9.0 as the supporting electrolyte
Aflatoxin Ep (V) Regression Sensitivity Linearity range LOD R2

equations (µA/µM) (x 10-8 M / ppb) (x10-8 M / ppb)
B1 -1.22 y = 5.4363x + 3.7245 0.5436 2 – 32 / 6.24 – 99.84 0.5 / 1.56 0.9989
B2 -1.23 y = 5.5385x + 3.2224 0.5539 2 – 32 / 6.29 – 100.63 0.796 / 2.50 0.9980
G1 -1.15 y = 5.5899x + 3.7080 0.5900 2 – 32 / 6.56 – 104.92 1.0 / 3.28 0.9992
G2 -1.17 y = 5.7132x + 1.1682 0.5713 2 – 32 / 6.60 – 105.61 0.758 / 2.50 0.9989
Notes:
Ep = Peak potential for aflatoxins at concentration of 0.1 µM
y = mx + c; y = nA, x = x 10-8 M, c = nA
LOD = limit of detection

146
a) The concentration giving a signal three times the standard error plus the
y-intercept divided by the slope of calibration curve (Miller and Miller,
1993; Sturm et al., 1997; Yilmaz et al., 2001: Arranz et al., 2003;
Skrzypek et al., 2005 and Nouws et al., 2005). This calculation is used
by International Union of Pure and Applied Chemistry (IUPAC)
(IUPAC, 1978).
b) The analyte concentration giving a signal equal to a blank signal plus

two or three (Conesa et al., 1996) standard deviations of the blank.
c) The concentration giving a signal equal to three times (Zhang et al.,

1996) or 3.3 times (Perez-Ruiz et al., 2004) the standard deviation of the
blank signal divided by the slope from the calibration curve. The latter
used 12 blank determinations to calculate standard deviation of the
blank.
d) The concentration giving a signal to noise ratio of 3:1 (3 S/N) (Yardimer

and Ozaltin, 2001; Khodari et al., 1997; Wang et al., 1997; Liu et al.,
2000; Ibrahim et al., 2002; Al-ghamdie et al., 2004 and Farghaly et al.,
2005).
e) The value of three fold standard deviation from seven determinations of

the analyte at the concentration corresponding to the lowest point on the
appropriate straight line of the calibration curve which was evaluated by
R2 value as reported by Barek et al. (1999).
f) The concentration giving 3 times standard deviation of intercept of the

calibration divided by a slope of the calibration curve as reported by Gao
et al. (2004) and El-Desoky and Ghoneim (2005).
147
g) The lowest concentration of sample that give the significantly different

signal from the blank after standard addition of lower concentration
(Barek et al., 2001a).
h) Use of the robust regression method where the detection limit is dealt as
a hypothesis test in relation to the presence of analyte in an unknown
sample by using the experimental information provided by the
calibration set (Ortiz et al., 1993).
The results of the determinations of aflatoxins using several methods according

to Barek et al. (2001a), Barek et al. (1999), Zhang et al. (1996) and Miller and Miller
(1993) are shown in Table 3.17. These methods were chosen due to its simplicity and
are widely used by many researchers. Examples of these calculations for AFB1 are
represented in Appendix S, T, U and V for each method respectively.
From the results, the LOD values obtained according to Barek et al. (1999),
Barek et al. (2001a) and Zhang et al. (1996) are not significantly different as was
proven by ANOVA test which is reported in Appendix W (Youmen, 1973). The
Method 1 (according to Barek et al., 2001a) was selected for the determination of the
LOD of all aflatoxins throughout this study since it gives the lowest LOD value as
compared to that obtained by other methods.
3.2.2.3 Determination of Limit of Quantification (LOQ)
The limit of quantification (LOQ) is the lower limit for precise quantitative
measurements (Miller and Miller, 1993; Chan et al., 2004). It is determined by the
formula of (LOD / 3) x 10 as reported by Ozkan et al. (2003) and Ghoneim et al.
(2004). A value of YB + 10SB has been suggested for this limit (Miller and Miller,
1993; Rodriguez et al., 2005) where YB is a blank signal and SB is the standard deviation
of blank. The LOQ vaules for all aflatoxins obtained by the proposed methods are listed
in Table 3.18.
148
Table 3.17 LOD values for determination of aflatoxins obtained by various methods
Method
Aflatoxin 1 2 3 4
x 10-8 M ppb x 10-8 M ppb x 10-8 M ppb x 10-8 M ppb
B1 0.50 1.56 0.64 2.00 0.75 2.35 1.14 3.56
B2 0.78 2.50 0.89 2.80 0.91 2.86 1.47 4.62
G1 1.00 3.28 1.06 3.50 1.10 3.60 2.09 6.84
G2 0.76 2.50 0.92 3.02 0.86 2.84 2.48 8.18
Notes:
Method 1: According to Barek et al. (2001a)
Method 2: According to Barek et al. (1999)
Method 3: According to Zhang et . (1996)
Method 4: According to Miller and Miller (1993)

149
Table 3.18 LOQ values for determination of aflatoxins obtained by various methods
Method
Aflatoxin 1 2 3 4
x 10-8 M ppb x 10-8 M ppb x 10-8 M ppb x 10-8 M ppb
B1 1.67 5.20 2.13 6.67 2.50 7.83 3.80 11.87
B2 2.60 8.33 2.97 9.33 2.03 9.53 4.90 15.40
G1 3.33 10.93 3.53 11.67 3.67 7.87 12.00 22.80
G2 2.53 8.33 3.07 10.07 2.87 9.47 8.27 27.27
Notes:
Method 1: According to Barek et al. (2001a)
Method 2: According to Barek et al. (1999)
Method 3: According to Zhang et al. (1996)
Method 4: According to Miller and Miller (1993)

150
3.2.2.4 Interference studies
Possible interference from various metal ions and organic substances were
investigated by spiking with excess amount of these interfering substances to the BRB
at pH 9.0 containing 0.10 µM of each aflatoxin under optimum experimental conditions.
The experiments showed that the presence of up to 10-fold of Al3+, Cu 2+, Ni2+, Pb 2+,
Zn 2+, ascorbic acid, β-cyclodextrin and cysteine did not affect much the signal measured
which indicates that there was no serious interference to the voltammetric determination
of all studied aflatoxins. For example, the effect of Zn ion to the Ip of all aflatoxins is
shown in Figure 3.55. Voltammogram of AFB2 with addition of this ion is shown in
Figure 3.56(i). The interference study by Zn2+ was further carried out by initially
reacted all aflatoxins with Zn2+ in BRB at pH 3.0 before being added into the
voltammetric cell containing BRB at pH 9.0. Voltammograms of complex AFB2-Zn
with increasing concentration of Zn2+ are shown in Figure 3.56(ii). Figure 3.57 shows
the Ip of all aflatoxins which were complexed with Zn2+ decreased drastically with the
addition of 1.0 µM Zn2+. This effect indicates that Zn2+ interferes with the
measurement of aflatoxins in acid condition.
70
60
50
AFB1
40
Ip (nA)
AFB2
30 AFG1
AFG2
20
[Aflatoxin] = 0.1 uM
10
0
0 0.2 0.4 0.6 0.8 1 1.2
[Zn(II)] / uM
Figure 3.55 Ip of all aflatoxins with increasing concentration of Zn2+ up to 1.0 µM

151
(i) (ii)
Figure 3.56 Voltammograms of (i) 0.10 µM AFB2 and (ii) 0.1 µM AFB2-Zn
complex with increasing concentration of Zn2+ ( a = 0, b = 0.75 µM, c = 1.50 µM, d =
2.25 µM and e = 3.0 µM). Blank = BRB at pH 9.0. Experimental conditions; Ei = -1.0
V, Ef = -1.4 V, Eacc = -0.60 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.
.
70
60
50
AFB1
40
Ip (nA)
AFB2
30 AFG1
AFG2
20
10
0
0 1 2 3 4
[Zn] / uM
Figure 3.57 Ip of all aflatoxins-Zn complex after reacting with increasing

concentration of Zn 2+ in BRB at pH 3.0. Measurements were made in BRB at pH 9.0
within 15 minutes of reaction time. Ei = -1.0 (except AFG1 = -0.95 V), Ef = -1.4 V, Eacc
= -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.
152
Similarly, studies carried out using UV-VIS spectrometric technique also

showed that absorbance of 1.0 ppm AFB2 decreases with increasing concentration of
Zn 2+ (from 0.15 to 0.60 ppm) in acidic condition as shown in Figure 3.58. The same
patterns were also found for other aflatoxins. Their absorbances decreased linearly with
increasing concentration of Zn2. From this study, it can be concluded that Zn2+ reacted
with all aflatoxins in acidic medium but does not in BRB at pH 9.0.
0.12
0.1
0.08
AFB1
AFB2
Abs
0.06
AFG1
AFG2
0.04
0.02
[aflatoxins ] = 1.0 ppm
0
0 0.15 0.3 0.45 0.6
[Zn(II)] / ppm
Figure 3.58 Absorbance of all aflatoxins with increasing concentration of Zn2+ in

BRB at pH 3.0 within 15 minutes of reaction time.
For Zn 2+, studies using initial potential (Ei) at - 0.25 V instead -1.0 V was
conducted. The aim was to investigate the response of Zn peak which appeared at -0.98
V with increasing Zn2+ concentration and the result is shown in Figure 3.59. The figure
illustrates that the Zn 2+ peak increases while no significant change was observed on Ip
and Ep of AFB2 which show that the peak appeared at the Ep of -0.98 was from the
Zn 2+.
153
Figure 3.59 Voltammograms of 0.10 µM AFB2 with increasing concentrations of

Zn 2+ (from 0.10 to 0.50 µM) in BRB at pH 9.0. Ei = -0.25 V, Ef = -1.4 V, Eacc = -0.6 V,
For interference study by organic substance, ascorbic acid (Figure 3.60) was
selected due to its high content in food as a natural micronutrient and plays many
physiological roles as reported by Perez-Ruiz et al. (2004).
OH HO
HO
O OH
O
Figure 3.60 Chemical structure of ascorbic acid
Ascorbic acid may interfere with the measurement of aflatoxins because of its
reducing property due to the presence of the ene-1, 2- diol system (Verma et al., 1991).
Its structure has a double bond which conjugates to the ketone group, similar to the
154
aflatoxins chemical structures which may reduce at HMDE in BRB at pH 9.0 and
interfere the measurement of aflatoxins. The result of this study is shown in Figures
3.61 and 3.62 indicate that no significant change was observed by increasing the
concentration of ascorbic acid up to ten-fold of the aflatoxins concentration.
70
60
50
40
Ip (nA)
AFB1
30 AFB2
AFG1
20
AFG2
10
0
0 0.2 0.4 0.6 0.8 1 1.2
[ascorbic a cid] / uM
Figure 3.61 Ip of all aflatoxins with increasing concentration of ascorbic acid up to

1.0 µM. Concentrations of all aflatoxins are 0.1 µM.
Figure 3.62 Voltammograms of 0.1 µM AFB2 with increasing concentration of

ascorbic acid.
155
Interference studies were also carried out with β-cyclodextrin. Cyclodextrins

(CDs) are cyclic oligosaccharides with a hydrophilic outer surface and lipophilic central
cavity. CDs are formed with a various numbers of glucose units such as α-, β- and γ-
CD, formed by 6, 7 and 8 glucose units, respectively, are present in the greatest amount.
The pKa values of α-, β- and γ-CD are 12.33, 12.20 and 12.08, respectively and the
species are charged in strong alkaline solution (Fang et al., 1998). In aqueous solutions,
CDs are able to solubilise lipophilic moiety of the compound molecule into the central
cavity without any covalent bond. Such complexes are called inclusion complexes
(Loftson and Brewster, 1996).
Due to its inherent interesting inclusion properties which are exhibited both in
solid state and in aqueous solution as reported by Fang et al. (1998) and its capability of
forming complexes with aflatoxins, β-CD was selected in this study. In optimum pH of
supporting electrolyte for the determination of aflatoxins (pH 9.0), the charged β-CD
may react with aflatoxins. The result of this study is as in Figure 3.63 and the
voltammograms of AFB2 with increasing concentration of β-CD in Figure 3.64 shows
that not much interference was observed with increasing concentration of β-CD up to
ten-fold of concentration of aflatoxins.
70
60
50
40 AFB1
Ip (nA)
AFB2
30 AFG1
20 AFG2
[Aflatoxins] = 0.1 uM
10
0
0 0.2 0.4 0.6 0.8 1
[B-CD] / uM
Figure 3.63 Ip of all aflatoxins with increasing concentration of β- cyclodextrin up to

1.0 µM.
156
Figure 3.64 Voltammograms of 0.10 µM AFB2 with increasing concentration of β-

cyclodextrin.
Another organic compound which was used in this interference study was L-
cysteine (Figure 3.65).
OH
H 2N
SH
Figure 3.65 Chemical structure of L-cysteine
The result of this study is given in Figure 3.66 which shows that Ip of all
aflatoxins decreased slightly when cysteine was added. Cysteine is one of the organic
compound that show a high activity for electrocatalytic adsorption on mercury and the
most modified electrode as reported by Shahrokhian and Karimi (2004). The result
obtained may be due to the accumulation of L-cysteine on the surface electrode as the
concentration of cysteine was increased, which made the electrode surface saturated,
hence, limiting the adsorption of aflatoxins.
157
70
60
50
40 AFB1
Ip (nA) AFB2
30 AFG1
AFG2
20
[Aflatoxins] = 0.1 uM
10
0
0 0.2 0.4 0.6 0.8 1
[L-cyste ine ] / uM
Figure 3.66 Ip of all aflatoxins with increasing concentration of cysteine up to 1.0 µM
From this interference study, all studied metal ions and organic compounds
neither significantly decreased nor enhanced the Ip of all aflatoxins in BRB at pH 9.0.
Attempts have been made to enhance the Ip of aflatoxins by performing mercury
electrode modification using poly-L-lysine (PLL) as described by Ferreira et al. (1999).
However the aflatoxin peak does not increase and this may be due to unsuitable medium
for reaction between aflatoxins and PLL to take place. For example, Ip of AFB2 was
enhanced about 15% in BRB at pH 5.0 however at pH 9.0 the Ip was decreased to
almost 35% from its original value as shown in Appendix X.
3.3 Square-Wave Stripping Voltammetry (SWSV) of Aflatoxins
Since aflatoxins can be adsorbed on the mercury surface as has been proven by
CV and DPCSV studies, another technique of adsorptive stripping method for
quantitative measurement of aflatoxins such as SWSV was developed. By using strong
adsorptive phenomenon and by accumulation of aflatoxins at a HMDE prior to SWSV
measurement, higher sensitivities can be readily achieved in order to obtain a much
lower level of detection for aflatoxins. Compared with other pulse techniques, SWSV is
faster and the entire potential scan can be completed in approximately 2 s on a single
drop of mercury electrode (Jelen et al., 1997). In this study, SWSV was used in
combination with an HMDE to study the Ip of aflatoxins in various BRB pH medium
using optimum parameters that have been obtained using DPCSV technique. The
158
influences of SWSV instrumental variables such as frequency and voltage step on the
SWSV responses have been evaluated. Overall results show that, compared with
DPCSV technique, SWSV produces much higher Ip for all aflatoxins at the same
concentration. Finally the proposed method was applied to the determination of
aflatoxin in groundnut samples.
3.3.1 SWSV Determination of AFB2
Previous optimised parameters that were obtained by DPCSV have been applied
to the determination of 0.10 µM AFB2 using SWSV technique which produces a single
and well developed peak at Ep of -1.30 V with Ip of 230 nA. The Ip was about 380 %
(3.8 times) higher as compared with that obtained by DPCSV which was only 60 nA as
shown in Figure 3.67.
Figure 3.67 Voltammograms of 0.10 µM AFB2 obtained by (a) DPCSV and (b)
SWSV techniques in BRB at pH 9.0. Parameters for DPCSV: Ei = -1.0 V, Ef = -1.4 V,
Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV and for SWSV: Ei =
-1.0 V, Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, frequency = 50 Hz, voltage step = 0.02 V,
amplitude = 50 mV and υ = 1000 mV/s.
159
3.3.1.1 Optimisation of Experimental and Instrumental SWSV Parameters
3.3.1.1a Influence of pH of BRB
Using optimised parameters as stated in Figure 3.67 for SWSV, the pH of BRB
was varied between 6.0 and 13.0. Ip of 0.10 µM AFB2 showed maximum value at pH
9.0 as shown in Figure 3.68. This result was the same as found using CV and DPCSV
techniques where BRB at pH 9.0 is the best supporting electrolyte for the determination
of aflatoxins. Voltammogram of AFB2 in differenct pH is shown in Figure 3.69.
300
250
200
I p (nA)
150
100
50
0
5 6 7 8 9 10 11 12 13 14
pH of BRB
Figure 3.68 Influence of pH of BRB on the Ip of 0.10 µM AFB2 using SWSV

technique. The instrumental parameters are the same as in Figure 3.67.
160
Figure 3.69 Voltammograms of 0.10 µM AFB2 in different pH of BRB. Parameter

conditions; Ei = -1.0 V, E f = -1.4 V, Eacc = -0.6 V, tacc = 80 s, voltage step = 0.02 V,
amplitude = 50 mV, frequency = 50 Hz and scan rate = 1000 mV/s
3.3.1.1b Effect of Instrumental Variables
With regard to the instrumental variables, the relationship between Ip and Ei was
studied for 0.10 µM AFB2, and Ei was varied from 0 to -1.2 V. It was found that a
maximum amount of 250 nA was obtained for Ei = -1.0 as shown in Figure 3.70. The Ip
was slowly decreased when the value of Ei was more negative than -1.0 V.
.
300
250
200
Ip InA)
150
100
50
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
- Ei (V)
Figure 3.70 Effect of Ei to the Ip of 0.10 µM AFB2 in BRB at pH 9.0

161
The influence of Eacc to Ip of AFB2 was investigated where the Eacc was varied between
0 to -1.4 V. The maximum value of Ip was obtained at -0.8 V (265 nA) as shown in
Figure 3.71. This value was selected for subsequent experiments
300
250
200
I p (nA)
150
100
50
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
- Eacc (V)
Figure 3.71 Effect of Eacc to the Ip of 0.10 µM AFB2 in BRB at pH 9.0
The effect of tacc on the Ip was studied where tacc was varied from 0 to 200 s.
The result is shown in Figure 3.72 which reveals that there is linearity up to 100 s
according to equation; y = 3.020x + 20.143 (n=6) with R2 = 0.9950, after which it
increased slower and levelled off at about 140 s. After 160 s, Ip decreased which may be
due to the desorption of the aflatoxins back to the bulk solution. Thus, the tacc of 100 s
appeared to be the optimum tacc for the pre-concentration prior to stripping.
A linear increase in Ip of AFB2 was observed when frequency was varied from
25 to 125 Hz as shown in Figure 3.73. The frequency value of 125 Hz was adopted as
the optimum value.
162
400
350
300
250
Ip (nA)
200
150
100
50
0
0 40 80 120 160 200 240
t acc (s)
Figure 3.72 Relationship between Ip of 0.10 µM AFB2 while increasing tacc
1000
800
600
Ip (nA)
400
200
0
0 50 100 150
Frequency (Hz)
Figure 3.73 Effect of frequency to the Ip of 0.10 µM AFB2 in BRB at pH 9.0
The Ip of AFB2 depends linearly on the square root of frequency as shown in

Figure 3.74 which indicates that AFB2 adsorbed on the mercury electrode as reported
by Komorsky-Lovric et al. (1992). This result is in agreement with that obtained by
previous CV and DPCSV experiments.
163
1000
800
600
I p (nA)
400
y = 107.91x - 424.7
200
R2 = 0.9919
0
3 5 7 9 11
(Frequency)1/2
Figure 3.74 Linear relationships between Ip of AFB2 and square root of frequency
The Ip of the AFB2 increases linearly with variation voltage step from 10 mV to
30 mV but after this value the Ip decreases (Figure 3.75). Hence, 30 mV was selected as
the optimum voltage step for further experiments.
1000
800
600
Ip (nA)
400
200
0
0 0.01 0.02 0.03 0.04 0.05
Voltage step (V)
Figure 3.75 Influence of square-wave voltage step to Ip of 0.10 µM AFB2.

164
Regarding the influence of the square wave amplitude, the maximum value for
Ip was observed at 0.05 V when the amplitude increases from 0.025 to 0.10V as shown
in Figure 3.76. The Ep of AFB2 was ideally linear and shifted to a more negative
direction with increasing amplitude according to the equation; y = 1.2x -1.36 (n=4, R2 =
1.0) as shown in Figure 3.77. Since the square wave frequency, voltage step and scan
rate are interrelated, for frequency of 125 Hz and voltage step of 0.03 V, the scan rate is
3750 mV/ s.
1000
750
Ip (nA)
500
250
0
0 0.025 0.05 0.075 0.1 0.125
Amplitude (V)
Figure 3.76 Influence of square-wave amplitude to Ip of 0.10 µM AFB2.
1.34
1.32
1.3
Ep (-V)
1.28
1.26
y = -1.2x + 1.36
1.24
R2 = 1
1.22
0 0.025 0.05 0.075 0.1 0.125
Amplitude (V)
Figure 3.77 Relationship of SWSV Ep of AFB2 with increasing amplitude

165
Accordingly, the optimum experimental and instrumental parameters of the

proposed SWSV procedure for the determination of AFB2 are Ei = -1.0 V, Ef = -1.4 V,
Eacc = -0.80 V, tacc = 100 s, frequency = 125 Hz, voltage step = 0.03 V, amplitude = 75
V, scan rate = 3750 mV/s in BRB at pH 9.0 as the supporting electrolyte. Using these
parameters, the Ip for 0.10 µM AFB2 was 913 nA with Ep at -1.28 V an increase of
220% before being optimised. Figure 3.78 shows the Ip of 0.10 µM AFB2 obtained
under optimised and non-optimised SWSV parameters compared with that obtained
under optimised DPCSV parameters.
1050
900
750
600
Ip (nA)
450
300
150
0
a b c
Figure 3.78 The Ip of 0.10 µM AFB2 obtained under (a) non-optimised and (b)
optimised SWSV parameters compared with that obtained under (c) optimised DPCSV
parameters.
The voltammograms of AFB2 are shown in Figure 3.79. As compared to the

DPCSV, the Ip value for the optimised parameters for SWSV was 15 times higher. This
high sensitivity is one of the SWSV characteristics that was also found by other
researchers such as reported by Radi et al. (2001), Guiberteau et al. (2001), El-Hady et
al. (2004), Gazy et al. (2004), Farghaly and Ghandour (2005) and Pacheco et al. (2005).
166
Figure 3.79 Voltammograms of 0.10 µM AFB2 obtained under (a) non-optimised

and (b) optimised SWSV parameters compared with that obtained under (c) optimised
DPCSV parameters.
3.3.2 SWSV Determination of Other Aflatoxins
Using the optimum SWSV parameters, the Ip of AFB1, AFG1 and AFG2 at
concentration of 0.10 µM have been measured in BRB at pH 9.0. The results are shown
in Table 3.19. When compared with the Ip that were obtained by DPCSV, overall
results show that it increases about 15 times for AFB1 and AFB2 while for AFG1 and
AFG2 about 11 times as shown in Figure 3.80. Their voltammograms are shown in
Figure 3.81 which also includes DPCSV voltammograms for comparison.
167
Table 3.19 Ip and Ep for all aflatoxins obtained by SWSV in BRB at pH 9.0 (n=5)
Aflatoxin Ip (nA) / (RSD) Ep (V)
AFB1 838.8 ± 5.72 (0.68%) -1.30
AFB2 825.4 ± 11.51 (1.39%) -1.30
AFG1 633.0 ± 3.16 (0.50%) -1.24
AFG2 674.2 ± 11.86 (1.76%) -1.24
1000
800
DPCSV
600
I p (nA) SWSV
400
200
0
AFB1 AFB2 AFG1 AFG2
Figure 3.80 Ip of 0.10 µM aflatoxins obtained using two different stripping

voltammetric techniques under their optimum parameter conditions in BRB at pH 9.0.
168
(i) (ii)
(iii) (iv)
Figure 3.81 Voltammograms of (i) AFB1, (ii) AFB2, (iii) AFG1 and (iv) AFG2
obtained by (b) DPCSV compared with that obtained by (c) SWSV in (a) BRB at pH
9.0.
3.3.3 Calibration Curves and Method Validation
After optimisation of the experimental condition, calibration curves were

prepared by measuring the Ip of each aflatoxin with different concentration. For
example, voltammograms of AFB1 with varying concentrations are shown in Figure
3.82.
169
Figure 3.82 SWSV voltammograms for different concentrations of AFB1 in BRB at

pH 9.0. The broken line represents the blank; (a) 0.01 µM, (b) 0.025 µM, (c) 0.05 µM,
(d) 0.075 µM, (e) 0.10 µM, (f) 0.125 µM and (g) 0.150 µM. Parameter conditions; Ei =
-1.0 V, Ef = -1.4 V, Eacc = -0.8 V, tacc = 100 s, frequency = 125 Hz, voltage step = 0.03
V, pulse amplitude = 75 mV and scan rate = 3750 mV/s.
The calibration curves for all aflatoxins are shown in Figures 3.83 to 3.86. The
LOD were determined by standard addition of low concentration of analyte (AFB1,
AFB2, AFG1 and AFG2) until a response was obtained that is significantly different
from the blank sample (Barek et al., 2001a). LOQ were calculated using the formula
LOD/3 x 10. Figure 3.87 shows the comparison of the LOD values obtained by SWSV
compared with those obtained by DPCSV. The obtained LOD and LOQ for all
aflatoxins are lower than those obtained by DPCSV technique which suggests that the
SWSV is much better than DPCSV in terms of sensitivity.
170
1200
1000
800
Ip (nA) 600
y = 70.97x + 76.588
400
R2 = 0.9984
200
0
0 5 10 15 20
-8
[AFB1] x 10 M
Figure 3.83 Calibration curve for AFB1 obtained by SWSV method.
1200
1000
800
Ip (nA)
600
400
y = 61.97x + 243.36
200
R2 = 0.9938
0
0 2.5 5 7.5 10 12.5 15
-8
[AFB2] x 10 M
Figure 3.84 Calibration curve for AFB2 obtained by SWSV method
1000
800
600
Ip (nA)
400
y = 51.046x + 164.79
200
R2 = 0.9912
0
0 5 10 15
[AFG1] x 10-8M
Figure 3.85 Calibration curve for AFG1 obtained by SWSV method.

171
1000
800
600
Ip (nA)
400
y = 49.816x + 157.18
200 R2 = 0.9906
0
0 5 10 15
-8
[AFG2] x 10 M
Figure 3.86 Calibration curve for AFG2 obtained by SWSV method.
3.5
3
2.5
LOD (ppb)
2 DPCSV
1.5 SqWSV
0.5
0
AFB1 AFB2 AFG1 AFG2
Figure 3.87 LOD for determination of aflatoxins obtained by two different stripping
methods.
Furthermore, this technique provides a shorter time analysis due to the fast scan
rate (3750 mV/s). Table 3.20 summarises the characteristics of the calibration plots
established using optimum SWSV parameters for all aflatoxins.
172
Table 3.20 Analytical parameters for calibration curves for AFB1, AFB2, AFG1 and
AFG2 obtained by SWSV technique in BRB at pH 9.0 as the supporting electrolyte.
Aflatoxin Ep (V) Regression Sensitivity Linearity range LOD LOQ R2

Equations (µA/µM) (x 10-8 M / ppb) (x 10-8 M (x 10-8
/ ppt) M/
ppb)
B1 -1.30 y = 70.97x + 76.59 7.097 1 – 15 / 3.12 – 46.73 0.125 / 0.42 / 0.9984

393 1.31
B2 -1.30 y = 61.97x + 243.36 6.197 1– 12.5 / 3.14 – 39.31 0.15 / 468 0.50 / 0.9938
1.56
G1 -1.24 y = 51.046x + 64.79 5.105 1– 12.5 / 3.28 – 40.98 0.15 / 492 0.50 / 0.9912
1.64
G2 -1.24 y = 49.816x + 57.18 4.982 1– 12.5 / 3.30 – 41.25 0.15 / 500 0.50 / 0.9906
1.67
Notes;
Ep = Peak potential for aflatoxins at concentration of 0.1 µM
y = mx + c; y = nA, x = x 10-8 M, c = nA
LOQ = limit of quantification

173
Reproducibility was evaluated by performing ten measurements of aflatoxins

with concentration of 0.10 µM on the same day and within 3 days for intra and inter-
days measurements respectively. The results are shown in Table 3.21. For all
measurements of all aflatoxins, the RSD were between 0.30% to 1.92% which indicates
that the proposed method is reproducible and gives high precision. Voltammograms of
0.1 µM AFB1, AFB2, AFG1 and AFG2 for intra-day precision (n=10) are shown in
Appendix Y.
Table 3.21 Result of reproducibility study for 0.1 µM aflatoxins in BRB at pH 9.0
Aflatoxins Ip (nA) for intra-day Ip (nA) for inter-day (± SD, n = 10) (RSD)
( ± SD, n = 10) (RSD)
Day 1 Day 2 Day 3
AFB1 833 ± 10.02 832 ± 8.77 833 ± 10.87 833 ± 9.07

(1.20%) (1.05%) (1.30) (1.09)
AFB2 816 ± 14.88 817 ± 14.24 812 ± 15.62 816 ± 13.92

(1.82%) (1.74%) (1.92%) (1.71%)
AFG1 630 ± 4.38 634 ± 2.07 628 ± 2.55 631 ± 4.32

(0.70%) (0.33%) (0.41%) (0.68%)
AFG2 669 ± 9.92 674 ± 11.86 665 ± 4.83 674 ± 8.41

(1.48%) (1.76%) (0.73%) (1.25)
For assessing accuracy of the proposed method, an amount of 5 x 10 -8 M and 10

x 10-8 M of each aflatoxin were spiked into voltammetric cell and their respective Ip
were measured. From the obtained Ip, the actual concentration found was calculated
according to the respective calibration plot. The results are summarised in Table 3.22
which show that the recoveries for 5 x 10-8 M and 10 x 10-8 M of all aflatoxins are
between 93 to 106% which indicate that the proposed method is of a very high
accuracy. Voltammograms of 10 x 10 -8 M of all aflatoxins (n= 3) are shown in
174
Appendix Z. From the method validation, the results show that the proposed SWSV
method is satisfactorily precise and accurate, gives very low detection limit and
provides very short time analysis. This method was applied for analysis of aflatoxin
content in groundnut samples together with the DPCSV method.
Table 3.22 Application of the proposed method in evaluation of accuracy of the

SWSV method by spiking the aflatoxin standard solutions.
Aflatoxin Amount Obtained Ip Amount Recovery ± RSD of

added (nA) found SD (%) recovery
( x 10 -8 M) (± SD, n =3) ( x 10 -8 M) (%)
(± SD)
AFB1 5.0 442.33 ± 2.56 5.15 ± 0.03 103.00 ± 0.60 0.58
10.0 839.00 ± 5.29 10.60 ± 0.07 106.00 ± 0.70 0.66
AFB2 5.0 566.30 ± 3.79 5.19 ± 0.03 103.64 ± 0.58 0.56
10.0 820.67 ± 6.81 9.39 ± 0.07 93.90 ± 0.70 0.75
AFG1 5.0 424.33 ± 5.03 5.04 ± 0.06 100.87 ± 1.20 1.19
10.0 639.07 ± 5.51 9.57 ± 0.80 95.70 ± 0.80 0.84
AFG2 5.0 417.33 ± 2.08 5.24 ± 0.03 104.80 ± 0.53 0.51
10.0 664.00 ± 1.00 10.17 ± 0.02 101.70 ± 0.20 0.20

175
3.4 Stability Studies of Aflatoxins
3.4.1 10 ppm Aflatoxin Stock Solutions
10 ppm aflatoxin stock solutions which were prepared in benzene: acetonitrile

(98:2) and stored in the dark under refrigeration at temperature of 14 0 C were
monitored for their stability by measuring the absorbance using UV-VIS
spectrophotometer every month for twelve months. This study was carried out to
determine the shelf life of aflatoxins stock solution in benzene: acetonitrile kept under
the dark and cool conditions. The obtained concentrations of all aflatoxins monitored
for the period of twelve months at their respective wavelengths are shown in Table 3.23.
UV-VIS spectrums for each freshly prepared AFB1, AFB2, AFG1 and AFG2 are shown
in Appendix AA.
Table 3.23 Average concentration of all aflatoxins within a year stability studies
Aflatoxin λ range (nm) Average concentration

within a year ± SD (ppm)
(RSD)
AFB1 346 – 348 10.63 ± 0.32 (3.01%)
AFB2 349 – 351 10.73 ± 0.42 (3.91%)
AFG1 355 – 357 10.68 ± 0.44 (4.12%)
AFG2 355 – 357 10.58 ± 0.40 (3.78%)
The results show that within a year, all aflatoxins which were stored in the cool and
dark conditions are stable and their concentrations are maintained at 10 ppm with RSD
of less than 5.0 %.
176
Figures 3.88 to 3.90 show the UV-VIS spectrums for all aflatoxins at 0, 6 and 12
months storage time respectively. There are no significant changes in wavelengths for
maximum absorbance of all aflatoxin within the stipulated period of time.
Figure 3.88 UV-VIS spectrum of 10 ppm of all aflatoxins in benzene: acetonitrile

(98%) at preparation date.

(98%) after 6 months of storage time.
177

(98%) after 12 months of storage time.
The maximum absorbance obtained for all aflatoxins are due to the presence of
UV chromophore which is a carbonyl group (C=O) in the aflatoxin molecules. This
group has a transition electronic level between non-bonding (n) and Π* antibonding
which generally occurs at 250 to 300 nm. Due to the conjugation of carbonyl group
with a double bond in benzene ring in the aflatoxin structure, this transition takes place
at longer wavelength which is more than 300 nm as stated in Table 3.23 (Fifield and
Haines, 2000). The result of this study shows that all aflatoxin stock solutions which
were stored in the cool and dark conditions have a shelf life of at least one year and can
be used for further voltammetric study
The same aflatoxin stock solution was exposed to ambient condition, for
example, AFB1, the obtained UV-VIS spectrum is different where the wavelength for
the maximum absorbance was shifted from 348 nm to 335 nm as shown in Figure 3.91
indicating that the chemical structure of AFB1 may be damaged and cannot be used for
voltammetric study.
178
Figure 3.91 UV-VIS spectrums of 10 ppm AFB1 (a) kept in the cool and dark
conditions and (b) exposed to ambient conditions for 3 days.
This can be clearly seen when a lower concentration (1 ppm) of the same
solution was studied and compared with a new fresh stock solution of the same
concentration (Figure 3.92). The result indicates that aflatoxin stock solutions must be
protected from light and kept in cool condition for longer shelf life. The spectrum of
AFB1 prepared from damaged stock solution shows extra peak at λ of 330 nm while for
AFB1 which was prepared from freshly prepared stock solution has only a single peak
at the same λ as obtained for the stock solution.
Figure 3.92 UV-VIS spectrums of 1 ppm AFB1 in BRB solution prepared from (a)
freshly prepared and (b) damaged 10 ppm AFB1 stock solution
179
The diluted solutions were kept in the dark and cool conditions for 2 weeks and
their absorbance were re-measured. The result is shown in Figure 3.93 which shows
that AFB1 from damaged stock produced lower absorbance and at the same time, λ was
shifted to lower value from 335 nm to 320 nm compared to the result that was obtained
at the preparation date while the AFB1 from freshly prepared stock shows no significant
change in its absorbance and λ values. These results indicate that AFB1 solution in
BRB prepared from damaged stock is less stable compared to that one prepared from
the fresh stock solution.
Figure 3.93 UV-VIS spectrums of 1 ppm AFB1 in BRB solution prepared from (a)
freshly prepared and (b) damaged 10 ppm AFB1 stock solution after 2 weeks of storage
in the dark and cool conditions.
3.4.2 1 ppm Aflatoxins in BRB at pH 9.0
4.4.2.1 Month to Month Stability Studies
1.0 ppm aflatoxins prepared in BRB at pH 9.0 which were stored in the freezer
at temperature of -4.0 0 C were monitored for their stability within 6 months from the
preparation date using the DPCSV technique. The aim of this study was to determine
the shelf life of 1.0 ppm aflatoxin in BRB at pH 9.0 kept under the cool and dark
conditions. 0.1 µM of each aflatoxin was spiked in 10 ml BRB at pH 9.0 as the
180
supporting electrolyte and the Ip and Ep of respective aflatoxin were observed from 0 to
6 month storage time. The result is shown in Figure 3.94. Voltammograms of all
aflatoxins obtained after 0 to 6 month storage time are shown in Appendix AB. The
result shows that after three months storage time, the Ip of AFG1 and AFG2 decreased
to about 60 % from original Ip measured at first day of preparation while the Ip of AFB1
and AFB2 still maintained about 75% of their respective Ip. Based of this result, the
stability order of these aflatoxins is suggested as in the order of AFB2 > AFB1 > AFG2
> AFG1. Based on their chemical structures, aflatoxin with a double bond in the
terminal furan ring is less stable in BRB at pH 9.0 as compared to aflatoxin that has a
single bond in their furan ring.
The finding also reveals that AFG1 and AFG2 can be used up to 2 months while
AFB1 and AFB2 up to 3 months. After these periods of time, the respective aflatoxin
should be disposed and the new standard solution should be freshly prepared for the rest
of experiment.
100
80
Percentage (%)
AFB1
60 AFB2
40 AFG1
AFG2
20
0
0 1 2 3 4 5 6 7
Storage time in freezer (months)
Figure 3.94 Percentage of Ip of 0.1 µM aflatoxins in BRB at pH 9.0 at different

storage time in the cool and dark conditions.
181
4.4.2.2 Hour to Hour Stability Studies
Each aflatoxin (0.1 µM) was spiked in BRB at pH 9.0 and monitored by the
DPCSV technique from 0 to 8 hours exposure to ambient conditions. The purpose of
this study was to evaluate the stability of aflatoxins in BRB under ambient conditions
up to 8 hours. The period of 8 hours was selected due to the normal working hours. A
plot of percentage of all aflatoxins versus exposure time is illustrated in Figure 3.95.
Voltammograms of all aflatoxins which were exposed from 0 to 8 hours in BRB at pH
9.0 are shown in Appendix AC.
100
80
Percentage (%)
AFB1
60 AFB2
40 AFG1
AFG2
20
0
0 1 2 3 4 5 6 7 8 9
Exposure time (hrs)
Figure 3.95 Percentage of Ip of all aflatoxins in BRB at pH 9.0 exposed to ambient

condition up to 8 hours of exposure time.
The result shows that AFB1 and AFB2 measured in BRB at pH 9.0 were stable
within 8 hours of exposure time where the Ip for both aflatoxins were reduced only 15%
from initial Ip. In contrast, AFG1 and AFG2 gave significant reduction in the Ip within
the same period of exposure time where the Ip of AFG1 and AFG2 were reduced to 38%
and 48% respectively. The Ep of all aflatoxins were shifted to more negative direction
with increasing exposure time as shown in Appendix AC. The experiment reveals that
AFG1 and AFG1 were less stable in BRB at pH 9.0 as compared to AFB1 and AFB2.
182
Based on their chemical structures, the most reactive functional groups susceptible for
ease of attack by chemical reagents are the two lactones rings of AFG1 and AFG2 and
cyclopentanone rings of AFB1 and AFB2. AFG1 and AFG2 were less stable due to the
easier opening of the lactone rings in strong alkali as compared to the cyclopentanone
ring as reported by Jaimez et al. (2000). The other functional group of this aflatoxin is
less readily attacked by any chemical reagents except for the double bond of terminal
furan ring in AFB1 and AFG1 which are susceptible to attacks by certain reagents such
as TFA (Beebe, 1978; Cespedes and Diaz, 1997; Dhavan and Choudry, 1995; Akiyama
et al., 2001; Erdogan, 2004), iodine (Davis and Diener, 1980; Tuinstra and Haasnoot,
1983; Beaver and Wilson, 1990) or bromine in special conditions as shown in Figure
3.96 (Kok et al., 1986).
O
O
HO
O OCH3
O
O T FA
O
0
30 m in, 50 C O
O
O
I2
I O
40 s, 60 0 C
O O CH3 I
O
O OCH3
O
B r2
4 s , 20 0 C
O
O
Br O
Br
O OCH3
O
Figure 3.96 Reactions of 8, 9 double bond furan rings in AFB1 with TFA, iodine
and bromine under special conditions (Kok et al., 1986).
183
As mentioned earlier, 1 ppm AFB1 standard solution from the “damaged” AFB1
stock solution was prepared in BRB at pH 9.0. Beside UV-VIS spectrophotometric
determination, it was measured by the DPCSV technique and found that the Ip was only
4.0 nA as compared to the freshly prepared from fresh stock solution which gave a peak
current of 60 nA as shown in Figure 3.97. The result indicates that almost all the AFB1
molecules present in BRB solution were already damaged.
Figure 3.97 Voltammograms of 0.1 µM AFB1 obtained in (a) BRB at pH 9.0 which
were prepared from (b) damaged and (c) fresh stock solutions.
Another possible reason why aflatoxins were damaged when exposed to ambient
condition is due to the fact that aflatoxins are not stable to light. Experiments were
performed to verify this statement by running the same experiment as previously
mentioned except by wrapping the voltammetric cell with aluminum foil. The results
are shown in Figure 3.98 to 3.101 which indicate that in the absence of a lighting
system, Ip decreased much faster due to unexplained reason. The result of this
experiment shows that the light was not a main factor that caused instability of
aflatoxins in BRB at pH 9.0.
184
70
60
(a)
50
Ip (nA)
40 (b)
30
20
10
0
0 2 4 6 8 10
Exposure time (hrs)
Figure 3.98 Ip of 0.1 µM AFB1 obtained in BRB at pH 9.0 from 0 to 8 hours in (a)
light exposed and (b) light protected.
70
60
(a)
50
(b)
40
Ip (nA)
30
20
10
0
0 2 4 6 8 10
Exposure time (hrs)
Figure 3.99 Ip of 0.1 µM AFB2 obtained in BRB at pH 9.0 from 0 to 8 hours in (a)
185
70
60
50
40
Ip (nA)
(a)
30
(b)
20
10
0
0 2 4 6 8 10
Exposure time (hrs)
Figure 3.100 Ip of 0.1 µM AFG1 obtained in BRB at pH 9.0 from 0 to 8 hours in (a)
70
60
50
40
Ip (nA)
30
(a)
20
(b)
10
0
0 2 4 6 8 10
Exposure time (hrs)
Figure 3.101 Ip of 0.1 µM AFG2 obtained in BRB at pH 9.0 from 0 to 8 hours in (a)
186
3.4.2.3 Stability Studies In Different pH of BRB
0.1 µM of each aflatoxin was measured in four different pH of BRB (6.0, 7.0,
9.0 and 11.0) and the measurements was made in ambient conditions from 0 to 3 hours
of exposure time. The purpose of this experiment was to study the stability of all
aflatoxins when exposed to ambient condition in slightly acidic, neutral and basic
medium in a period of three hours. The results of these studies are shown in Figures
4.103. At 0 hr exposure time, the Ip of all aflatoxins in BRB at pH 6.0 (Figure 3.102a),
7.0 (Figure 3.102b), and 11.0 (Figure 3.102d) were lower as compared in BRB at pH
9.0 (Figure 3.102c) as these pH were not the optimum pH for the voltammetric analysis
of aflatoxins. In acidic medium, all aflatoxins were stable within 3 hours exposure time
which shows that no reaction has occurred between acid and aflatoxins that can damage
the chemical structures of the aflatoxins. This may be due to the formation of the
phenolate ions which existed in the resonance state as shown in Figure 3.103
(Heathcote, 1984). However, in strong basic medium (pH 11), Ip for all aflatoxins
decreased with longer exposure time and the peak current of AFG1 and AFG2 totally
disappeared after 2 and 3 hours respectively. In strong basic medium, hydroxyl ion can
slowly react with cyclopentanone and lactone groups of the aflatoxins. AFG1 and
AFG2 show faster decreasing value in the Ip compared to AFB1 and AFB2 due to the
readiness of the lactone group being attacked in strong basic condition. Figures 3.104,
3.105, 3.106 and 3.107 show the voltammograms of 0.1 µM AFB2 and AFG2 in BRB
at pH 6.0 and 11.0 for 0 to 3 hours of exposure time respectively.
187
70 70
pH 6.0 pH 7.0
60 60
50 50
AFB1 AFB1
Ip (nA)
40 40
I p (nA)
AFB2 AFB2
30 AFG1 30 AFG1
AFG2 AFG2
20 20
10 10
0 0
0 1 2 3 0 1 2 3
Exposure time (hrs) Exposure time (hrs)
(a) (b)
70 50
pH 9.0
60 pH 11.0
40
50 AFB1 AFB1
I p (n A) 30 AFB2
I p (nA)
40 AFB2
30 AFG1 20 AFG1
20 AFG2 AFG2
10
10
0 0
0 1 2 3 0 1 2 3
(c) (d)
Figure 3.102 Peak heights of 0.1 µM aflatoxins in BRB at pH (a) 6.0, (b) 7.0, (c) 9.0
and (d) 11.0 exposed to ambient conditions up to three hours of exposure time.
O
O
O_
O
O
O_
O O O
OCH3 OCH3 OCH3
Figure 3.103 Resonance forms of the phenolate ion (Heathcote, 1984)

188
Figure 3.104 Voltammograms of 0.1 µM AFB2 in BRB at pH 6.0 from 0 to 3.0 hrs of
exposure time.
Figure 3.105 Voltammograms of 0.10 µM AFB2 in BRB at pH 11.0 (b) from 0 to 3.0
hrs exposure time.
189
Figure 3.106 Voltammograms of 0.10 µM AFG2 in BRB at pH 6.0 from 0 to 3.0 hrs
of exposure time.
Figure 3.107 Voltammograms of 0.10 µM AFG2 in BRB at pH 11.0 from 0 to 3.0 hrs
of exposure time.
190
The same studies were carried out using UV-VIS spectrophotometer for the
same period of exposure time which showed that the absorbance of all aflatoxins in
BRB at pH 11.0 decreased with increasing exposure time as shown in Figure 3.108.
Overall results are in agreement with that found by voltammetric techniques. UV-VIS
spectrums for AFB2 and AFG2 in BRB pH at 6.0 and 11.0 are shown in Appendix AD.
0.16 0.12
pH 6.0 pH 7.0
0.14 0.1
0.12 AFB1 AFB1
0.1 0.08
AFB2 AFB2
Abs
Abs
0.08 0.06
AFG1 AFG1
0.06 0.04
0.04 AFG2 AFG2
0.02 0.02
0 0
0 1 2 3 0 1 2 3
(a) (b)
0.14 0.07
pH 9.0 pH 11.0
0.12 0.06
0.1 AFB1 0.05 AFB1
0.08 AFB2 0.04 AFB2
Abs
Abs
0.06 AFG1 0.03 AFG1

0.04 AFG2 0.02 AFG2
0.02 0.01
0 0
0 1 2 3 0 1 2 3
(c) (d)
Figure 3.108 Absorbance of 1.0 ppm aflatoxins in BRB at pH of (a) 6.0, (b) 7.0, (c)
9.0 and (d) 11.0 from 0 to 3 hrs of exposure time.
191
3.4.2.4 Stability Studies in 1.0 M HCl and 1.0 M NaOH
The objective of this study was to investigate the stability of all aflatoxins in
acid and basic medium within certain period of time where the stability of aflatoxins in
1.0 M HCl and 1.0 M NaOH respectively were carried out. In each case, 0.1 µM of
aflatoxin was dissolved in 1.0 M HCl and NaOH respectively before being spiked into
the voltammetric cell for voltammetric measurement. The measurement was taken
every hour in a period of 6 hours as shown in Figures 3.109 and 3.110. Ip for all the
aflatoxins show no significant difference when placed in 1.0 M HCl up to 6 hours as
compared to that obtained with 1.0 M NaOH. For aflatoxins left in 1.0 M NaOH, the Ip
of AFG1 and AFG2 were significantly reduced as compared to AFB1 and AFB2. The
voltammograms of AFB1 and AFG1 in 1.0 M HCl and 1.0 M NaOH for a period of 6
hrs are shown in Appendix AE. No peak was observed after 5 hours of reaction for
AFG1 and AFG2. It can be concluded that up to 6 hrs of reaction time, aflatoxins are
stable in 1.0 M HCl medium compared with 1.0 M NaOH. This outcome confirmed the
results obtained by previous stability studies where all aflatoxins in acidic media are
more stable than those in basic media.
80
70
60
AFB1
50
Ip (n A )
AFB2
40
AFG1
30
AFG2
20
10
0
0 1 2 3 4 5 6
Reaction time (hrs)
Figure 3.109 The peak heights of 0.10µM aflatoxins in 1.0 M HCl from 0 to 6 hrs of
reaction time.
192
70
60
50 AFB1
Ip (nA)
40 AFB2
30 AFG1
20 AFG2
10
0
0 1 2 3 4 5 6
Reaction time (hrs)
Figure 3.110 The peak heights of 0.10 µM aflatoxins in 1.0 M NaOH from 0 to 6 hrs
of reaction time.
3.5 Voltammetric Analysis of Aflatoxins in Real Samples
Both the proposed DPCSV and SWSV techniques with their optimum
parameters were applied to the determination of aflatoxins in 15 groundnut real
samples. The extraction and clean-up methods for aflatoxin in real samples which have
been previously applied by other researchers (Garden et al., 2001; Pena et al., 2002;
Chemistry Department, 2002) were tried in order to obtain the highest yield of
aflatoxins with the minimum matrix effect. From this study the most reliable technique
was chosen for the analysis of the real samples. In addition, a standard addition method
was applied for the determination of aflatoxin content in order to overcome any matrix
effect (Sanna et al., 2000; Guiberteau et al.,2001; Ghoneim et al., 2003; Rodriguez et
al., 2004). This method also provides the highest precision because the uncertainties
introduced by the sample injection are avoided (Chan, 2004). The results which were
obtained by these proposed techniques were compared with those obtained by the
HPLC which is currently an accepted technique for the routine analysis of aflatoxin
compounds in Malaysia (Chemistry Dept., 2000).
193
3.5.1 Study on the Extraction Techniques
Three different extraction techniques, labelled as Technique 1, 2 and 3 were

applied in the extraction of groundnut. The results in Figures 3.111a to 3.111c showed
the Technique 3 gives a well defined peak current for the aflatoxin as compared to other
techniques.
(a) (b)
(c)
Figure 3.111 Voltammograms of real samples after extraction by Technique (a) 1, (b)
2 and (c) 3 with addition of 10 ppb AFB1 standard solution in BRB at pH 9.0 as a
blank.
194
For Technique 3, a standard addition of 10 ppb AFB1 into the sample solution
produced a higher Ip of 20.5 nA with the Ep at -1.24 V. However, for the Technique 1,
Ip of 10 ppb AFB1 standard solution was only 5 nA and no peak was observed for
AFB1 which was spiked in real sample solution obtained by the Technique 2 even
though the concentration of standard solution was increased 5 times. This may be due
to high matrix effect that eliminates the analyte response. Based on this result, the
Technique 3 was selected for extraction and clean-up processes of aflatoxin from
groundnut samples throughout this analysis of real samples.
4.5.1 Analysis of Blank
The results of these analyses are shown in Figures 3.112a and 3.112b which
show no peak was observed for the blank samples which indicate that the reagents used
in extraction and clean-up steps did not produce any peak within the scanned potential
range.
(a) (b)
Figure 3.112 Voltammograms of blank in BRB at pH 9.0 obtained by (a) DPCSV and
(b) SWSV methods.
195
Baseline for the blank however, is higher as compared to the BRB at pH 9.0.
This may be due to the effect of organic compounds which were used in the extraction
process such as methanol and chloroform present in the blank solution which could not
be removed completely during nitrogen purging. The voltammetric measurement was
repeated after two weeks for the same solution and no significant changes in their
voltammograms were observed.
Further investigation which involved the addition of 10 ppb AFB2 in the blank
sample to observe any effect on Ip of AFB2 was carried out with the DPCSV and SWSV
methods. Voltammograms of the blank sample with AFB2 are shown in Figure 3.113
and Table 3.24 shows the Ip of AFB2 for both cases. Since the Ip of 10 ppb AFB2
measured in the presence and absence of the blank sample is not significantly different,
it may be concluded that the blank sample does not interfere with the determination of
aflatoxin in the real sample.
(a) (b)
Figure 3.113 DPCSV (a) and SWSV (b) voltammograms of 10 ppb AFB2 (i) in
presence of a blank sample (ii) obtained in BRB at pH 9.0 (iii) as the supporting
electrolyte.
196
Table 3.24 The Ip and Ep of 10 ppb AFB2 in the presence and absence of a blank
sample
DPCSV (n=3) SWSV (n=3)

Sample
Ip (nA) Ep (V) Ip (nA) Ep (V)
10 ppb AFB2 in 21.23 ± 0.21 -1.25 454 ± 2.65 -1.34

BRB at pH 9.0 (0.98%) (0.58%)
10 ppb AFB2 in 20.40 ± 0.20 -1.25 443 ± 3.21 -1.34

BRB at pH 9.0 + (0.99%) (0.72%)
blank sample
3.5.3 Recovery Studies of Aflatoxins in Real Samples
This study was carried out by spiking three different concentration levels (3 ppb,
9 ppb and 15 ppb) into the eluate of ground nut sample followed by the extraction and
cleaning-up processes. 15 ppb was the highest concentration used in this study since
this is the highest permissible level for total aflatoxin that should be present in
groundnut sample as imposed by the Malaysian Government. The voltammetric
measurements have been made continuously for three days to observe the recovery of
aflatoxins in real samples which was kept in the freezer at 4° C. Figure 3.114 shows the
DPCSV voltammograms of real samples added with 3 ppb, 9 ppb and 15 ppb AFB1 in
BRB at pH 9.0 for day one. For other aflatoxin such as AFG1 their voltammograms are
shown in Appendix AF. The same study was carried out by the SWSV method and for
example, the SWSV voltammograms of AFB1 are shown in Appendix AG.
197
(i) (ii)
(iii)
Figure 3.114 DPCSV voltammograms of real sample (b) added with 3 ppb (i), 9 ppb
(ii) and 15 ppb AFB1 (iii) obtained in BRB at pH 9.0 (a) as the blank on the first day of
measurement.
198
Figure 3.115 shows the percentage of recovery for all aflatoxins within 3 days
obtained by the DPCSV techniques. For those obtained by the SWSV technique, their
figures are shown in Appendix AH.
120 120
100 100
80 AFB1 80 AFB1
% recovery
% recovery
AFB2 AFB2
60 60
AFG1 AFG1
40 AFG2 40 AFG2
20 20
0 0
Day 1 Day 2 Day 3 Day 1 Day 2 Day 3
(a) (b)
120
100
80 AFB1
% recovery
AFB2
60
AFG1
40 AFG2
20
0
Day 1 Day 2 Day 3
(c)
Figure 3.115 Percentage of recoveries of (a) 3 ppb, (b) 9 ppb and (c) 15 ppb of all
aflatoxins in real samples obtained by the DPSV method for one to three days
measurements.
The results show that at first day of measurement using both techniques, the
recoveries of 3 ppb, 9 ppb and 15 ppb of all aflatoxins were from 85 % to 102 % which
indicate that the recovery is good. An example on how the recovery is calculated is
represented in Appendix AI. The European Committee for Standardisation (CEN
Report, 1999) stated that the percentage recovery for aflatoxins in real samples should
199
be 50 to 120%. Based on these criteria, the recoveries of total aflatoxins are acceptable
using this developed method. These recovery values slightly decreased with longer
keeping time which may be due to the matrix effect. Based on this finding, it is
suggested that the recovery study should be performed on the same day of the sample
preparation.
3.5.4 Analysis of Aflatoxins in Real Samples
In this study, all real samples were analysed using the developed DPCSV and
SWSV methods. For all samples, only the AFB1 was found in this analysis. The
DPCSV and SWSV voltammograms of the real samples and with added 10 ppb AFB1
standard solution are shown in Figures 3.116 to 3.121. Examples of real samples with
no aflatoxin, small and large traces of aflatoxin are shown in samples S11, S07 and S10
respectively. The DPCSV voltammograms of these samples are shown in Figure 3.116,
3.118 and 3.120 respectively. The sample S11 shows no traces of aflatoxin, S07
contains 5.90 ppb and S10 contains 31.93 ppb aflatoxin. As comparison, the HPLC
chromatograms for real samples (S07 and S10) are shown in Appendix AJ.
Figure 3.116 DPCSV voltammograms of real sample, S11 (b) with the addition of 10
ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. Parameter conditions; Eacc = -0.6 V,
tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV.
200
Figure 3.117 SWSV voltammograms of real sample S11 (b) with the addition of 10
tacc = 100 s, scan rate = 3750 mV/s, frequency = 125 Hz, voltage step = 0.03 V and
Figure 3.118 DPCSV voltammograms of real sample S07 (b) with the addition of 10
tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV.
201
Figure 3.120 DPCSV voltammograms of real sample S10 (b) with the addition of 10
tacc = 80 s, scan rate = 50 mV/s and pulse amplitude = 80 mV
202
ppb AFB1 (c) in BRB at pH 9.0 (a) as the blank. . Parameter conditions; Eacc = -0.8 V,
The total aflatoxin contents in all 15 samples are shown in Table 3.25. The
results that were obtained by the HPLC technique are also listed which are not
significantly different. In our study using the both proposed techniques, out of 15
analysed samples, 2 samples (13%) were contaminated with more than 15 ppb of total
aflatoxins, 8 samples (53%) were not contaminated and 34% were contaminated with
less than 15 ppb of aflatoxin. Higher levels of aflatoxins were observed in groundnuts
which have been kept for two weeks in the laboratory (S04 and S10) before being
analysed compared to the same samples that were analysed immediately (S01 and S02).
This indicates that the storage time also can affect the level of aflatoxins in groundnut
samples. Due to that reason other samples were analysed immediately. Appendix AK
shows an example on how the aflatoxin content in the S13 was calculated.
203
Table 3.25 Total aflatoxin contents in real samples which were obtained by the
DPCSV, SWSV and HPLC techniques (average of duplicate analysis)
Total aflatoxin (ppb)

Samples
DPCSV SWSV HPLC
S01 12.73 13.92 14.34
S02 7.12 8.36 8.83
S03 n.d n.d 0.50
S04 21.46 29.72 19.08
S05 9.90 8.95 5.34
S06 n.d n.d n.d
S07 5.90 8.17 3.67
S08 n.d n.d n.d
S09 n.d n.d 1.84
S10 31.93 35.65 36.00
S11 n.d n.d 0.42
S12 n.d n.d 1.75
S13 10.76 9.21 8.25
S14 n.d n.d n.d
S15 n.d n.d n.d
n.d: not detected

204
CHAPTER IV
CONCLUSIONS AND RECOMMENDATIONS
4.1 Conclusions
The voltammetry technique which is one of the electrochemical methods was

successfully studied through various methods such as cyclic voltammetry (CV),
differential pulse cathodic stripping voltammetry (DPCSV) and square-wave stripping
voltammetry (SWSV) for determination of aflatoxin compounds. The objectives of this
research were to study the electroanalytical properties of aflatoxins and developing
voltammetric methods for the determination of these compounds in food samples such
as groundnuts. This study met these objectives in terms of the determination of
electroanalytical properties of aflatoxins and has also proven the hypothesis regarding
these properties for the studied compounds such as the compounds that are actively
adsorbed on mercury electrode and undergone totally irreversible reduction reaction.
This study was also successful in developing the DPCSV and SWSV methods for
qualitative and quantitive determinations of aflatoxins in standard solution and real
groundnut samples. The study also proved that aflatoxins which are organic compounds
can be determined by voltammetric techniques with convenient quantitation down to
10-9 M (or ppt) concentration level
The voltammetric studies of aflatoxins reveals that the proposed DPCSV and
SWSV methods are free from inteference effect by studied metal ions and organic
compounds. Both techniques are successful in the determination of aflatoxin as
205
individual rather than in mixed condition. An attempt to analyse them simultaneously

was not possible due to the fact that the location of the peak potentials of all aflatoxins
are very close each other. Harvey (2000) suggested that to get separate peaks for mixed
analytes, the difference of their respective peak potential should be more than 0.08 V.
All studied aflatoxins (AFB1, AFB2, AFG1 and AFG2) undergo reduction
reactions at the mercury electrode with totally irreversible reaction in BRB at pH 9.0.
The respective DPCSV and SWSV peak currents increase linearly with increasing
concentration for all aflatoxins in the range of 0.02 to 0.32 µM and from 0.01 to 0.125
µM. The peak potentials for all aflatoxins in this solution are located within -1.20 V
and they are shifted towards the less negative direction with increasing concentration of
aflatoxins. The aflatoxins that are adsorbed on the mercury electrode produce higher
peak current with longer accumulation time until a limit is reached due to the saturation
of the electrode surface.
The DPCSV technique that was successfully developed for the determination of
aflatoxins was one which gives a detection limit of around 2.0 ppb for all aflatoxins.
This is an acceptable LOD for the determination of aflatoxin in groundnut samples.
The optimum parameter conditions for the DPCSV method of the aflatoxins are Ei = -
1.0 V (except for AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, scan rate = 50
mV/s and pulse amplitude = 80 mV/s. A SWSV technique was also successfully
developed for the analysis of aflatoxins which gives a lower LOD of around 0.5 ppb.
The optimum parameter conditions for the SWSV for all aflatoxins are Ei = -1.0 V
(except for AFG1 = -0.95 V), Ef = -1.4 V, Eacc = -0.8 V, t acc = 100 s, scan rate = 3750
mV/s, frequency = 125 Hz, voltage step = 0.03 V and pulse amplitude = 50 mV/s. From
the statistical analysis of the both proposed techniques, the methods are considered
sensitive, accurate, precise, rugged, robust and provide shorter analysis time.
From the stability studies of aflatoxins, it can be concluded that aflatoxins stock
solution in acetonitrile: benzene (98%) should be kept in the dark and cool conditions
for longer shelf life. For aflatoxin standard solutions prepared in BRB at pH 9.0 and
206
stored in the same conditions as for the stock solutions, AFG1 and AFG2 could be used
up to 2 months while AFB1 and AFB2 up to 3 months. All afaltoxins are stable in 1.0
M HCl within 6 hours compared to that in 1.0 M NaOH.
Both proposed technique are successfully developed and applied in the

determination of aflatoxins in groundnuts. The results that were obtained by both
techniques are not significantly different than those obtained by the HPLC. As
compared to the latter technique, the proposed techniques gave an advantage in terms of
analysis time where the voltammetric analysis need less than 2 minutes or even less
than 30 s for SWSV technique to complete the analysis of one sample while the HPLC
takes approximate 15 min. However, the HPLC is able to separate and analyse all
aflatoxins simultaneously compared to the proposed techniques.
Finally, it can be concluded that the proposed DPCSV and SWSV methods
which are sensitive, accurate, precise, fast, rugged, robust and low cost methods were
successfully developed for the determination of aflatoxins in groundnuts samples. Both
methods have a great potential as an alternative method for this application in the
future.
4.2 Recommendations
In this study, the mercury electrode was applied throughout the experiment. Due
to the oxidation of mercury taking place at the oxidation potential of more than 0 V to
form mercury (I), the study of aflatoxins were restricted in the range of 0 to -1.5 V.
Further study is suggested by using alternative electrodes such as carbon paste, carbon
paste with chemical modified electrode, gold, platinum or glassy carbon electrode
which has a larger potential window compared to the mercury electrode. Another
working electrode such as bismuth film electrode (BFE) can be further investigated for
this analysis due to its performance that has been proven to compare favorably to or
even surpass to that of mercury electrode (Hutton et al., 2005; Economou, 2005).
207
A study on reagents that can complex with aflatoxins is suggested in order to

make the simultaneous determination of aflatoxins in real sample possible. The use of
surface active reagents such as alginic acid, Triton X-100 or alkaline phosphatase that
can be adsorbed on mercury electrode (Wang, 2000) and inhibit one of the aflatoxins
can be studied so as to observe their effect on the peak current of aflatoxins either as in
standard solution or simulation samples.
A study of the effect of storage condition and the storing time duration of
groundnut samples to the level of aflatoxins using the DPCSV and SWSV techniques is
suggested. The results from this study can provide valuable information on good
practice in storing groundnuts at home and for obtaining a data base regarding the
storing time of groundnuts to avoid any contamination by aflatoxins. The stability of
aflatoxins which are treated at various temperatures and exposure time to ultra violet
(UV) light is suggested. The reason for this study is to determine the degree of
temperature and exposure time to UV light that can totally diminish the aflatoxins.
Finally, in this study, the DPCSV and SWSV techniques were applied in
analysed of groundnut samples but for the future works, both techniques can be
explored for analysis of aflatoxins in other types of samples such as rice, cooking oil,
cigarettes tobacco, local and imported meats, bread, fish and vegetables due to higher
consumption by the Malaysian population. Some reports have been published regarding
these analyses (Aycicek et al., 2005; Edinboro and Karnes, 2005; Shenasi and Candlish,
2002; Arrus et al., 2004; AbdulKadar et al., 2004; Erdogan, 2004; Gowda, 2004) but
unfortunately none of them are related to the local environment in Malaysia except a
paper by Ali et al. (2005) which focused on the analysis of aflatoxins in commercial
traditional herbal medicines used in Malaysia and Indonesia.
208
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255
APPENDIX A
Relative fluorescence of aflatoxins in different solvents
Aflatoxin Methanol Ethanol Chloroform
B1 0.6 (430 nm) 1.0 (430 nm) 0.20 (413 nm)
B2 5.3 (430 nm) 2.7 (430 nm) 0.25 (413 nm)
G1 1.0 (450 nm) 1.4 (450 nm) 6.2 (430 nm)
G2 8.7 (450 nm) 4.7 (450 nm) 6.8 (430 nm)

256
APPENDIX B
257
APPENDIX C
258
APPENDIX D
Calculation of concentration of aflatoxin stock solution
Formula used for calculation of concentration of aflatoxin stock solution by UV-

VIS spectrometric technique
[Aflatoxin] / µg/ml = Abs x MW x 1000

ε
Where
Abs = absorbance value
MW = molecular weight
ε = molar absorbtivity (cm-1 M-1)
Parameters used in this calculation;
Aflatoxin MW Solvent ε Λ (nm)
AFB1 312 19,800 353
AFB2 314 Benzene:acetonitrile 20,900 355

(98:2)
AFG1 328 17,100 355
AFG2 330 18,200 357
Example: AFB1
Abs = 0.640 MW = 312 ε = 19,800
[AFB1] = 0.640 x 312 x 1000 = 10.08 µg / ml = 10.08 ppm

19,800
259
APPENDIX E
Extraction procedure for aflatoxins in real samples
Extraction procedure for aflatoxins in real samples: Chemistry Dept. Penang

Branch, Ministry of Science, Technology and Innovation;
1. Groundnut with shell: remove shell of entire sample. Coarse grind.

Remove 50 g and regrind this portion to finer size for drawing analytical sample.
2. Transfer 25 gm analytical sample into a waring blender and add 125 ml

of MeOH-0.1N HCl (100:25) and blend for 2 min (timing). Stand for 5 min
(timing).
3. Filter through whatman No. 1 paper or equivalent. Complete filtration as

soon as possible
4. Collect 50 ml of filtrate in a stoppered container.

(Equivalent weight: 125 ml / 25 g sample)
5. Pipette 20 ml of filtrate into a stoppered or screw cap bottle

(Equivalent weight: 20 ml / 4 g sample)
6. Add exactly 20 ml of 15% ZnSO4 solution. Stopper or cap tightly and

shake vigorously for 30 sec. Filter through diatomaceous earth into another
container. (Equivalent weight: 40 ml / 4 g sample)
7. Pipette 20 ml of this filtrate into a small separating funnel and add

exactly 5 ml of chloroform. Stopper or cap tightly and shake vigorously for 30
sec. (Equivalent weight: 20 ml / 2 g sample).Equivalent weight: 5 ml chloroform
/ 2 g sample)
8. Stand for a few minute to let layers separate. After separated, draw
chloroform layer into a suitable container and pipette 1 ml extract accurately
into amber bottle for preparation of final sample before injecting into
voltammetric cell.
260
APPENDIX F
Calculation of individual aflatoxin in groundnut sample
Aflatoxin, ng/g (ppb) =
P x C x 1000 µl x 125 ml x 1
P’ 100 µl V W
where;
P = peak height of sample (nA)
P’= peak height of standard (after substract with peak height of sample) (nA)
C = amount of aflatoxin injected into voltammetric cell (ng)
V = effective volume;
= 20 x volume of chloroform used for sample preparation = 20 x 1 = 2 ml

2 total volume of chloroform added 2 5
W = weight of sample (25 g)
===> P x C x 1000 µl x 125 ml x 1

P’ 100 µl 2 ml 25 g
===> P x C x 25 ( for injected volume = 100 µl)

P’
261
For other injection volume;
Volume (µl) Formula
200 P x C x 12.5
P’
300 P x C x 8.33
P’
400 P x C x 6.25
P’
Notes;
From first equation;
1000 µl = Volume of final solution prepared in BRB at pH 9.0
100 µl = Injection volume of sample
125 ml = Volume of solvent for mixing and blending groundnut

262
APPENDIX G
Cyclic voltammograms of AFB1, AFG1 and AFG2 with increasing of their

concentrations
Figure G-1 Effect of increasing AFB1 concentrations on the Ip of cathodic cyclic

voltammetric curves in BRB at pH 9.0. (a) 1.30 µM, (b) 2.0 µM (c) 2.70 µM and (d)
3.40 µM. Parameters conditions are Ei = 0, Elow = -1.5 V, Ehigh = 0, scan rate = 200
mV/s.
263
Figure G-2 Effect of increasing AFG1 concentrations on the Ip of cathodic cyclic

mV/s.
Figure G-3 Effect of increasing AFG2 concentrations on the Ip of cathodic cyclic

mV/s.
264
APPENDIX H
Dependence of the peak heights of AFB1, AFG1 and AFG2 on their concentrations
120
90
Ip (nA)
60
30 y = 22.451x + 10.989
R2 = 0.9899
0
1 1.5 2 2.5 3 3.5 4
[AFB1] / uM
Figure H-1 Dependence of the Ip of AFB1 on concentration of AFB1 in BRB

solution at pH 9.0.
265
120
Ip (nA) 90
60
y = 24.756x + 13.072
30
R2 = 0.9812
0
1 1.5 2 2.5 3 3.5 4
[AFG1] / uM
Figure H-2 Dependence of the Ip of AFG1 on concentration of AFG1 in BRB

solution at pH 9.0.
120
100
80
Ip (nA)
60
40
y = 28.757x + 1.4407
20 R2 = 0.9914
0
1 1.5 2 2.5 3 3.5 4
[AFG2] / uM
Figure H-3 Dependence of the Ip of AFG2 on concentration of AFG2 in BRB

solution at pH 9.0.
266
APPENDIX I
Repetitive cyclic voltammograms and their peak height of AFB2, AFG1 and AFG2
in BRB at pH 9.0
Figure I-1 Repetitive cathodic cyclic voltammograms of 1.3 µM AFB1 in BRB

solution at pH 9.0. Parameter conditions are Ei = 0, Elow = -1.5 V, Ehigh = 0 and scan
rate = 200 mV/s
267
70
60
50
40
Ip (nA)
30
20
10
0
1 2 3 4 5
No of cycle
Figure I-2 Increasing Ip of AFB1 cathodic peak obtained from repetitive cyclic
voltammetry
Figure I-3 Repetitive cathodic cyclic voltammograms of 1.3 µM AFG1 in BRB

rate = 200 mV/s
268
80
70
60
50
Ip (nA)
40
30
20
10
0
1 2 3 4 5
No of cycle
Figure I-4 Increasing Ip of AFG1 cathodic peak obtained from repetitive cyclic
voltammetry
Figure I-5 Repetitive cathodic cyclic voltammograms of 1.3 µM AFG2 in BRB

rate = 200 mV/s
269
70
60
50
40
Ip (nA)
30
20
10
0
1 2 3 4 5
No of cycle
Figure I-6 Increasing Ip of AFG2 cathodic peak obtained from repetitive cyclic
voltammetry
270
APPENDIX J
Voltammetric plot E p-log υ for the reduction of AFB1, AFG1 and AFG2 in BRB at
pH 9.0
1350
1340
1330
1320
1310
Ep (-mV)
1300
1290
1280
1270 y = 61.415x + 1171.6
1260 R2 = 0.9987
1250
1240
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8
log v
Figure J-1 The voltammetric plot Ep – log υ for the reduction of 1.3 µM AFB1 in
BRB solution at pH 9.0
271
1280
1270
1260
1250
Ep (-m V)
1240
1230
1220
1210 y = 48.484x + 1134.8
R2 = 0.9978
1200
1190
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8
log v
Figure J-2 The voltammetric plot Ep – log υ for the reduction of 1.3 µM AFG1 in
1290
1280
1270
1260
Ep (-mV)
1250
1240
1230
y = 49.297x + 1146
1220 R2 = 0.9984
1210
1200
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8
log v
Figure J-3 The voltammetric plot Ep – log υ for the reduction of 1.3 µM AFG2 in
272
APPENDIX K
Plot of peak height versus scan rate for AFB1, AFG1 and AFG2 in BRB at pH 9.0
80
70
60
50
Ip (nA)
40
30
20
10
0
0 100 200 300 400 500 600
v ( mV/sec)
Figure K-1 Plot of Ip versus scan rate (υ) for 1.3 µM AFB1 in BRB at pH 9.0
273
110
100
90
80
Ip (n A) 70
60
50
40
30
20
10
0
0 100 200 300 400 500 600
v ( mV/s )
Figure K-2 Plot of Ip versus scan rate (υ) for 1.3 µM AFG1 in BRB at pH 9.0
70
60
50
40
Ip (nA)
30
20
10
0
0 100 200 300 400 500 600
v ( mv/sec)
Figure K-3 Plot of Ip versus scan rate (υ) for 1.3 µM AFG2 in BRB at pH 9.0
274
APPENDIX L
Voltammograms of AFB2 with increasing concentrations
Figure L-1 Cathodic stripping voltammograms of increasing concentration of AFB2

in (a) BRB at pH 9.0, (b) 0.02 µM (c) 0.06 µM (d) 0.10 µM (e) 0.14 µM, (f) 0.18 µM
(g) .22 µM (h) 0.26 µM and (i) 0.32 µM. Parameter conditions; Ei = -1.0 V, Ef = -1.4
V, Eacc = -0.60 V, t acc = 80 s, υ = 50 mV/s and pulse amplitude = 80 mV.
275
APPENDIX M
Voltammograms of 0.1 µM and 0.2 µM AFB2 obtained on the same day

measurements
Figure M-1 Cathodic stripping voltammograms of 0.1µM AFB2

obtained in the same day (n= 8) with RSD = 2.83% Eacc = -6.0 V, tacc= 80 s,
υ = 50 mV/s and pulse amplitude = 80 mV
Figure M-2 Cathodic stripping voltammograms of 0.2 µM AFB2

obtained in the same day (n= 8) with RSD = 0.72%. Eacc = -6.0 V, t acc= 80 s,
υ = 50 mV/s and pulse amplitude = 80 mV
276
APPENDIX N
Voltammogramms of AFB2 at inter-day measurements
Figure N-1 Cathodic stripping voltammograms of 0.1µM AFB2

obtained at day 1 ( n= 8) with RSD = 2.83% .Eacc = -6.0 V, tacc= 80 s, υ = 50
mV/s and pulse amplitude = 80 mV
277

obtained at day 2 (n= 8) with RSD = 2.39% Eacc = -6.0 V, tacc= 80 s, υ = 50

obtained at day 3 (n= 8) with RSD = 1.31% Eacc = -6.0 V, tacc= 80 s, υ = 50
278
APPENDIX O
F test for robustness and ruggedness tests
a) Two tailed F test was used to observe any significant different of variance by using
small variation of a few important parameters such pH of buffer solution, Eacc and tacc..
Optimum parameters Variation
pH of BRB = 9.0 8.5 and 9.5
Eacc = -0.60 V -0.59 V and -0.61 V
tacc = 80 s 75 and 85 s
0.1 µM AFB2 (n =5):
For optimum and small variation in pH of BRB (as an example)
pH n Ip SD
9.0 5 60.62 0.88
8.5 5 58.82 0.79
F = (0.88)2 / (0.79)2 = 0.7744/ 0.6241 = 1.24 (< F tabulated at 95 % confidential level;

9.60).
No significant different for pH 9.0 and 8.5 at 95% confidential level.

279
pH n Ip SD
9.0 5 60.62 0.88
9.5 5 59.30 0.44
F = (0.88)2 / (0.44)2 = 0.7744 / 0.1936 = 4.00 (< F tabulated at 95 % confidential level;

9.60).
No significant different for pH 9.0 and 9.5 at 95% confidential level.
b) Two tailed F test was used to observe any significant different of variance by
using different voltammetric analyser which are BAS and Metrohm under optimum
parameters (AFB1 as an example).
AFB1
Voltammetric analyser n Ip SD
Metrohm 5 59.88 0.94
BAS 5 58.28 2.79
F(4,4) at 95% confidence level = 9.60

F = (2.79)2 / (0.94)2 = 7.78 / 0.88 = 8.84 (< F tabulated; 9.60)
No significant difference between the results obtained for 0.1 µM AFB1 by two types of
voltammetric analyser.
280
APPENDIX P
Voltammograms of AFB1 with increasing concentration
Figure P-1 Cathodic stripping voltammograms of increasing concentration of AFB1

in (a) BRB at pH 9.0, (b) 0.02 µM (c) 0.04 µM (d) 0.14 µM (e) 0.18 µM, (f) 0.22 µM
(g) 0.26 µM (h) 0.30 µM and (i) 0.32 µM. Parameter conditions; Ei = -1.0 V, Ef = -1.4
281
APPENDIX Q
Voltammograms of AFG1 with increasing concentrations
Figure Q-1 Cathodic stripping voltammograms of increasing concentration of AFG1

in (a) BRB at pH 9.0, (b) 0.02 µM, (c) 0.04 µM, (d) 0.06 µM, (e) 0.08 µM, (f) 0.10
µM, (g) 0.14 µM, (h) 0.18 µM, (i) 0.26 µM (j) 0.30 µM and (k) 0.32 µM. Parameter
conditions; Ei = -0.95 V, Ef = -1.4 V, Eacc = -0.60 V, t acc = 80 s, υ = 50 mV/s and pulse
amplitude = 80 mV.
282
APPENDIX R
Voltammograms of AFG2 with increasing concentrations
Figure R-1 Cathodic stripping voltammograms of increasing concentration of AFG2

in (a) BRB at pH 9.0, (b) 0.02 µM, (c) 0.04 µM, (d) 0.06 µM, (e) 0.10 µM, (f) 0.14
µM, (g) 0.18 µM, (h) 0.26 µM, (i) 0.28 µM and (j) 0.30 µM. Parameter conditions; Ei
= -1.0 V, Ef = -1.4 V, Eacc = -0.60 V, t acc = 80 s, υ = 50 mV/s and pulse amplitude = 80
mV.
283
APPENDIX S
LOD determination according to Barek et al. (2001a)
By standard addition of lower concentration of analyte (AFB1, AFB2, AFG1 and

AFG2) until obtaining the sample response that is significantly difference from blank
sample
a) AFB1; 0.5 x 10-8 M or 1.56 ppb gave Ip = 4.3 nA at Ep = -1.250 V
Figure S-1 Voltammogram of 0.5 x 10 -8 M of AFB1 in BRB at pH 9.0

284
b) AFB2; 0.78 x 10-8 M or 2.5 ppb gave Ip = 5.7 nA at Ep = -1.260 V
Figure S-2 Voltammogram of 0.78 x 10-8 M of AFB2 in BRB at pH 9.0
c) AFG1; 1.0 x 10-8 M or 3.28 ppb gave Ip = 4.34 nA at Ep = -1.160 V
Figure S-3 Voltammogram of 1.0 x 10 -8 M of AFG1 in BRB at pH 9.0

285
d) AFG2; 0.76 x 10-8 M or 2.5 ppb gave Ip = 6.19 nA at Ep = -1.190 V
Figure S-4 Voltammogram of 0.76 x 10-8 M of AFG2 in BRB at pH 9.0
From the results, LOD for AFB1, AFB2, AFG1 and AFG2 are 0.5 x 10-8 M (1.56 ppb),
0.78 x 10-8 M (2.50 ppb), 1.0 x 10-8 M (3.28 ppb) and 0.76 x 10-8 M (2.50 ppb),
respectively.
286
APPENDIX T
LOD determination according to Barek et al. (1999)
The limit of detection is calculated as threefold standard deviation from seven analyte
determinations at the concentration corresponding to the lowest point on the appropriate
calibation curve.
AFB1 (as an example);
concentration for lowest point on calibration curve is 2.0 x 10-8 M.
Ip values from seven determinations of this concentration;
11.76, 12.00, 12.40, 11.80, 11.85, 11.92, 11.92
Mean = 11.95 Standard deviation = 0.2142
0.2142 x 3 = 0.642 x 10-8 M or 2.00 ppb
LOD for determination of AFB1 = 0.64 x 10-8 M or 2.00 ppb.

287
APPENDIX U
LOD determination according to Zhang et al. (1996)
The LOD is the concentration giving a signal equal to three times the standard deviation
of the blank signal divided by slope from calibration curve.
AFB1 as an example
Regression equation for calibration curve is y = 5.4363x + 3.7245

Slope = 5.4363
Ip for blank from seven measurements;
40.86, 42.24, 40.22, 38.53, 42.52, 41.04, 41.64
standard deviation of blank (SDblk) = 1.3533
3SDblk / m = (3 x 1.3533) / 5.4363 = 0.7468 x 10-8 M or 2.35 ppb
LOD for determination of AFB1 = 0.75 x 10-8 M or 2.35 ppb.

288
APPENDIX V
LOD determination according to Miller and Miller (1993)
LOD is the concentration giving a signal three times the standard error plus the y-
intercept divided by the slope of calibration curve
Regression equation for peak height of analyte with their concentrations is;
yi = mx + b
where; yi = peak height

m = slope
b = y-intercept
y value for calculation of standard error is y’i = mx + b where x is the concentration of

analyte.
Standard error is calculated based on following equation;
Sy/x = √( ∑ (yi – yi’)2 ) / n -2
LOD = (3 x Sy/x ) / m
289
As example, LOD for AFB1 is calculated as below;
Regression equation for AFB1; y = 5.4363x + 3.7245
[AFB1] = x Peak height = y y i’ = mx + b y – y i’ (y – yi’)2

2 11.86 14.597 -2.737 7.491
4 23.77 25.470 -1.70 2.89
6 36.34 36.342 -0.002 4 x 10-6
10 60.4 58.088 2.312 5.345
14 82.12 79.833 2.287 5.230
18 103 101.578 1.422 2.022
22 125.1 123.323 1.777 3.158
26 145.6 145.068 0.532 0.283
30 165.2 166.814 -1.614 2.605
32 175.4 177.686 -2.286 5.226
∑ (yi – yi’ )2 34.250
(x in 10 -8 M and peak height is in nA)
n = 10, m = 5.4363, ∑ (yi – yi’)2 = 34.250
Sy/x = √34.250/ 8 = 2.069
LOD = (3 x 2.069) / 5.4363 = 1.142 x 10-8 M or 3.56 ppb
====> LOD for AFB1 is 1.14 x 10-8 M or 3.56 ppb.

290
APPENDIX W
ANOVA test (Youmens, 1973)
Table W-1 LOD (in ppb) of aflatoxins obtained from three different method
Aflatoxin B1 B2 G1 G2 Totals
Method 1 1.56 2.50 3.28 2.50 9.84
2 2.00 2.80 3.50 3.02 11.32
3 2.35 2.86 3.60 2.84 11.65
Totals 5.91 8.16 10.38 8.36 32.81
Four sums of squares were calculated in order to make up the ANOVA table. They are
total, sample, method and error sums of squares. The sums of squares were calculated
in five steps as follows;
a) Calculation of C = (∑ Xij)2 / kn = (32.81)2 / 12 = 89.71

b) Calculation of total sum of squares = ∑ (Xij)2 – C = 93.63 – 89.71 = 3.92
c) Calculation of sample or block sum of squares = 1 ∑2j – C = 1 x 279.15 – 89.71
n 3
= 3.34
d) Calculation of method or process sum of squares =1 ∑2i– C

k
= 1 x 360.69 – 89.71 = 0.46

4
291
e) Calculation of error sum of squares
Error sum of squares = total sum of squares – (block sum of squares + process sum
of squares)
= 3.92 – (3.34 + 0.46) = 0.12
The ANOVA table was constructed as follows:
Table W-2 Analysis of variance
Source Degrees of freedom (f) Sum of squares (SS) Mean squares

(SS / f = MS)
Total kn – 1 = 11 3.92
Block n–1=2 3.34
Process k–1=3 0.46 0.153
Error kn – n – k + 1 = 6 0.12 0.02
f) Calculation of F
F = process mean square / error mean square
= 0.153 / 0.02 = 7.65
g) Test of null hypothesis
Tabular F from Fisher’s F table is 8.94 for f1 = 6 and f2 = 3. This is larger than
the calculated value of F. The hypothesis was not disproved, hence the
experiment indicates that the methods are not giving significantly different of
LOD of aflatoxins at 95% probability level.
This ANOVA test indicates that all three methods can be selected in
determination of LOD of aflatoxins using proposed technique.
292
APPENDIX X
Peak height of AFB2 obtained from modification of mercury electrode with PLL
Table X-1 Ip of 10 ppb all aflatoxins in BRB at pH 9.0 in presence and absence of
poly-L-lysine (PLL)
No PLL With PLL

Aflatoxin
Ip (nA) E p (V) Ip (nA) Ep (V)
AFB1 15.70 -1.26 10.22 -1.22
AFB2 14.83 -1.26 12.20 -1.26
AFG1 18.77 -1.19 16.20 -1.19
AFG2 17.60 -1.22 13.90 -1.22
60
50
40
Ip (nA)
No PLL
30
W ith PLL
20
10
0
5 6 9 11
pH of BRB
Figure X-1 Ip of 0.1 µM AFB2 (31.4 ppb) in different pH of BRB with and without
PLL coated on mercury electrode.
293
APPENDIX Y
SWSV voltammograms of AFB1, AFB2, AFG1 and AFG2 in BRB at pH 9.0
Figure Y-1 SWS voltammograms of AFB1 in BRB at pH 9.0 (n =10). Experimental

parameters; Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.8 V, tacc = 100 s, υ = 3750 mV/s,
frequency = 125 Hz, voltage step = 0.03 s and amplitude = 50 mV. Blank is
represented by broken line.
Figure Y-2 SWS voltammograms of AFB2 in BRB at pH 9.0 (n=10). All

experimental conditions are the same as in the Figure Z-2.
294
Figure Y-3 SWS voltammograms of AFG1 in BRB at pH 9.0 (n=10). All

experimental conditions are the same as in the Figure Z-2 except for Ei = -0.95 V.
Figure Y-4 SWSV voltammograms of AFB2 in BRB at pH 9.0 (n=10). All

experimental conditions are the same as in the Figure Z-2.
295
APPENDIX Z
SWSV voltammograms of 0.1 µM of AFB1, AFB2, AFG1 and AFG2 in BRB at pH

9.0
Figure Z-1 SWSV voltammograms of 0.10 µM of AFB1 (n =3) in BRB at pH 9.0. Ei

= -1.0 V, Ef = -1.4 V, Eacc = -0.8 V, t acc = 100 s, υ = 3750 mV/s, frequency = 125 Hz,
voltage step = 0.03 V and amplitude = 50 mV.
Figure Z-2 SWSV voltammograms of 0.10 µM of AFB2 (n = 3) in BRB at pH 9.0.

All experimental parameters are the same as in Figure Z-1.
296
Figure Z-3 SWSV voltammograms of 0.10 µM of AFG1 (n=3) in BRB at pH 9.0.

All experimental parameters are the same as in Figure Z-1 except for Ei = -0.95 V.
Figure Z-4 SWSV voltammograms of 0.10 µM of AFG2 (n =3) in BRB at pH 9.0.

All experimental parameters are the same as in Figure Z-1.
297
APPENDIX AA
UV-VIS spectrums of 10 ppm AFB1, AFB2, AFG1 and AFG2 stock solutions
Figure AA-1 UV-VIS spectrums (n=3) of 10 ppm AFB1 in benzene: acetonitrile

(98%)
Figure AA-2 UV-VIS spectrums (n=3) of 10 ppm AFB2 in benzene: acetonitrile

(98%)
298
Figure AA-3 UV-VIS spectrums (n=3) of 10 ppm AFG1 in benzene: acetonitrile

(98%)
Figure AA-4 UV-VIS spectrums (n=3) of 10 ppm AFG2 in benzene: acetonitrile

(98%)
299
APPENDIX AB
Voltammograms of AFB1, AFB2, AFG1 and AFG2 obtained from 0 to 6 months

storage time in the cool and dark conditions
Figure AB-1 Voltammograms of 0.10 µM AFB1 in BRB at pH 9.0 obtained from

difference storage time of 0 to 6 months in the dark and cool conditions. DPCSV
parameter conditions: Ei = -1.0 V, E f = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s
and pulse amplitude = 80 mV.
300
Figure AB-2 Voltammograms of 0.10 µM AFB2 in BRB at pH 9.0 obtained from

parameter conditions are the same as in Figure AB-1.
Figure AB-3 Voltammograms of 0.10 µM AFG1 in BRB at pH 9.0 obtained from

parameter conditions are the same as in Figure AB-1 except for Ei = -0.95 V.
301
Figure AB-4 Voltammograms of 0.10 µM AFG2 in BRB at pH 9.0 obtained from

parameter conditions are the same as in Figure AB-1.
302
APPENDIX AC
Voltammograms of AFB1, AFB2, AFG1 and AFG2 obtained from 0 to 8 hours

exposure time
Figure AC-1 Voltammograms of 0.10 µM AFB1 in BRB at pH 9.0 exposed to normal

laboratory conditions from 0 to 8 hrs. Experimental conditions: Ei = -1.0 V, Ef = -1.4
303
Figure AC-2 Voltammograms of 0.10 µM AFB2 in BRB at pH 9.0 exposed to normal

laboratory conditions from 0 to 8 hrs. Experimental conditions are the same as in
Figure AC-1.
Figure AC-3 Voltammograms of 0.10 µM AFG1 in BRB at pH 9.0 exposed to normal

Figure AC-1 except for Ei = -0.95 V.
304
Figure AC-4 Voltammograms of 0.10 µM AFG2 in BRB at pH 9.0 exposed to normal

Figure AC-1.
305
APPENDIX AD
UV-VIS spectrums of AFB2 and AFG2 in BRB at pH 6.0 and 11.0
Figure AD-1 UV-VIS spectrums of 1.0 ppm AFB2 in BRB at pH 6.0 from 0 to 3 hrs
exposure time
Figure AD-2 UV-VIS spectrums of 1.0 ppm AFB2 in BRB at pH 11.0 from 0 to 3 hrs
exposure time
306
Figure AD-3 UV-VIS spectrums of 1.0 ppm AFG2 in BRB at pH 6.0 from 0 to 3 hrs
exposure time
Figure AD-4 UV-VIS spectrums of 1.0 ppm AFG2 in BRB at pH 11.0 from 0 to 3 hrs
exposure time
307
APPENDIX AE
Voltammograms of AFB1 and AFG1 in 1.0 M HCl and 1.0 M NaOH
Figure AE-1 DPCSV voltammograms of AFB1 in 1.0 M HCl from 0 to 6 hours
Figure AE-2 DPCSV voltammograms of AFB1 in 1.0 M NaOH from 0 to 6 hours.

308
Figure AE-3 DPCSV voltammograms of AFG1 in 1.0 M HCl from 0 to 6 hours.
Figure AE-4 DPCSV voltammograms of AFG1 in 1.0 M NaOH from 0 to 4 hours.

309
APPENDIX AF
DPCS voltammograms of real samples added with various concentrations of AFG1
(i) (ii)
(iii)
Figure AF-1 DPCSV voltammograms of real samples (b) added with 3 ppb (i), 9 ppb
(ii) and 15 ppb (iii) AFG1 obtained in BRB at pH 9.0 (a) as the blank on the first day
measurement.
310
APPENDIX AG
SWSV voltammograms of real samples added with various concentrations of

AFB1
(i) (ii)
(iii)
Figure AG-1 SWSV voltammograms of real samples (b) added with 3 ppb (i), 9 ppb
(ii) and 15 ppb (iii) AFB1 obtained in BRB at pH 9.0 (a) as the blank on the first day
measurement.
311
APPENDIX AH
Percentage of recoveries of 3 ppb and 9 ppb of all aflatoxins in real samples

120
100
80 AFB1
% recovery
AFB2
60
AFG1
40 AFG2
20
0
Da y 1 Day 2 Da y 3
Figure AH-1 Percentage of recoveries of 3 ppb of all aflatoxins in real samples

obtained by SWSV method for one to three days of measurements.
120
100
80 AFB1
% recovery
AFB2
60
AFG1
40 AFG2
20
0
Da y 1 Day 2 Da y 3
Figure AH-2 Percentage of recoveries of 9 ppb of all aflatoxins in real samples

obtained by SWSV method for one to three days of measurements.
312
APPENDIX AI
Calculation of percentage of recovery for 3.0 ppb AFG1 added into real sample.
From voltammograms of real sample added with 3.0 ppb AFG1;
Peak height = 5.85 nA, 6.53 nA, 6.09 nA Average = 6.16 nA
Peak height for 10 ppb AFG1 in BRB pH 9.0 = 20.60 nA*. So for 1 ppb = 2.060 nA
For peak height = 6.16 nA ===> (6.16 / 2.060) x 1.0 ppb = 2.99 ppb
Injected AFG1 = 3.0 ppb

From the above calculation, found AFG1 = 2.99 ppb
Recovery (%) = (2.99 / 3.0) x 100 % = 99.67%.
Summary:
AFG1 added = 3. 0 ppb
AFG1 found = 2.99 ppb
% recovery of AFG1 in real sample = 99.67 %
*
Notes; For other aflatoxins, the peak heights for 10 ppb of AFB1, AFB2 and AFG2 in
BRB pH 9.0 is 21.93 nA, 21.33 nA and 20.23 nA respectively.
313
APPENDIX AJ
HPLC chromatograms of real samples: S10 and S07
Figure AJ-1 HPLC chromatogram for S10 which contains 36.00 ppb total aflatoxins.
Figure AJ-2 HPLC chromatogram for S07 which contains 3.67 ppb total aflatoxins.
314
APPENDIX AK
Calculation of aflatoxin in real sample; S13
1 st analysis
From voltammograms of real sample ;
From voltammograms of real sample added with 10 ppb AFB1 standard solution.
From equation as stated in Appendix F;

Aflatoxin content = [(1.79) / (22.63 – 1.79)] x 12.5 = 10 .74 ppb
2 nd analysis
From voltammograms of real sample ;
From voltammograms of real sample added with 10 ppb AFB1 standard solution.
From equation as stated in Appendix F;

Aflatoxin content = [(1.80) / (22.67 – 1.80)] x 12.5 = 10 .78 ppb
Average for duplicate analysis = (10.74 + 10.78) / 2 = 10.74 ppb
Total aflatoxins content in S13 = 10.76 ppb

315
APPENDIX AL
List of papers presented or published to date resulting from this study
1. Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Cylic voltammetry study of
AFB2 at the mercury electrode. Paper presented at SKAM -16, Kucing, Sarawak, 9 –
11th September 2003.
2. Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Cylic voltammetry study of
AFB2 at the mercury electrode. Malaysian J. Anal. Sci. In press.
3. Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Differential pulse stripping
voltammetric technique for determination of AFB2 at the mercury electrode. J. Collect.
Czech. Chem. Common. In press
4. Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Stability studies of

aflatoxin G1 (AFG1) using differential pulse stripping voltammetric technique. Paper
presented at Symposium Life Science II, USM Penang. 31st to 3rd April 2004.
5. Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Developement of

differential pulse stripping voltammetric (DPCSV) technique for determination of
AFG1 at the mercury electrode. Chemical Analysis (Warsaw). In press
6. Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Cyclic voltammetry study
of AFG1 at the mercury electrode. Paper presented at KUSTEM 3rd Annual Seminar on
Sustainability Science and Management, Kuala Terengganu, Terengganu. 4 – 5th May
2004.
7. Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Stability studies of

aflatoxins using differential pulse stripping voltammetric (DPCSV) technique. Paper
presented at SKAM-17, Kuantan, Pahang, 24 – 26th August 2004.
316
8 Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Stability studies of

aflatoxins using differential pulse stripping voltammetric (DPCSV) technique.
Malaysian J. Anal. Sci. In press.
9. Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Voltammetric

determination of aflatoxins: Differential pulse voltammetric (PCSV) versus Square-
wave stripping voltammetry (SWSV) techniques. Paper presented at KUSTEM 4 th
Annual Seminar on Sustainibility Science and Management, Kuala Terengganu, 2nd –
3 rd May 2005.
10. Yaacob, M.H., Mohd. Yusoff, A.R. , Ahamad, R. and Misni, M. Determination
of the aflatoxin B1 in groundnut samples by differential pulse cathodic stripping
voltammetry (DPCSV) technique. Paper presented at Seminar Nasional Kimia II,
Universiti Sumatera Utara, Medan, Indonesia. 14th April 2005.
11. Yaacob, M.H., Mohd. Yusoff, A.R. Ahamad, R., and Misni, M. Development of
differential pulse cathodic stripping voltammetry (DPCSV) technique for the
determination of aflatoxin B1 in groundnut samples. J Sains Kimia. 9(3): 31-36.
12. Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Application of differential
pulse cathodic stripping voltammetry (DPCSV) technique in studying stability of
aflatoxins. J Sains Kimia. 9(3): 64-70.
13. Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Square-wave cathodic
stripping voltammetric (SWSV) technique for determination of aflatoxin B1 in
groundnut samples. Paper presented at SKAM-18, JB, Johor. 12 – 14th September
2005.
14. Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. Square-wave cathodic
stripping voltammetric (SWSV) technique for determination of aflatoxin B1 in
groundnut samples. Malaysian J. Anal. Sci. In press.
317
APPENDIX AM
ICP-MS results on analysis of BRB at pH 9.0
Figure AM-1 ICP-MS results on analysis of BRB at pH 9.0

Introduction Experimental Results & Discussion Problem Future Works
PRESENTED AT
1st ASSESMENT
SEMINAR.
3/1/2007 1st ASSESSMENT REPORT 1

STRIPPING VOLTAMMETRIC STUDY FOR DETERMINATION
OF AFLATOXIN COMPOUNDS
MOHAMAD HADZRI YAACOB (PS023001)
SUPERVISOR:
AP DR ABDUL RAHIM HJ MOHD YUSOFF
CO-SUPERVISOR:
PROF RAHMALAN AHAMAD
TOPICS FOR DISCUSSION
z INTRODUCTION
AFLATOXIN COMPOUNDS AFLATOXINS
RESEARCH BACKGROUND
OBJECTIVES
SCOPE OF RESEARCH
z EXPERIMENTAL
z RESULTS AND DISCUSSION
z CONCLUSION
z PROBLEMS
z SUGGESTIONS FOR FUTURE
WORKS
z FLOW CHART OF FUTURE
PLANNING
z ACKNOWLEDGEMENT

INTRODUCTION
z AFLATOXINS:
Mycotoxin group
Produced by two fungi;
Aspergillus flavus and

Aspergillus parasiticus
Types of aflatoxins:
Raw peanuts, peanuts products,

AFB1
AFB2 wheat, maize, vegetable oils, dried
AFG1 fruites, barley, pear, cocoa, coffee,
AFG2 herbs, spices, wine, cottonseeds.
AFM1 Cow milk and its products, eggs

AFM2 and meat product

INTRODUCTION …cont
z AFLATOXINS:
Occurs naturally in commodities used for animal and human
Carcinogenic, teratogenic and mutagenic
IARC (1988): Class I carcinogenic materials
Dangerous to health: will cause aflatoxicosis disease.
Effect the economy of commodities export countries

INTRODUCTION…cont
z AFLATOXINS
Its growth depends on temperature and medium (acidity)
Contamination of feed through infection and seed damaged by insects
Enter into human body through consumption of contaminated food

and skin absorption.
Regulatory level in Malaysia:

Raw peanuts < 15 ppb
Milk < 0.5 ppb
Others < 5 ppb

INTRODUCTION… cont
z CHEMICAL STRUCTURES O
O
O
O
O
O
O O
O O
O
O
AFB1 AFB2
O O
O O
O O
O O
O
O
O
O O
O
AFG1 AFG2
RESEARCH BACKGROUND
z Very dangerous to human health
z Its actual amount must be determined by

fast, accurate and relatively low cost
technique / method.
z Analytical level must be at ppb or ppt ( In

Malaysia: HPLC with LOD:2.0 ppb and
analysis time: 20 mins)

RESEARCH BACKGROUND…cont
Methods that have been used:

z TLC ( 0.03 – 5.0 ppb)
z HPLC (UVD, AD or FD) ( 0.014 – 7.0 ppb)
z Enzyme linked immunosorbant assay (ELISA):
( 0.01 – 5.0 ppb)
z Immunoaffinity chromatography (IAC):
( 0.2 – 2.0 ppb)
z Micellar electrokinetic’s capillary chromatography (MECC):
( 0.02 – 0.09 ppb)
z Differential pulse polagoraphy (DPP): ( 25 ppb)
LOD of aflatoxins depends on the type of:

aflatoxins, samples and extraction techniques

RESEARCH BACKGROUND… cont
z Problems/disadvantages:
TLC Longer analysis time, accuracy problem,
aflatoxins absorption problem
HPLC Longer analysis time, need a lot of toxic

organic solvents, high cost for instruments,
and maintenance
ELISA Reagents /kits are costly. Suitable for screen
test.
RIA Antibodies are costly, using dangerous
radioisotopes, need special precaution for
dealing with radioisotopes and its waste. It
has certain half life.

RESEARCH BACKGROUND… cont
VOLTAMMETRY
Until now, no study has been performed
using voltammetric techniques either cyclic
voltammetry, cathodic/anodic stripping
voltammetry or absorption stripping
Voltammetric technique has been chosen as an alternative method

to be studied for determination of aflatoxins

RESEARCH BACKGROUND …cont
z Voltammetric technique: sensitive, high

accuracy, low cost and easy to use

OBJECTIVES OF RESEARCH
(1) (2)
TO STUDY
TO DEVELOPE A
ELECTROCHEMICAL
DETECTION METHOD FOR
PROPERTIES OF
DETERMINATION OF
AFLATOXINS (AFB1, AFB2,
AFLATOXINS IN FOOD
AFG1 AND AFG2) USING
SAMPLES WHICH IS
VOLTAMMETRIC
SENSITIVE, SIMPLE,
TECHNIQUE TOGETHER
ACCURATE AND
WITH CONTROL GROWTH
ECONOMIC TECHNIQUE.
MERCURY ELECTRODE
(CGME) AS THE WORKING
ELECTRODE
CV DPSV

SCOPE OF STUDY
z Using cyclic voltammetry (CV):-
9 Determine the type of aflatoxins reaction at

mercury electrode when anodic or cathodic
scanning is performed.

SCOPE OF STUDY…cont
9 Identify the peak or peaks obtained either

oxidation peak or reduction peak or both for
developing a technique for analysis of
aflatoxins.

z Using voltammetric technique:
¾ study the effect of pH of electrolyte

solution on reduction/oxidation
aflatoxin peaks.
¾ determine the best electrolyte for the

process of reduction / oxidation of
aflatoxins at mercury electrode.

Parameters optimisation to obtain

maximum peak height and linear with
addition of aflatoxins concentration.
Use optimum parameters for
determination of AFB2, AFB1, AFG1 and
AFG2

PLANNING ACTIVITIES
Activity 2002
M J J O S O N D
1) Literature survey
2) Cyclic voltammetric study of AFB2
3) Stripping voltammetric study of

AFB2
4) Parameters optimisation:
4a) using high concentration
4b) using low concentration

PLANNING ACTIVITIES…cont
Activity 2003
J F M A M J J O S O N D
1) Determination of
AFB1, AFB2, AFG1
and AFG2
1a) Prepare
calibration curve
1b) Determine
analytical parameters
2) Interference study
3) Complexing of
aflatoxins study

PLANNING ACTIVITIES…cont
Activity 2004
1) Determination of mixing
aflatoxins
2)Determination of aflatoxins
in artificially contaminated
samples
in real samples
4)Determination by HPLC
5) Study using different

working electrodes
5) Comparison study
6) Thesis writting up
Activity 2005
1) Analysis of contaminated
food samples

STUDY FLOW CHART
AFLATOXIN COMPOUNDS
AFB1 AFB2 AFG1 AFG2
Differential pulse stripping

Cyclic voltammetric, anodic/cathodic Voltammetric, anodic / cathodic
Complexing with metals

Direct measurement (Zn, Pb, Cu) or Modified electrode /
Organic materials Screen printed electrode
(Cystein)
Method optimisation

STUDY FLOW CHART…cont
Optimise;E acc, tacc,

Ei, Ef, scan rate,
pulse amplitude
Method validation
Limit of detection Linearity Precision Accuracy
By By
experiment graph Intra-day Inter-day

STUDY FLOW CHART…cont
Method testing
Mixing aflatoxins std Artificially contaminated

Real samples
(AFB1,AFB2,AFG1,AFG2) samples
Compare the results with that obtained by HPLC
Method is tested at different laboratory / analyser
Method is accepted

EXPERIMENTAL: INSTRUMENTS AND REAGENTS
z INSTRUMENTS: BAS CGME

(consist of Ag/AgCl as RE, Pt
wire as AE and HMDE as WE)
interface with BAS C50W
Voltammetric Analyser
z Electrolyte solutions: BRB 0.04M

at various pH (2.0,3.0, 4.0, 5.0,
6.0, 7.0, 8.0, 9.0,10.0,11.0, 12.0
dan 13.0). 0.1M HCl for pH 1.0
z Standard solutions: AFB1,

AFB2, AFG1 dan AFG2
10 ppm (10ml) – SIGMA
z Techniques: CV dan DPCSV

EXPERIMENTAL FLOW CHART
10 ML BRB SOLUTION NITROGEN GAS IS FLOWED IN AFLATOXINS

SOLUTION, REDISSOLVED IN BRB SOLUTION
SPIKE INTO 20 ML
VOLTAMMETRIC CELL
SOLUTION IS
DEGASSED
SOLUTION IS BLANK SOLUTION +

BLANK SOLUTION
SCANNED AFLATOXIN
BLANK SAMPEL
VOLTAMMOGRAM OBTAINED
VOLTAMMOGRAM VOLTAMMOGRAM

z CV STUDY ( [AFB2] = 1.3 x 10-6M in BRB pH 9.0)
Ip for AFB2 = 30 nA
Ep = -1315 mV
No anodic peak
Irreversible reaction
Scan rate = 200mV/s

1 = cathodic direction 2 = anodic direction

RESULTS AND DISCUSSION…cont
z CV : to confirm the cathodic peak

z [AFB2] = 1.3 x 10-6M in BRB pH = 9.0
Ip AFB2 = 35 nA
Ep = -1311 mV
No anodic peak
Irreversible reaction
1. Anodic direction
2. Cathodic direction

z CV : to confirm the cathodic peak

z By [AFB2] standard addition and cathodic scanning in BRB pH 9.0
(a) 1.3 uM 30nA, -1315 mV
(b) 2.0 uM 47 nA, -1313 mV
(c) 2.7 uM 67 nA, -1306 mV
(d) 3.4 uM 79 nA, -1303 mV
100
80
Ip (nA)
60
40 y = 26.541x - 3.035
20 R2 = 0.9895
0
0 1 2 3 4
[AFB2] X 10-6M

z Repeatitive cathodic 60
No of Ep
scanning using single 50
cycle (-mV)
i p c a th o d i c (n A )
mercury drop, 1.3 x 10-6M 40
30 1 1314
AFB2 in BRB pH = 9.0 20
3 1310
10
5 1309
0
1 3 5 7 9 7 1307
no of cycle
9 1304
Adsorption process at mercury electrode

O
Diffusion current is influenced

O
AFB2
O by adsorption process at
mercury electrode
O O

z Effect of scan rate (V) on Ip and Ep of AFB2
(1) Log Ep vs log V
1.9
1.7
1.5
Linear with m >0.5:
1.3
lo g E p
1.1
0.9 y = 0.5097x + 0.4115

ÎDiffusion current produced
0.7
R2 = 0.995 by reduction process of
0.5 aflatoxins which is influenced
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 by adsorption process at
log V
mercury electrode.

z Effect of scan rate (V) on Ip and Ep of AFB2

(2) Ep vs log V
1340
1330
1320
Linear :
E p (-m V )
1310
1300 Ep AFB2 changing with
1290 y = 40.065x + 1223.3
R2 = 0.9936
Increasing of V.
1280
1270
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 ÎIrreversible reaction
log V

z Effect of scan rate (V) on Ip Linear : two different regions

of AFB2 due to different scan rate
(3) Ip vs V
(1) 100 – 500 mV/s (fast) :
70
m = 0.076 R = 0.9917
60
50 (2) 20 – 100 mV/s (slow):

40
m = 0.264 R = 0.9979
Ip (n A )
30
20
10 Linear for both regions: AFB2 is

0 adsorped at mercury electrode.
0 50 100 150 200 250 300 350 400 450 500 550
Î Reduction and adsorption
V (mv/s)
processes at electrode are
influenced by the rate of
reaction which prefers the slow
reaction.

z AFB2 reduction
reaction: 1. CV produces only
cathodic peak.
: Irreversible
2. Graph Ep vs log V: linear
: Diffusion Cathodic Stripping Volltammetric is selected

current is 1. graph log Ep vs log V: linear,
influenced by m = >0.5
adsorption
process with 2. graph Ip vs V: linear with
higher m at slow V compare
prefer the slow to the m at fast V
reaction 3. I increases with repeatitive
cathodic scanning.
p

(1)
z Differential Pulse Cathodic
Stripping Voltammetric -1256mV, 5nA
(DPCSV) technique
(1)Scan [AFB2] = 1.0 nM
Ei = 0, Ef = -1500 mV, Eacc =0 mV
T acc = 0, v = 50 mv/s, pulse
amplitude: 100 mV (in BRB pH
=9.0)
(2)Scan [AFB2] = 1.0 nM -1244mV, 25nA

Ei = 0, Ef = -1500 mV, Eacc =0 mV
T acc = 30s, v = 50 mv/s, pulse
amplitude: 100 mV (In BRB pH = (2)
9.0)
Accumulation time greatly effect on
peak height (Ip) of AFB2

z Study effect of pH of supporting electrolyte ( BRB )

Aims:
a) To study the pH of BRB that gives maximum Ip of AFB2
without giving any extra peak.
b) To study either hidrogen ion is greatly involved in the

reduction reaction of AFB2 or not.
[AFB2] = 2.0 uM, BRB with pH = 2.0 to 13.0, use 0.1M HCl
for pH = 1.0
Method: DPCSV
Parameters:
Ei = 0, Ef = -1500 mV, Eacc = 0, t acc = 15 s, v = 50 mV/s
[AFB2] = 2.0 uM, BRB with pH = 2.0 to 13.0, 0.1 M HCl for pH=1.0
Method: DPCSV
Parameters:
Ei = 0, Ef = -1500 mV, Eacc = 0, t acc = 15s, v = 50 mV/s
pH =9.0, optimum pH
Î Ip = 48 nA, Ep = -1192 mV
60
50
40
pH < 5.0, > 12.0 : no AFB2 peak
Ip (nA)
30
20
10 pH: 5.0 – 9.0: Ip increased
0
4 6 8 10 12
pH: 9.0 – 12.0: Ip decreased
pH
The reaction is effected by pH of

=Î proton ion is greatly involve in
BRb pH 9.0 is selected
reduction reaction of AFB2.
z Voltammograms of AFB2 with changing pH
pH = 4.0
pH = 7.0
No peak
( - 1168 mV, 42 nA)

z Voltammograms of AFB2 with changing pH
Interfered by blank
( BRb pH = 9.0 )
pH = 9.0 ( -1192 mV, 48 nA) pH = 11.0 (-1193 mV, 32 nA)

z Effect of pH of BRB on Ep of AFB2

1250 pH: 5.0 – 8.0: linear:
R = 0.9777
E p (-m V )
1200
pH: 9.0 – 12.0: nearly constant
1150
1100
Ep shifted towards more negative
4 6 8 10 12 14
direction
pH
=Î At or before optimum pH (9.0),

reaction is effected by H+ ion

z Parameters optimisation:
z Scan rate (v)
z Accumulation potential ( Eacc)
z Accumulation time (tacc)
z Scanning potential window( Ei and Ef)
z Pulse amplitude

z Parameters optimisation steps
Using two different concentrations of AFB2
(1) High: (2) Low:
[AFB2] = 2.0 uM [AFB2] = 0.06 uM
Optimum parameters for determination

of AFB2 using DPCSV technique

Initial parameters:
Ei = 0,Ef = -1500 mV,Eacc = 0, tacc = 15 s, V = 20 mv/s and
P.A = 100 mV. [AFB2] = 2.0 uM
Effect of Hg oxidation
-1240 mV , 29 nA
H2 overpotential
Interfered by blank
( BRb pH 9.0 )

z Effect of scan rate (v)

Optimum V : 40 mv/s
50
40 Ip = 36 nA, Ep = -1256 mV
Ip (nA)
30
20
Initial Ip : 29 nA
10
0
0 20 40 60 80 100 120 1) At higher v, Ip decreased due to
Scan rate (mV/s) uncomplete reaction.
1320
2) Higher v caused Ep linearly

Ep (-mV)
1290
1260
( R2 = 0.9868) shifted to more
1230
0 30 60 90 120
negative direction.
Scan rate (mV/s)
reduction reaction of AFB2 at

V = 40 mV/s mercury electrode prefered slow
reaction

z Effect of accumulation time (tacc)
Optimum tacc : 40s

60
Ip = 45 nA, Ep = -1256 mV
40 Initial Ip : 36 nA
Ip (n A )
20
Tacc > 40s: Ip decreased due to
0 the formation of saturated
0 20 40 60 layer at mercury electrode.
2. Amount of analyte (AFB2)
Accumulation time (s)
in the bulk solution is lower
due to the adsorption process
at mercury electrode

z Effect of accumulation potential(Eacc)

Optimum Eacc: -800 mv
Ip = 51nA, Ep = -1248 mV
40
Ip (nA )
20
When:
0 Eacc = Ei ( 0 mV): Ip lower
0 400 800 1200 1600 Eacc = Ef ( -1500 mV): no peak
Accumulation potential (-mV) was observed
At Eacc = -800 mV, AFB2 maximum adsorped at mercury electrode

z Effect of initial potential

Optimum Ei: -1000 mv
Ip = 53 nA, Ep = -1256 mV
60
50
Scanning from Ei =-1000 mV increasing
Ip (n A )
40
30 Ip of AFB2 because it can minimised
20
10 the effect of blank (which give a peak
0 around -950mV)
0 300 600 900 1200
Ei (-mV) Ei at -1000 mV will save scanning time
Ei = -1000 mV and Ef = -1500 mV is selected as an opimum

potential window.

z Effect of pulse amplitude (P.A)

Optimum P.A: 100 mv
Ip = 53 nA, Ep = -1256 mV
80 Initial Ip : 53 nA (same P.A)
60
Ip (nA )
40
Not selected eventhough Ip
increased because:
20
0
a) Very small increment of Ip, only
0 30 60 90 120 150
1nA ( 54nA)
Pulse amplitude (mV)
b) Peak broader and produces

higher noise level
P.A =100 mV is selected as the optimum P.A

Initial parameters
2.0 µM AFB2 Ei = 0, Ef = -1500 mV, Eacc = 0, tacc = 15 s,

in BRB pH 9.0 scan rate = 20 mv/s and P.A = 100 mV
Ip = 29 nA, Ep = -1240 mV
Ei = -1000 mV, Ef = -1500 mV,

Eacc =-800 mV, tacc = 40 s, Optimum parameters
Scan rate = 40 mV/s and
P.A = 100 mV,
Ip = 53 nA (↑83%) , Ep = -1208 mV

Using lower concentration of AFB2
Using previous optimum parameters

Ei = -1000 mV, Ef = -1500 mV,
0.06µM AFB2
Eacc =-800 mV, tacc = 40 s,
In BRB pH 9.0
s.rate = 40 mV/s and P.A = 100 mV
13.9nA, -1232 mV
Reoptimisation steps
Obtained new optimum
parameters
Ei = -1000 mV, Ef = -1400 mV, 38.18 nA, ( ↑200%), -1220mV
( Final optimum parameters)

Before optimised: -1232 mV, 13.90 nA

Ei = -1000 mV, Ef = -1500 mV,
-1220 mV, 38.18 nA
After optimised:
Ei = -1000 mV, Ef = -1400 mV,
Optimum parameters for

determination of AFB2
[AFB2] = 0.06 µM

z Preparation of calibration curve ( AFB2 in BRB pH 9.0)
200
150
ip (nA)
100
y = 5.55x + 3.6435
50 R2 = 0.9978
0
0 10 20 30 40
[AFB2] X10-8M
Ei = -1000 mV, Ef = -1400 mV,

Linear range: 0.02 – 0.32 µM
s.rate = 50 mV/s and P.A = 80 mV LOD: 0.0159 µM ( 5.0 ppb)
Sensitivity: 0.555 µA / µM
Ep AFB2 (0.1 uM) = -1230 mV

z From calibration curve:

1) Precision :
Determine the AFB2 with concentrations of (1) 0.06 µM and (2)

0.10 µM. Calculate relative standard deviation (RSD) for each
determination.
( repeatibility ) ( RSD < 5%)
2) Accuracy:
Determine the recovery by scanning a known amount of AFB2

and calculate the actual amount of AFB2 using regression
equation which was obtained from the calibration curve. The
obtained value is compared with amount of spiked AFB2.
(recovery)
z Precision:
(2)
(1)
[AFB2] =0.06 µM, n = 8, RSD = 2.80% [AFB2] =0.20 µM, n = 8, RSD = 2.53%
RSD = < 5%: Proposed method is high precision

z Precision: (inter-day)
[AFB2] = 0.1 uM Day 2
Day 1
Day 3
Day 1: 72.70 +/- 0.521 ( 0.72% ) , n = 8
Day 2: 72.22 +/- 0.533 ( 0.74% ) ,,
Day 3: 72.91 +/- 0.706 ( 0.97% ) ,,
Average: 72.61 +/- 0.587 ( 0.81% ) ,,
High precision for intra and inter day measurements

z Accuracy (recovery) n = 3
No of Amount Peak Amount Recovery (%)
experiment added current found (RSD)
(x 10-8M) (nA) (x 10-8M)
51.70 8.66
1 10.0 52.77 8.84 86.83 +/- 1.46
51.03 8.55 ( 1.68%)
134.4 23.56
2 25.0 133.3 23.36 94.26 +/- 0.81
135.6 23.77 (0.86%)

z Application to determine AFG2, AFB1 and AFG1
AFG2, AFB1 and AFG1 :

CV Î In BRB pH 9.0, all aflatoxins are reduced at mercury electrode
produced single cathodic peak and the reactions are irreversible. Diffusion
currents are effected by adsorption process at electrode surface
=AFB2
DPCSV Î used the same optimum parameters as used for AFB2 to

determine AFG2, AFB1 dan AFG1

Voltammograms of AFB1 (A), AFB2 (B), AFG1 (C) and AFG2 (D)
obtained from individual measurement at concentration of 0.1 uM
in BRB pH 9.0
Ei =-1000 mV ( except for AFG1 = -950 mV), Ef = -1400 mV,

Eacc = -600 mV, t acc = 80 s, v = 50 mV/s and p.amplitude = 80 mV
(A) AFB1 = -1220 mV ( 62.57 nA ), (B) AFB2 = -1230 mV ( 63.79 nA )

(C) AFG1 = -1150 mV ( 62.29 nA ), (D) AFG2 = -1170 mV ( 63.14 nA )
Saturation effect at electrode surface

z AFG2:
200
300
250 150
200
Ip (nA)
Ip (nA)
100
150
y = 6.3464x + 9.8329
100 R2 = 0.999
50
50
0 0
0 5 10 15 20 25 30 35
0 10 20 30 40 50 60 70 80 90
[AFB2]X10-8M
[AFG2] / X10-8M
y = 6.346x + 9.8329 ( R =0.9990)

Linear range: 0.024 uM – 0.288 uM
LOD: 0.0160 uM (5.28 ppb)
Sensitivity: 0.635 uA/uM
Ei = -1000 mV, Ef = -1400 mV,

Ep= -1170mV
z AFB1 250
300
200
Ip (nA)
250 150
200 100
Ip (n A )
y = 4.4296x + 3.0034
150 50 2
R = 0.9972
100
0
50
0 10 20 30 40 50
0
0 10 20 30 40 50 60 70 80 90 100 [AFB1]X10-8M
[AFB1]X10-8M
Y = 4.4296x + 3.0034 ( R =0.9972)
Linear range : 0.0321 uM – 0.5778 uM
LOD: 0.0161 uM ( 5.00 ppb)
Parameters for AFB1 = AFG2 = AFB2
Ep = -1220 mV

z AFG1 160
140
200 120
175
100
Ip (n A)
150
125 80
Ip (nA)
100 60
75 40 y = 5.5958x + 3.7725
50 20 R2 = 0.998
25
0
0
0 5 10 15 20 25 30
0 10 20 30 40 50 60
[AFG1] X 10-8M [AFG1]X10-8M
y = 5.5958x + 3.7725
Linear range: 0.02 uM – 0.260 uM
LOD: 0.0150 uM ( 4.92 ppb)
Ei = -950 mV, Ef = -1400 mV,
s.rate = 50 mV/s and P.A = 80 mV Ep= -1150mV

z Voltammograms of AFG1
160
140
120
100
Ip (n A)
80
60
40 y = 5.5958x + 3.7725
20 R2 = 0.998
0
0 5 10 15 20 25 30
[AFG1]X10-8M
Ei = -950 mV, Ef = -1400 mV, y = 5.5958x + 3.7725

Eacc =-600 mV, tacc = 80 s, R = 0.9980
Ep AFG1 (0.1 uM) = -1150 mV

z
Aflatoxin AFB1,
E (mV)AFB2,
* AFG1 dan
Y = mx + c AFG2: Sensitivity LOD Linear range R2
p
(y = nA, x = x 10-8M, (uA/uM) ( x 10-8M)/ (x 10-8M) /
c = nA) ppb ppb
AFB1 -1220 Y = 4.4296x + 3.0034 0.443 1.606/ 3.00 – 45.00/ 0.9972

5.00 10 - 150
AFB2 -1230 Y =5.5500x + 3.6435 0.555 1.580/ 2.00 – 32.00/ 0.9976

5.00 6 - 100
AFG1 -1150 Y = 5.5958x + 3.7725 0.560 1.500/ 2.00 – 26.00/ 0.9980

4.92 6 - 90
AFG2 -1170 Y = 6.3464x + 9.8329 0.635 1.600/ 2.00 – 29.00/ 0.9990

5.28 6 -96
* [aflatoxins] = 0.1 uM

CONCLUSION
z From the CV study, AFB1, AFB2, AFG1

and AFG2 have reduced at a mercury
electrode which produced diffusion
current that controlled by adsorption
process at electrode surface. The
reduction reaction was totally irreversible
and prefered the slow reaction.

CONCLUSION…cont
z From the DPCSV, in BRB pH 9.0, AFB1,

AFB2, AFG1, AFG2 have reduced at
peak potentials of -1220 mV, -1230 mV, -
1150 mV and -1170 mV ( versus
Ag/AgCl) respectively.
z The proposed method provides high
precision and accuracy for determination
of AFB2.

PROBLEMS
1. Blank BRB pH 9.0 gives a peak ( around -950 mV to -1100 mV)

with unconsistent Ip (15 nA – 40 nA)
(a) (c)
(b)
Ei = 0, Ef = -1500 mv Ei = 0, Ef = -1500 mv Ei = -1000 mv, Ef = -1400 mv

Eacc = 0 tacc = 20 s Eacc = 0 tacc = 20 s Eacc = -600 mv tacc = 80 s

PROBLEMS…cont
2. Initial analysis of mixing aflatoxins sample:

: AFB2 + AFG2 Î 1 overlap peak
: AFB1+ AFB2 Î 1 overlap peak
3. Initial analysis of real sample (uncontaminated
green nut powder ):
The sample supress AFB2 standard peak
almost 50%. ( recovery: 50%)

FUTURE WORKS
z Further study on suitable technique to

reduce LOD / enhance sensitivity.
z Analysis of accuracy ( recovery) at inter-
day for AFB2.
z Analysis of precision and accuracy at
intra-day and inter-day for AFG2, AFB1
dan AFG1

FUTURE WORKS…cont
ÎComplexing of aflatoxin for the

selectivity of analysis.
ÎStudy interference effect;
1. Analysis the mixing of aflatoxins
sample.
2. Standard aflatoxin solution will be

mixed with other ketone compounds.
FUTURE WORKS…cont
z Study the stability of aflatoxins ( shelf life

study) and the effect of exposure time to the
aflatoxins peak current ( aflatoxins will be left
in voltammetric cell subject to the laboratory
conditions for 8 hrs and measurement will be
carried out for every hour)
z Study other type of working electrodes such as

carbon paste and screen printed electrodes.
z Robustness test of the proposed method.

FUTURE WORKS…cont
z Analysis an artificially and real

contaminated samples.
z Compare the results with that obtained

using accepted technique such as
HPLC-FD.

FUTURE WORKS…cont
Activity 2003
1) Determination of
AFB1, AFB2, AFG1
and AFG2
1a) Prepare
calibration curve finished
1b) Determine
analytical parameters
2) Interference study
3) Complexing of
aflatoxins study

FUTURE WORKS…cont
Activity 2004
1) Determination of mixing
aflatoxins
in artificially contaminated
samples
in real samples
4)Determination by HPLC
5) Study using different

working electrodes
5) Comparison study
Activity 2005
1) Analysis of contaminated
food samples

WHAT EXPERTS SAID ABOUT VOLTAMMETRY?
Prof Barek (2001):
“Polarographic and voltammetric methods can play a useful role

in the analysis as an alternative to chromatographic and
spectrophotometric techniques, however, their relationship to
other methods is complimentry rather than competitive”
Prof Wang (1999):

“Stripping analysis to the 21th century: faster, smaller,
cheaper, simpler and better”.
Hopefully this will come true!!!
Jaroslav Heyrovsky (1890-1967)

ACKNOWLEDGEMENT
SUPERVISOR:
AP DR ABDUL RAHIM MOHD YUSOFF
CO-SUPERVISORS:
1. PROF RAHMALAN AHAMAD

2. DR ABD. RAHIM YAACOB
STAFF OF CHEMISTRY DEPT, UTM

CHEM. DEPT, MOSTE ( EN.RODWAN )
ELECTROANALYSIS GROUP,
CHEM. DEPT, UTM
USM ( STUDY LEAVE UNDER

ASHES)

THANK YOU
Wassalam
Mohd Hadzri,
Hadzri,
PPSains Kesihatan
20 Ogos 2003/
22 Rejab 1424H



LOD DETERMINATION

LOD of an analyte is the concentration
which gives an instrumental signal (y)
significantly different from the blank or
background signal
( Miller J. C. and Miller J. N. 1993)

LOD DETERMINATION…cont
z By Barek’s and Miller’s methods

z Barek’s method: a) experiment
b) graph
1a) Experiment:
By standard addition of lower
concentration of analyte until obtaining
the sample response that is significantly
difference from blank signal
z 1b)By graph: The limit of determination

was calculated as the tenfold standard
deviation from seven analyte
determinations at the concentration
corresponding to the lowest point on
the appropriate calibration straight line
z Miller’s method: By graph; 3SD/b

By Barek’s Method By Miller’s

Aflatoxin Method
Experiment Graph Graph; 3SD/b
x 10-8 M ppb x 10-8 M ppb x 10-8 M ppb
AFB1 1.606 5.00 2.160 6.70 45.36 141.5
AFB2 1.580 5.00 1.837 5.77 28.59 89.0
AFG1 1.500 4.92 2.050 6.72 23.88 78.33
AFG2 1.600 5.28 1.345 4.44 27.47 90.67

BLANK PEAK PROBLEM
z Attempt to illiminate;
1. Used dionised water from different lab;
(Analytical, biological, FKKKSA,
Biotechnology) and R.O water from
personal used….failed

BLANK PEAK PROBLEM…cont
2. Add 1.0 M HCl … failed

3. Add 1.0 M EDTA…failed
4. Used different voltammetry model
(Metrohm) peak still existed

STABILITY STUDY OF AFLATOXINS
z AFB1, hour to hour stability study
Stability study of AFB1 in BRb pH=9.0

Ip = 58.53 +/- 0.62 nA
60
[AFB1] = 10 x 10-8M ( 1.06%)
58
56
Ip (n A )
54
52
50
0 1 2 3 4 5 6 7 8
Standing time (hrs)

STABILITY STUDY OF AFLATOXINS…cont
z AFB2, hour to hour stability study
Stability study of AFB2 in BRB pH 9.0

Ip = 72.74 +/- 0.94
73.5
[AFB2] =12 x 10-8M
( 1.29 % )
73
72.5
Ip (nA)
72
71.5
71
70.5
70
1 2 3 4 5 6 7 8 9
Standing time (hrs)

CV OF AFB1
z Cyclic voltammogram of AFB1 in BRB pH 9.0,

scan rate 200 mV/s
Ip = 50 nA, Ep = -1305 mV

CV OF AFG1
z Cyclic voltammogram of AFG1 in BRB pH 9.0,

scan rate 200 mV/s
Ip = 48 nA, Ep = -1245 mV

CV OF AFG2
z Cyclic voltammogram of AFG2 in BRB pH 9.0,

scan rate 200 mV/s
Ip = 52 nA, Ep= -1254 mV

VOLTAMMOGRAMS OF AFB2
z DPSV voltammograms of AFB2 in BRB pH

9.0 (blank), scan rate 50 mV/s
AFB2 ( 2.0 uM )
Blank, BRB pH 9.0

VOLTAMMOGRAMS OF STD AFB2 AND REAL SAMPLE
AFB2, 0.07 uM
( 35.83 nA, -1240 mV
AFB2 + REAL SAMPLE

( 16.90 nA, -1245 mV)
BLANK
USED OPTIMISED PARAMETERS FOR AFB2
REAL SAMPLE: EXTRACT FROM GREEN NUT POWDER

VOLTAMMOGRAM OF MIXED AFLATOXINS
z AFG2 + AFB2
0.1 uM AFG2 ( before mix)

( 64 nA, -1170 mV)
+ 0.1 uM AFB2 ( 61.5 nA, -1170mV)
+ 0.2 uM AFB2 (60.8 nA, -1200 mV)

VOLTAMMOGRAM OF MIXED AFLATOXINS…cont
z AFG2 + AFB2
+ 0.3 uM AFB2 + 0.4 uM AFB2

( 76 nA, -1210) ( 79 nA, -1210 mV)

VOLTAMMOGRAM OF MIXED AFLATOXINS…cont
0.1 uM AFG2 ( before mixed): -1170 mV, 64.0 nA

+ 0.05 uM AFB2: -1170 mV, 62.7 nA
+ 0.10 uM AFB2: -1170 mV, 61.5 nA
+ 0.15 uM AFB2: -1170 mV, 58.2 nA
+ 0.20 uM AFB2: -1200 mV, 60.8 nA
+ 0.25 uM AFB2: -1210 mV, 69.9 nA
+ 0.30 uM AFB2: -1210 mV, 76.0 nA
+ 0.35 uM AFB2: -1210 mV, 79.0 nA
+ 0.40 uM AFB2: -1210 mV, 79.0 nA


WILL BE
PRESENTED FOR
PhD VIVA
PRESENTATION
3/1/2007 PhD viva presentation

STRIPPING VOLTAMMETRIC
METHOD TO DETERMINE
AFLATOXIN COMPOUNDS
BY
MOHAMAD HADZRI YAACOB
PS 023001
INTRODUCTION
Mycotoxin group

Aspergillus flavus
AFB1, AFB2, AFG1, AFG2 Contaminated

corn
AFM1 and AFM2
INTRODUCTION…cont
Chemical structures;
O O
O O
AFB1 AFB2
O O
O O O O
O O
O O O O
AFG1 AFG2
O O O O
O O
O O
O O

OBJECTIVE
• To study electroanalytical properties of aflatoxin
compounds (AFB1, AFB2, AFG1 and AFG2) at
the working electrode.
• To develop a new alternative technique for high

sensitive determination of aflatoxin compounds

SCOPE OF STUDY
• Studies on the voltammetric behaviour of
aflatoxins using cyclic voltammetric (CV)
technique.
• Studies on the differential pulse cathodic and
anodic stripping voltammetry of all aflatoxins
and parameters optimisation
• Studies on the effect of increasing concentration
of the aflatoxins to their peak height

• Interference studies by metal ions and organic
compounds
• Studies on the square wave stripping voltammetry
technique for the determination of aflatoxin
compounds
• Application of the proposed methods for the
determination of aflatoxin compounds in real samples.
The results were compared with HPLC results
• Stability studies of aflatoxin solutions

• By CV technique;
30
¾ Britton Robinson 25
Buffer (BRB) at pH 9.0 20
I p (n A )
was the best supporting 15
10
electrolyte 5
0
5 6 7 8 9 10 11 12 13
For 0.6 µM AFB1; pH of BRB
pH = 9.0, Ip = 24.5 nA

• Suggested mechanism (Smyth et al.,1979)
O O
O O
O
O
2e -
2 2
OCH 3
OCH 3
2 H 2O
Dimer + 2 OH -

¾ All aflatoxin compounds are undergo irreversible

reduction reaction at mercury electrode
For 1.3 µM AFB2;

Ip = 30 nA
Ep = -1.315 V (Ag/AgCl)

¾ All aflatoxin compounds adsorb on the surface of the mercury

electrode.
60
50
40
I p (n A )
30
20
10
0
1 2 3 4 5
No of cycle
Repetitive CV; Ip increased with the number of cycle

• From DPCSV experiment

¾ Optimised parameters:
Ei = -1.0 V (except for AFG1; -0.95 V), Ef = -1.4 V, Eacc
= -0.6 V, tacc = 80 s, scan rate = 50 mV/s and pulse
amplitude = 80 mV.
0.06
0.06µM
µMAFB2 0.06 µM AFB2 in BRB at
AFB2 pH 9.0
(a) Unoptimised
(b)Optimised
¾ Method validation
AFB1 AFB2 AFG1 AFG2

LOD/ppb 1.56 2.50 3.28 2.50
LOQ/ppb 5.20 8.33 10.93 8.33
Linearity 6 – 100 6 – 100 6 – 105 6 – 105

Range/ ppb
R2 0.9989 0.9980 0.9992 0.9989

¾Method validation
Accuracy:
% Recovery > 95% Î High accuracy
Precision:
%RSD for n = 8 < 3.0% Î High precsion

Robustness;
Small influences of important conditions (pH of BRB,
Eacc and tacc ) were not significantly affect the peak
height Î Method is robust
Ruggedess;
No significant different in the peak heights obtained by
using two different voltammetric analysers (BAS vs
Metrohm) Î Method is rugged

• Interference studies;
Metal ions (Al, Cu, Pb, Ni and Zn) and organic
compounds (ascorbic acid, β-cyclodextrin and L-
cysteine) were not interfere up to 10 times
concentration as compared with aflatoxin
compounds

Using SWSV technique

• For all aflatoxins, the peak heights were
increased > 10 times Î More sensitive
• Analysis can be completed within a few second
Î Faster technique
• LOD and LOQ lower than DPCSV technique
Î More sensitive

• Voltammograms of aflatoxins (DPCSV vs SWSV)
AFB1 AFB2 AFG1 AFG2
Text:
3/1/2007 page no - 191 PhD viva presentation
• From stability studies;
10 ppm aflatoxin stock solutions (in benzene:acetonitrile,

98%) stored in the dark and cool condition: stable up to 1
year.
1 ppm aflatoxin standard solutions (in BRB pH 9.0 ) stored

in the dark and cool conditions: stable up to 3 months for
AFB1 and AFB2 while for AFG1 and AFG2 stable up to 2
months..

1 ppm aflatoxins in BRB pH 9.0 exposed to

normal laboratory condition; AFB1 and AFB2
were stable up to 8 hrs but for AFG1 and AFG2
were stable up to 3 hrs.
Aflatoxin prepared in basic condition were less

stable as compared to those prepared in acidic
and neutral media.

• Analysis real sample (groundnuts)
Followed extraction and clean-up procedure as applied by

Chem. Dept. MOSTI.
Recovery studies: Aflatoxins standard solutions were

spiked into the groundnut eluates;
80-99% for 1st day analysis, 80 -90% for 2nd day and 75-
85% for 3rd day analysis.
• Analysis of 15 samples by DPCSV, SWSV and HPLC (result: total
aflatoxins in ppb level)
Sample DPCSV SWSV HPLC Notes
S01 12.73 13.02 14.34 -ve
S02 7.12 8.36 8.83 -ve
S03 n.d n.d 0.50 -ve
S04 21.46 29.72 19.08 +ve

S05 9.90 8.95 5.34 -ve
S06 n.d n.d n.d -ve
S07 5.90 8.17 3.67 -ve
S08 n.d n.d n.d -ve

S09 n.d n.d 1.84 -ve
S10 31.93 35.63 36.00 +ve
S11 n.d n.d 0.42 -ve
S12 n.d n.d 1.75 -ve

S13 10.76 9.21 8.25 -ve
S14 n.d n.d n.d -ve
S15 n.d n.d n.d -ve
+ve : > 15 ppb ( 2 out of 15 samples: 13%)

CONCLUSIONS
The study has achieved the objective;
A sensitive, accurate, robust, rugged, fast and

low cost technique was successfully developed
and applied for the determination of aflatoxins
in groundnut samples
The hypothesis regarding electroanalytical

properties of aflatoxins was also proven.
SUGGESTIONS
• Use other types of the working electrode (CPE,
GCE or BFE)
• Study the reagents that can be complexed with
aflatoxins
• Study the effect of storage time and conditions
of groundnuts to the level of aflatoxins
• Analyse aflatoxins in other type of samples (rice,
bread, fish, vegetables and cigarretes tobacco)
using the proposed technique.

ACKNOWLEDGEMENT
• Supervisors: AP Dr Abdull Rahim Hj. Mohd.
Yusoff and Prof Rahmalan Ahamad
• USM: study leave and fellowship
• UTM: Short term grant (Vot No: 75152/2004)
• Chemistry Dept staff

ORAL PRESENTED AT
KUSTEM 3rd ANNUAL
SEMINAR ON
SUSTAINABLE SCIENCE
AND MANAGEMENT
(TERENGGANU; 4 - 5th
MAY 2004)
CYCLIC VOLTAMMETRIC STUDY OF
AFLATOXIN G1 (AFG1) AT THE MERCURY
ELECTRODE
BY
Mohd Hadzri Yaacob, Abd.Rahim Yusoff and

Rahmalan Ahamad
UTM, SKUDAI
• INTRODUCTION AFLATOXINS
• OBJECTIVE
• EXPERIMENTAL
• RESULTS AND
DISCUSSION
• CONCLUSION
• ACKNOWLEDGEMENT
INTRODUCTION
• AFLATOXINS:
Mycotoxin group.
Aspergillus flavus Aspergillus parasiticus
Types of aflatoxins: Raw peanuts, peanuts products, wheat,

AFB1, AFB2, AFG1 maize, corn, vegetable oils, dried fruites,
and AFG2: barley, pear, cocoa, coffee, herbs, spices,
wine, cottonseeds.
Cow milk and its products, eggs

AFM1 and AFM2 and meat product
• AFLATOXINS:
Occurs naturally in commodities used for animal and
human
Dangerous to health: causes aflatoxicosis disease.

AFLATOXIN G1 ( AFG1):
Chemical and physical properties
O O
C17H12O7
O O
O MW = 328
O
O
Chemical name: 3,4,7a, 10a-tetrahydro-5-methoxy-1H,

12H furo[3’,2’:4,5]furo[2,3-h]pyrano[3,4-c][l]-benzopyran-
1,12-dione
Yellowish crystal.
Odorless, tasteless and colorless solution ( in organic
solvent).
Chemical and physical properties…cont
Solubility: Dissolve in methanol, water:acetonitrile (9:1),

trifluoroactic acid, DMSO and acetone. In water: 10-20 mg /
liter
m.p; 244 – 246 0 C
Produces green color under UV light
UV abs (max) in methanol: 216, 242, 265 and 362 nm
IR spectrum ( in chloroform): 1760, 1695, 1630 and 1595

cm-1
Stability of AFG1
In crystal form: extremely stable in the absence of

light or UV radiation, even at T = > 1000C
Solution in water, DMSO, 95% acetone or ethanol

stable for 24 hrs under normal condition. Stable for
years if kept in cool and dark place
Stable in most food process; not destroy by normal

cooking process since it heat stable
ELECTROCHEMICAL PROPERTIES OF AFG1
Based on chemical structure:
Ketone groups in lactone ring can be reduced and

produce cathodic wave.
Unsaturated terminal furan ring can be reduced

and produce cathodic wave.
O O
Can forms
O O
anionic
radical
O
O
WHAT AND WHY CYLIC VOLTAMMETRY (CV)?
CV: Voltammetric technique that consist of scanning

linearly the potential of a stationary working electrode
( forward and reverse ) and the current flowing through
the cell is measured as a function of that potential.
Three parameters needed to be characteristiced; starting

potential of the scan, finishing / switching potential and
scan rate ( rate of change of potential with the time)
A plot of current as a function of applied potential is

called a voltammogram ( as in figure below)
Typical cyclic voltammogram for a reversible redox
process
O = oxidised form R = reduced form
Why CV?
1. To aquire qualitative information about
electrochemical reactions
2. Ability to rapidly provide information on;
@ thermodynamic of redox process
@ the kinetic of heterogeneous electron-transfer
@ coupled chemical reactions
@ adsorption processes at electrode surface
3. Offer a rapid location of redox potentials of the
electroactive species and convenient for evaluation
of the effect of media upon the redox process
Î Initial experiment performed in electroanalytical
study
OBJECTIVE
1) To get information about electrochemical

properties of aflatoxin G1 (AFG1) on the mercury
electrode.
2) From this information, other voltammetric
technique can be developed ( such as differential
pulse stripping voltammetric ) for quantitatively
determination of AFG1 ( AFG1 standard or AFG1
in real samples)
BAS C50W BAS CGME consist of

Voltammetric Ag/AgCl as RE, Pt wire as
Analyser AE, CGME as WE, 20 ml
cell and magnetic stirrer.
Reagents: AFG1 ( MERCK); 10 ppm in benzene:
acetonitrile (98%), 1 ppm: prepared in BRb pH 9.0.
EXPERIMENT
A) Cathodic and anodic scan of AFG1
1. 10 ml of supporting electrolyte (BRb pH = 9.0 ) in voltammetric

cell was degassed for at least 10 min.
2. Scanned using CV technique; Ei = 0 mV, E high = 0 mV, E low = -
1500 mV, scan rate = 200 mV/s to obtain voltammogram of blank.
3. The AFG1 standard solution was spiked into the cell (
concentration of AFG1 in the cell = 1.5 uM), was degassed for at
least 3 min and scanned follows the same procedure as 2.0 to
obtain voltammogram of AFG1 in BRb solution.
4. Experiment no 2 and 3 were repeated with different parameters (
Ei = -1500 mV, E high = 0 mV and E low = -1500 mV ). Other
parameters remain unchanged.
EXPERIMENT…Cont
B) Repeatitive scan
Repeatitive scan ( n = 5 ) for cathodic direction was
performed and the peak height of AFG1 was observed
C) Standard Addition of AFG1
Using the same parameters as no 2, the concentration of
AFG1 was added ( 1.5, 2.25 , 3.0, 3.75 and 4.5 M). The
peak current and peak potential of AFG1 was observed.
D) Effect of scan rate
The scan rate was changed ( 25, 50, 100, 150, 200, 250, 300,
400, 500, 700 mV/s ) while other parameters remain
unaltered. The peak current and peak potential of AFG1
was observed due to the various applied scan rate.
EXPERIMENT…Cont
E) Effect of pH of BRb solution
The pH of supporting electrolyte (BRb solution ) was

adjusted from 3.0 to 13.0 ( using 1.0 M NaOH or 1.0
M HCl ) and the AFG1 standard solution was
scanned in various pH of BRB. The peak current and
peak potential of AFG1 was observed.
A) Cathodic and anodic scan of AFG1
Ip = 45.2 nA, Ep = -1245 mV
Ei = 0 mV, E high = 0 mV, E low = -1500

mV, s.rate = 200 mV/s, [AFG1] = 1.5 uM
in BRb pH =9.0
Ip = 58.5 nA, Ep = -1243 mV
Ei = -1500 mV, E high = 0 mV, E low

= -1500 mV, s.rate = 200 mV/s,
[AFG1] = 1.5 uM
Both scan directions gave only single cathodic peak ( no anodic

wave ) ÎAFG1 undergoes irreversible reduction reaction at
mercury electrode.
RESULTS AND DISCUSSION… cont
B) Repeatitive cathodic scan ( n = 5 or 10 segments)
80
60
Ip (n A )
40
20
0
1 2 3 4 5
No of cycle
Peak heights of AFG1 increased with the number of scans with

no extra peak neither anodic nor anodic peak was observed.
Peak potentials of AFG1 were – 1245 to -1220 mV.
=Î Progressive adsorptive accumulation of AFG1 at the
mercury electrode surface
C) Standard Addition of AFG1
150
120
90
Ip (nA)
60
y = 24.756x + 13.072
30
R2 = 0.9812
0
1 1.5 2 2.5 3 3.5 4 4.5 5
[AFG1] / uM
Ip of AFG1 increased and peak

potentials shifted toward less
Ei = 0 mV, E high = 0 mV, E low = -1500 negative direction with
mV, s.rate = 200 mV/s, [AFG1] = 1.5, 2.25, increasing of AFG1
3.0, 3.75 and 4.5 uM in BRB pH =9.0 concentration.
The peak potential at –1245 mV was confirmed due to the

reduction of AFG1 in BRB pH =9.0.
D) Effect of scan rate
i) Log peak height versus log scan rate, [AFG1] = 1.5 uM
2.1
1.9
1.7
1.5
Lop Ip
1.3
1.1 y = 0.5623x + 0.446
0.9 R2 = 0.9919
0.7
0.5
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8
log sca n ra te
Linear relationship was observed between log Ip versus log

scan rate with the slope of 0.56 ( > 0.5 ) =Î Diffusion current
is influenced by an adsorption of AFG1 on the electrochemical
process at mercury electrode surface.
ii) Peak potential versus log scan rate, [AFG1] = 1.5 uM
1275
1260
1245
Ep (-mV)
1230
1215 y = 48.484x + 1134.8
R2 = 0.9978
1200
1185
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8
log scan rate
Plot of peak potential versus log scan rate is a linear graph ( R2 = 0.9978)
=Î confirmed that the reduction of AFG1 on the electrode surface is
totally irreversibe.
iii) Peak height versus scan rate, [AFG1] = 1.5 uM
110
100
90
1) Slow scan rate:
80
70 Ip = 0.2298x + 14.164 ( R2 = 0.9757, n = 5)
Ip (nA)
60
50
40
30
20 2) Fast scan rate:
10
0
0 50 100 150 200 250 300 350 400 450 500 550
Ip = 0.1598x + 19.18 ( R2 = 0.9892, n = 5)
Scan rate ( m V/s )
Based on the slopes of the regression equations for two different

regions, the reduction and adsorption of AFG1 on the electrode
surface is governed by the speed of reaction which preferred slow
reaction.
E) Effect of different pH of BRb solution, [AFG1] = 1.5 uM
i) Effect on peak height
60
50 At pH = 9.0, highest peak
40
Ip (nA )
current was observed

30
20
10
0
2 3 4 5 6 7 8 9 10 11 12 ii) Effect on peak potential
pH
1270
1250
1230
Peak height and peak potential
Ep (-mV)
1210
of AFG1 are pH dependent 1190
except for pH 6,7, 8 and 9, peak 1170
1150
potential are unchanged at -1245 2 3 4 5 6 7 8 9 10 11 12
pH
mV
CONCLUSION
1. AFG1 is reduced at mercury electrode. The

reduction reaction is totally irreversible reaction
which gives maximum peak height in BRb at pH
of 9.0. The diffusion current produced by this
reaction is affected by adsorption process of
AFG1 at the mercury electrode surface which
prefer a slow reaction.
2. All information on electrochemical properties of
AFG1 are useful for development of other
voltammetric technique for determination of
AFG1 in standard solution and real samples.
ACKNOWLEDGEMENT
SUPERVISOR:
CO-SUPERVISORS:
2. AP DR ABD. RAHIM YAACOB
ELECTROANALYSIS GROUP AND

STAFF OF CHEMISTRY DEPT
SCHOOL OF HEALTH SCIENCES, USM ,

HEALTH CAMPUS, KUBANG KRIAN,
KELANTAN
THANK YOU
Proposed mechanisme of the reduction of AFG1 that
may take place at mercury electrode as reported by
Smyth et al. (1979);
O O
O
ΘO
O O
2e- O O
2H2O
2 O
O Dimer + 2OH-
O O O O
ORAL PRESENTED AT
SYMPOSIUM LIFE SCIENCE II
(USM PENANG; 31st MAC – 3rd
APRIL 2004)
3/1/2007 2nd Life PostCon USM Penang 1

STABILITY STUDY OF AFLATOXIN G1
(AFG1) USING DIFFERENTIAL PULSE
STRIPPING VOLTAMMETRIC TECHNIQUE
BY

Rahmalan Ahamad
UTM, SKUDAI

• INTRODUCTION AFLATOXINS
• OBJECTIVE
• EXPERIMENTAL
• RESULTS AND
DISCUSSION
• CONCLUSION
• ACKNOWLEDGEMENT

INTRODUCTION
• AFLATOXINS:
Mycotoxin group.

wine, cottonseeds.


• AFLATOXINS:
human

O O
C17H12O7
O O
O MW = 328
O
O
Chemical name: 3,4,7a, 10a-tetrahydro-5-methoxy-1H,

12H furo[3’,2’:4,5]furo[2,3-h]pyrano[3,4-c][l]-benzopyran-
1,12-dione
Yellowish crystal.
solvent).


trifluoroactic acid, DMSO and acetone. In water: 10-20 mg /
liter
m.p; 244 – 246 0 C
Produces green color under UV light
UV abs (max) in methanol: 216, 242, 265 and 362 nm
IR spectrum ( in chloroform): 1760, 1695, 1630 and 1595

cm-1
Stability of AFG1

Solution in water, DMSO, 95% acetone or ethanol


cooking process since it heat stable

Stability of AFG1… cont
In strong alkali: lactone ring- susceptible to

alkaline hydrolysis
In acid: acid catalytic addition of water across the

double bond of the furan ring.
O O
O O
O
O

OBJECTIVE
To study the stability of AFG1 in;

a) Britton-Robinson buffer (BRB) pH 9.0; AFG1
prepared in BRB pH 9.0, measured in same
buffer, exposed to normal condition ( 0 to 6
hrs).
b) BRB pH 9.0: kept in cool and dark place ( 0 to
6 months )
c) AFG1 prepared in BRB pH 9.0, measured in
BRB with different pH, expose to normal
condition ( 0 to 3 hrs).
Using Differential pulse cathodic stripping
3/1/2007 voltammetric2ndtechnique
Life PostCon USM (DPCSV)
Penang 10
VOLTAMMETRY:
From Voltage- Amperometry
-Measure amount of current at different voltage
-Electroanalytical technique : sensitive, high accuracy, low cost
and easy to use
-2 important parameters; peak height (Ip) and peak potential (Ep)
-result: voltammogram ( e.g; Differential Pulse Cathodic Stripping
Voltammogram of AFG1)
[AFG1] = 0.1 uM = 30 ppb in

BRB pH 9.0
Ep = -1150 mV
Ip = 55.45 nA
Analysis date: 26th Jan 2004


Reagents: AFG1 ( MERCK); 10 ppm in benzene:
acetonitrile (98%), 1 ppm: prepared in BRB pH 9.0. BRB
pH ( 3.0 to 13.0)
10 ML BRB SOLUTION NITROGEN GAS IS FLOWED IN AFLATOXIN G1

SPIKE INTO 20 ML
VOLTAMMETRIC CELL
SOLUTION IS
DEGASSED

BLANK SOLUTION
SCANNED AFLATOXIN
BLANK SAMPEL

RESULTS
a) 0.1 uM AFG1 in BRB pH =9.0, exposed to normal
condition
Stability study of AFG1 in BRB pH 9
80
60
Ip (n A )
40
20
0
0 1 2 3 4 5 6 7 8
Standing time (hrs)
0 to 3 hrs: Peak current: 60.26 to 57.7 nA

4 hrs: 43.67 nA ( 78%) AFG1 stable up to 3 hrs in
BRB pH 9.0, exposed to
6 hrs: 33.56 nA ( 56%)
normal condition
8 hrs:
3/1/2007 29.02 nA ( 45%)
2nd Life PostCon USM Penang 14
b) 0.1 uM AFG1 from 1 ppm AFG1 in BRB pH 9.0 kept in cool and
dark place, measured in BRB pH 9.0
60
50
40
Ip (nA)
30
20
10
0
0 1 2 3 4 5 6
Storage time (month)
(a) 0 (b) 1 m (c) 2 m (d) 3 m

(e) 4 m (f) 5 m (g) 6 m
AFG1 in BRB pH 9.0,
0 to 3 m : 57.50 – 39.50 nA ( 70%) kept in cool and dark
place Îstable up to 3
4 m : 36.5 nA (60%) 6 m ( 25%) m keeping time

c) 0.1 uM AFG1 ( from 1 ppm AFG1 in BRB pH 9.0)
measured in different pH of BRB and exposed to normal
condition
70
60
Stability order for
50
pH = 6.0
AFG1 in different pH of
BRB exposed to
Ip (nA)
40 pH = 7.0
30 pH = 9.0
20
pH = 11.0 normal condition up to
10
3 hrs;
0
0 1 2 3
Standing tim e (hr s ) pH 11.0 < 9.0 < 6.0 < 7.0
In BRB pH = 6.0, 7.0 : peak current increases with time

In BRB pH = 9.0: peak current slowly decreases with
time
In BRB pH = 11.0 peak current sharply decreases with
time
3/1/2007where after 2 hrs noPostCon
2nd Life peakUSM
was observed
Penang 16
CONCLUSION
1) AFG1 stable up to 3 hrs in BRB pH 9.0 when

exposed to normal condition.
2) AFG1 in BRB pH 9.0, stable up to 3 m when
kept in cool and dark place
3)Stability order for AFG1 in different pH of BRB
exposed to normal condition up to 3 hrs;
pH 11.0 < 9.0 < 6.0 < 7.0

FURTHER WORKS
1. Stability study of AFG1 using different

concentration of AFG1 ( 0.05 uM and 1.0 uM)
2. Stability study of AFG1, keep in room
temperature and expose to light from 0 to 6
months.
3. Stability study of other aflatoxins (AFB1, AFB2
and AFG2)

ACKNOWLEDGEMENT
SUPERVISOR:
CO-SUPERVISORS:

SCHOOL OF HEALTH SCIENCES, USM

( STUDY LEAVE AND FELLOWSHIP
UNDER ASHES: May 2002 – May 2005)
MR RODWAN MAT AIL, CHEM

DEPT, MOSTE, PENANG.
THANK YOU
Wassalam
Mohd Hadzri,
Hadzri,
Sains Forensik,
Forensik,
PPSains Kesihatan
USM KELANTAN

ORAL PRESENTED AT
SKAM-17
(KUANTAN, 24-26th
AUGUST 2004)
STABILITY STUDIES OF AFLATOXINS
USING DIFFERENTIAL PULSE CATHODIC
STRIPPING VOLTAMMETRIC (DPCSV)
TECHNIQUE
BY

Rahmalan Ahamad
UTM, SKUDAI
INTRODUCTION
AFLATOXINS
C=O groups Electroactive
Electroactive species can be

property of determined
aflatoxins using
voltammetric
technique
INTRODUCTION…cont
• AFLATOXINS:
Mycotoxin group.

wine, cottonseeds.

• AFLATOXINS:
human

AFLATOXIN:
O O
O O
AFB1 AFB2
O O
O O
O O
O
O
C17H12O6, MW = 312 C17H14O6 MW = 314

O O O O
AFG1 AFG2
O O O O
O O
O O
O O
C17H12O7 MW = 328 C17H14O7 MW = 330

AFLATOXIN:

trifluoroactic acid, DMSO and acetone. In water: 10-20
mg / liter
Yellowish crystal

solvent).
AFLATOXIN:
AFB1 AFB2 AFG1 AFG2
m.point 2690 C 2870C 2450C 2370C
UV abs (max) 360 362 362 362

in methanol/
nm
IR spectrum 1760, 1684 1760, 1684 1760, 1695 1760, 1695

( in chloroform) 1632, 1598 1632, 1598 1630, 1595 1630, 1595
/ cm-1 1562 1562
Stability of aflatoxins

Solution in water, DMSO, 95% acetone or ethanol:


cooking process since it heat stable.
OBJECTIVE
To study the stability of aflatoxins which were;

a) exposed to normal laboratory condition for 8 hours in
BRb pH=9.0.
b) exposed to normal laboratory condition for 3 hours in
BRb pH=6.0, 7.0, 9.0 and 11.0
c) stored under cool temperature for 6 months in BRb
pH =9.0
using DPCSV technique
( a small portion of my research study in developing
voltammetric technique for determination of aflatoxins)

Reagents: AFB1, B2, G1, G2 ( MERCK); 10 ppm in
benzene: acetonitrile (98%), 1 ppm: prepared in BRb at
required pH.
EXPERIMENT
A) General procedure for DPCSV analysis

1. 10 ml of supporting electrolyte (BRb pH = 9.0 ) in
voltammetric cell was degassed for at least 15 min.
2. Scanned using DPCSV technique; Ei = -1000 mV, Ef = -
1400 mV, Eacc = -600 mV, tacc = 80 s, scan rate = 50
mV/s, pulse amplitude = 80 mV and rest time = 10 s to
obtain voltammogram of blank ( for all aflatoxins
except for AFG1, Ei = -950 mV)
3. The aflatoxins standard solution was spiked into the cell
( concentration of aflatoxins in the cell = 0.1 uM),
redegassed for at least 2 min and rescanned follows the
same procedure as no 2.0 to obtain voltammograms of
aflatoxins in BRb solution.
EXPERIMENT…Cont
B) Procedures for stability studies

1) 8 hours exposed to normal condition
Each aflatoxin was scanned every hour from 0 to
8 hours. ( aflatoxin in BRb pH =9.0 solution
remained in the voltammetric cell).
2) 3 hours exposed to normal condition
Each aflatoxin was scanned every hour from 0 to
3 hours in different pH of BRb ( 6.0, 7.0, 9.0 and
11.0)
3) Shelf life of aflatoxins ( 1 ppm) stored in freezer
Each aflatoxin ( 0.1 uM) was scanned every
month from 1st to 6th month storage period in
BRb pH =9.0
A) Voltammograms of aflatoxins
AFB1 [AFB1] = 0.1 uM / 31.2 ppb

In BRb pH =9.0
blank Ep = -1210 mV
Ip = 63.19 +/- 1.64 nA
n=8 RSD = 2.60%
AFB2 [AFB2] = 0.1 uM / 31.5 ppb

In Brb pH 9.0
blank
Ep = -1230 mV
Ip = 65.67 +/- 1.28 nA
n=8 RSD = 1.95%

AFG1 [AFG1] = 0.1 uM / 32.8 ppb

In BRb pH 9.0
Ep = -1150 mV
blank
Ip = 60.76 +/- 0.99 nA
n=8 RSD = 1.63%
[AFG2] = 0.1 uM / 33.0 ppb

AFG2 In BRb pH 9.0
Ep = -1170 mV
blank Ip = 58.88 +/- 0.88 nA
n=8 RSD = 1.50%

( STABILITY STUDIES)
A) 8 hours exposed to normal laboratory condition
70
60
Peak height (nA)

50 AFB1
40 AFB2
30 AFG1
20 AFG2
10
0
0 1 2 3 4 5 6 7 8 9
Exposure time (hrs)
Peak currents of AFB1 and AFB2 remained unchanged up to 8

hours but for AFG1 and AFG2 peak currents started decrease
after 1st hour exposure time. After 3 hours it gradually
decreased with AFG2 gave lower peak current compared to
AFG1.
ÎUp to 8 hrs, AFB1=AFB2 > AFG1, AFG2. AFG1> AFG2
Voltammograms of aflatoxins at 0 to 8 hours in BRb pH 9.0.
0.1 uM AFB1 0.1 uM AFB2

Voltammograms of aflatoxins at 0 to 8 hours in BRb pH 9.0...cont
AFG1 = 0.1 uM AFG2 = 0.1 uM

(STABILITY STUDIES)
B) In different pH of BRb ( pH 6.0 and (7.0)
50 70
pH = 6.0 pH = 7.0
45
60
40
35 50
P eak h eig h t (n A)
P eak h eig h t (n A)
AFB1 AFB1
30 40
AFB2 AFB2
25
AFG1 30 AFG1
20
AFG2 AFG2
15 20
10
10
5
0 0
0 1 2 3 0 1 2 3
In BRb pH = 6.0 In BRb pH = 7.0
In pH 6.0 and 7.0, all aflatoxins are stable within

3 hours in the cell.
(STABILITY STUDIES)
B) In different pH of BRb ( pH 9.0 and 11.0)

Stability of aflatoxins in BRB pH 9 Stability of aflatoxins in BRB pH 11
70 45
60 40
35
50
AFB1 30 AFB1
40
Ip (nA)
Ip (nA)
AFB2 25 AFB2
30 AFG1 20 AFG1
AFG2 15 AFG2
20
10
10
5
0 0
0 1 2 3 0 1 2 3
In BRb pH 9.0 In BRb pH11.0

In pH 9.0, all aflatoxins stable In pH 11.0, AFG1 and AFG2
within 3 hours in the cell. significantly decreased within
3 hours compared to AFB1
and AFB2.
(STABILITY STUDIES)
B) 6 months kept in freezer

AFG1
AFB2
80
70
60
Peak height (nA)

AFB1
50 AFB2
40 AFG1
AFG2
30
20
10
0 1 2 3 4 5 6 7
AFB1 AFG2
Stability order:
AFB1=AFB2>AFG2>AFG1
( in BRb pH =9.0)
(STABILITY STUDIES)
B) 6 months kept in freezer

80 100
70 90
60 80
Peak height (nA)
AFB1 AFB1
Percent left
50 AFB2 70 AFB2
40 AFG1 AFG1
60
AFG2 AFG2
30 50
0.1 uM in BRb pH = 9.0
20 40
10 30
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
Storage time in freezer (months) Storage time in freezer (months)
Peak height vs time Percent left vs time
3rd MONTH: AFB1 (72%), AFB2 (83%), AFG1 (66%), AFG2 (56%)
6th MONTH: AFB1 (50%), AFB2 (59%), AFG1 (42%), AFG2 (37%)
CONCLUSION
1. In BRb pH 9.0, AFB1 and AFB2 are stable up to

8 hours while AFG1 and AFG2 are stable up to 3
hours when exposed to normal condition in
voltammetric cell.
2. Within 3 hours exposed to normal condition, all
aflatoxins are stable in BRb pH 6, 7 and 9.0 but
in BRb pH 11.0, AFG1 is very unstable
compared to other aflatoxins. The proposed
stability order for aflatoxins in BRb pH 11.0 is:
AFB2 > AFB1 > AFG2 > AFG1
CONCLUSION…. cont
3. AFB1 and AFB2 solution in BRb pH 9.0 kept in freezer have

shelf life of 3 months while AFG1 and AFG2 only 2 months.
After this period of time, the new aflatoxins solution should
be prepared.
ACKNOWLEDGEMENT
SUPERVISOR:
CO-SUPERVISORS:


KELANTAN
THANK YOU
Stability of AFG1… cont
In strong alkali: lactone ring- susceptible to

alkaline hydrolysis
In acid: acid catalytic addition of water across the

double bond of the furan ring.
O O
O O
O
O
ELECTROCHEMICAL PROPERTIES OF AFG1
Ketone groups in lactone ring can be reduced and

produce cathodic wave.
Unsaturated terminal furan ring can be reduced

and produce cathodic wave.
O O
Can forms
O O
anionic
radical
O
O
Proposed mechanisme of the reduction of AFG1 that
may take place at mercury electrode as reported by
Smyth et al. (1979);
O O
O
ΘO
O O
2e- O O
2H2O
2 O
O Dimer + 2OH-
O O O
2H+ O
UV-VIS SPECTRUM OF AFB1
BRb pH 6.0 BRb pH 7.0

UV-VIS SPECTRUM OF AFB2

UV-VIS SPECTRUM OF AFG1

UV-VIS SPECTRUM OF AFG2

ORAL PRESENTED AT
SKAM-18
(JOHOR BAHRU, JOHOR ;
12 -14th SEPTEMBER 2005)
SQUARE-WAVE STRIPPING
VOLTAMMETRIC TECHNIQUE FOR THE
DETERMINATION OF AFLATOXIN B1 IN
GROUNDNUT SAMPLES
BY
MOHAMAD HADZRI YAACOB
AP DR ABDULL RAHIM MOHD YUSOFF
(UTM)
OBJECTIVE OF THIS STUDY
TO DEVELOP SWSV TECHNIQUE FOR
THE DETERMINATION OF AFB1
INTRODUCTION
• AFLATOXINS:
Mycotoxin group

Raw peanuts, peanuts products,

AFB1
AFB2 wheat, maize, vegetable oils, dried
AFG1 fruites, barley, pear, cocoa, coffee,
AFG2 herbs, spices, wine, cottonseeds.
AFM1 Cow milk and its products, eggs

AFM2 and meat product
Regulatory level in Malaysia:

Groundnuts < 15 ppb
Milk < 0.5 ppb
Others < 5 ppb
Chemical structure of AFB1
O
O
O O
(2,3,6a,9a-tetrahydro-4-methoxycyclo penta[c] furo

[3’,2’:4,5] furo [2,3-h][1] benzopyran-1,11-dione)
Produces blue colour under UV light

MW = 312
EXPERIMENTAL
INSTRUMENTS;
VA 757 Metrohm Voltammetry
analyser + MME Stand
Working electrode = HMDE
Ref electrode: Ag/AgCl, 3M KCl
REAGENTS;
a) Britton-Robinson buffer(BRB) pH = 9.0 and AFB1 std
b) Methanol, 0.1 N HCl, 15% ZnSO4, diatomoceus earth
powder, chloroform (for extraction and clean-up)
SAMPLES:
Groundnut from different shops
1) Scan a blank 2) Scan AFB1 std
10 ML BRB SOLUTION NITROGEN GAS IS FLOWED IN AFLATOXINS

SPIKE INTO 20 ML
VOLTAMMETRIC CELL
SOLUTION IS
DEGASSED

BLANK SOLUTION
SCANNED AFB1
BLANK SAMPEL
Method optimisation;
Study effect of
pH of BRB (6.0 to 13.0)
potential window (Ei and Ef ) (0.4 to -1.2 V)
Accumulation potential (Eacc) (0 to -1.4 V)
Accumulation time (tacc) (0 to 150 s)
Frequency (25 to 125 Hz)
Voltage step (0.01 to 0.04 V)
Amplitude (0.025 to 0.1 V)
Scan rate
to peak height (Ip) and peak potential (Ep)

of AFB1
Initial parameters (from optimum DPCSV parameters):
Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, scan

rate = 50 mV/s and pulse amplitude = 80 mV.
Supporting electrolyte = BRB pH 9.0. [AFB1] = 0.1 uM
Single cathodic peak

Ep = -1.23 V
Ip = 230 nA
Compare to DPCSV;
Ep = -1.24 V; Ip = 60 nA
a) Effect of pH of BRB (use 0.1 uM AFB1)
300 1.34
250 1.32
1.3
200
I p (n A )
1.28
Ep (-V)
150 1.26
100 1.24
50 1.22
1.2
0
1.18
5 6 7 8 9 10 11 12 13 14
5 6 7 8 9 10 11
pH of BRB
pH
1. pH 9.0 gave maximum Ip

2. Ep shifted towards negative direction at higher
pH Î Hydrogen ion involves in reduction process
Proposed mechanism;
a) pH 6-8 O O
O O
O
O
H H
-
2e
2H +
OCH 3
OCH 3
b) pH 9-12 O O
O O
O
O
2e-
2 2
OCH3
OCH3
2 H 2O
(Ref: Smyth et al. (1979): used DPP

+ 2 OH-
technique) D im e r
b) Effect of Ei
300
250
200
I p (n A )
150
100
50
0
0.2 0.4 0.6 0.8 1 1.2 1.4
Ei (-V)
Ei = -1.0 V gave highest Ip ( 285 nA) Î BRB pH 9.0

optimum Ei for further experiments
Ei = -1.0 V
c) Effect of Eacc
400
350
300
250
I p (n A)
200
150
100
50
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
Eacc (-V)
BRB pH 9.0
Eacc = - 0.8 V gave highest Ip (366 nA).
Ei= -1.0 V
Î Eacc - 0.8 V was selected as optimum Eacc
Eacc = -0.8 V
d) Effect of tacc
450
400
350
300
I p (nA)
250
200
150
100
50
0
0 40 80 120 160 200
Tacc (s)
BRB pH 9.0
100 s is optimum tacc. It gave Ei= -1.0 V
maximum Ip (400 nA) Eacc = -0.8 V
tacc = 100 s
e) Effect of frequency
1000
800
600
Ip (nA)
400
200
0
0 25 50 75 100 125 150
Frequency (Hz)
Frequency = 125 gave maximum BRB pH 9.0

Ip(820 nA). Ei= -1.0 V
Eacc = -0.8 V
> 125 Hz, not possible due to
tacc = 100 s
instrument limitation. Too fast.
Freq = 125 Hz
Ip is depend linearly on the square root of frequency
( 5 to 125 Hz);
y = 107.91x - 424.7 R 2 = 0.9919, n = 5.
Î AFB1 absorbed on the mercury electrode
1000
800
600
I p (nA)
400
y = 107.91x - 424.7
200
R2 = 0.9919
0
3 5 7 9 11
(Frequency)1/2
f) Effect of voltage step
1200
1000
800
I p (nA)
600
400
200
0
0 0.01 0.02 0.03 0.04 0.05
Voltage step (V)
BRB pH 9.0
Voltage step = 0.03 V which gave maximum Ip Ei= -1.0 V
(956 nA) was selected as the optimum v.step Eacc = -0.8 V
tacc = 100 s
Freq = 125 Hz
V.Step = 0.03 V
g) Effect of pulse amplitude
1200
1000
800
I p (nA)
600
400
200
0
0 0.025 0.05 0.075 0.1 0.125
Amplitude (V)
BRB pH 9.0
Amplitude = 0.05 V gave Ei= -1.0 V
maximum Ip (956 nA) Eacc = -0.8 V
tacc = 100 s
Freq = 125 Hz
V.Step = 0.03 V
Amplitude = 0.05 V
Square-wave frequency, voltage step and
scan rate are interrelated;
For frequency = 125 Hz and voltage step =
0.030 V Î scan rate = 3750 mV/s
Optimum parameters for the SWSV

determination of AFB1
BRB pH 9.0, Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.8 V, tacc =

100 s, Freq = 125 Hz, V.Step = 0.03 V, Amplitude = 0.05
V and scan rate = 3750 mV/s
Optimum SWSV parameters
Ip= 956 nA (Ep = -1.30 V) (b)
Non-optimum SWSV
Ip= 230 nA (Ep= 1.23 V)
Increases 4 times
Compare to optimum DPCSV
parameters
Ip= 60 nA ( Ep= -1.24 V).
Increases 16 times.
(a)
ÎSWSV more sensitive and faster
(scan rate = 3750 mV/s) compare
to DPCSV.
(* For DPCSV: 50 mV/s) (a) DPCSV (b) SWSV
Calibration curve for AFB1 and method validation
1200
1000
800
I p (n A )
600
y = 70.97x + 76.588
400
R2 = 0.9984
200
0
0 5 10 15 20
[AFB1] x 10-8 M
Linearity range: 0.01 µM to 0.15

µM / 3.12 ppb to 46.8 ppb AFB1
LOD; 0.125 x 10-8 M / 0.39 ppb /
390 ppt
* By DPCSV; LOD: 0.5 x 10-8 M / 1.56 ppb.
Precision / repetibility;(0.1 µM / 31.2 ppb) (n =5)
Ip ± SD / nA (RSD) Ep (V)
Day 1: 932 ± 7.92 (0.85%) -1.30
Day 2: 933 ± 7.74 (0.83%) -1.30
Day 3: 933 ± 7.84 (0.84%) -1.30
SWSV voltammograms of AFB1 (n=5): Day 1

Accuracy / Recovery:
0.05 uM: 99.67 ± 0.48% (n=3) -1.30 V
0.10 uM: 100.47 ± 0.34% (n=3) -1.30 V
(a) (b)
Voltammograms of (a) 0.05 and (b) 0.10 uM AFB1 (n=3)

Analysis of AFB1 in groundnut sample
1) Extracted and cleaned-up: follows Chemistry

Dept, MOSTI procedure.
2) Recovery studies – AFB1 in groundnut eluate
Result;
Amount added / ppb Amount found /ppb Recovery (%), n=3
3.00 2.82 ± 0.02 94.00 ± 0.67

9.00 8.21 ± 0.14 91.22 ± 1.56
15.00 13.88 ± 0.30 92.53 ± 2.00
Recovery: > 90 % is considered good recovery.

FLOW CHART OF SAMPLE ANALYSIS
EXTRACTION
SAMPLE
AND CLEAN UP
(FOLLOW CHEM DEPT, MOSTI
PROCEDURE)
INJECT 200 ul PREPARE FINAL

SAMPLE INTO CELL SOLUTION IN BRB
pH = 9.0
(+10 Ml BRB pH = 9.0)
SCAN USING STD ADDITION OF RESCAN

SWSV TECHNIQUE 10 ppb OF AFB1 USING SWSV
TECHNIQUE
Voltammogram of real sample + 10 ppb AFB1,
compare with HPLC result
(c)
(b)
(a)
(a) Blank (b) 200 ul real sample (c) real

sample + 10 ppb afb1
SWSV; 36.30 ppb HPLC; 36.00 ppb

Voltammogram of real sample + 10 ppb afb1 compare with HPLC
result

SWSV; ND HPLC; ND
No peak for AFB1: Not detected.

Results; AFB1 in groundnut samples (duplicate analysis)
No of sample By SWSV/ ppb By HPLC/ppb

1 ND ND
2 ND ND
3 ND ND
4 9.21 8.25
5 13.92 14.34
6 36.30 36.00
t test: No significant different between both

techniques (tcal = 0.51 < tcri (p : 0.05) = 2.57)
CONCLUSION
SWSV technique for the determination of

AFB1 in groundnut sample was
successfully developed. The technique
is precise, accurate, high sensitive, fast
and low cost technique.
ACKNOWLEDGEMENT
SUPERVISORS:

SHORT TERM GRANT: 75152/2004

KELANTAN
MR RADWAN, CHEM DEPT. MOSTI,

PENANG BRANCH.
CV result for 1.3 uM AFB1 in BRB pH 9.0
Single cathodic peak at Ep = -1.310 V Î

Irreversible reduction reaction at mercury electrode
FORMULA FOR CALCULATION AMOUNT
OF AFB1 IN GROUNDNUT SAMPLE;
ppb aflatoxin =
P’/ P x C x *12.5 ( for injection vol = 200 ul)
( P’ = Ip of sample (nA), P = Ip of std (nA)

C = conc of injected std, 10 ppb)
*For other injection volume;

100 ul = x 25 300 ul = x 8.33
400 ul = x 6.25 500 ul = x 5
Voltammogram of real sample+ 10 ppb afb1, compare
with HPLC result
(c)
(b)
(a)

SWSV; 36.30 ppb
Afb1 (ppb) = 94.3 x 10 x 12.5 = 36.30 ppb HPLC; 36.00 ppb

419 – 94.3
ices, herbs, 1.1 Ep of AFB2 chang
n
lo
duces blue 0.9
y = 0.5097x + 0.4115
rincreasing
e scan ra
hanol : 360 0.7 R2 = 0.995
w
p
ÎReduction
a of AF
0.5
ctor. 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 eiirriversible
r reactio
log scan rate
t
d
h
g
4.3) Ip vs V a
r
ta
70
h
L
p
60
m
e
hi
50
en
40 rn
( e
Ip (n A )
30 c
u
Ra
gassed with 20 ÎAFB2
u
m
2
r was adso
CTION 4.
oxin, dangerous a) DPCSV voltammogr
Type I carcinogen
ctures;
O O
O O
O O O O
O
ip = 61.71 nA ip = 59.97n
OCH3 O
OCH3
b) SQWSV voltammogr
AFG1 AFG2
: measures
s applied. Simple,
y and low cost
ip = 833 nA ip = 816nA
RIMENT
c) DPCSV and SQWSV
46 processor and
de electrode (MME)
Paper will be published in
Malaysian Journal of Analytical Science
1
SQUARE WAVE CATHODIC STRIPPING VOLTAMMETRIC TECHNIQUE FOR
DETERMINATION OF AFLATOXIN B1 IN GROUND NUT SAMPLE
Mohamad Hadzri Yaacob1, Abdull Rahim Hj. Mohd. Yusoff2 and Rahmalan Ahamad2
1
School of Health Sciences, USM, 16150 Kubang Krian, Kelantan, Malaysia
2
Chemistry Dept., Faculty of Sciences, UTM, 81310 Skudai, Johor, Malaysia
Abstract An electroanalytical method has been developed for the detection and determination of the
2,3,6a,9a-tetrahydro-4-methoxycyclo penta[c] furo[3’,2’:4,5] furo [2,3-h][l] benzopyran-1,11-dione
(aflatoxin B1, AFB1) by a square wave cathodic stripping voltammetric (SWSV) technique on a hanging
mercury drop electrode (HMDE) in aqueous solution with Britton-Robinson Buffer (BRB) at pH 9.0 as
the supporting electrolyte. Effect of instrumental parameters such as accumulation potential (Eacc),
accumulation time (tacc), scan rate (v), square wave frequency, step potential and pulse amplitude were
examined. The best condition were found to be Eacc of -0.8 V, tacc of 100 s, v of 3750 mV/s, frequency of
125 Hz, voltage step of 30 mV and pulse amplitude of 50 mV. Calibration curve is linear in the range of
0.01 to 0.15 uM with a detection limit of 0.125 x 10-8 M. Relative standard deviation for a replicate
measurements of AFB1 (n = 5) with a concentration of 0.01 uM was 0.83% with a peak potential of -1.30
V (against Ag/AgCl). The recovery values obtained in spiked ground nut elute sample were 94.00 +/-
0.67 % for 3.0 ppb, 91.22 +/- 1.56 % for 9 ppb and 92.56 +/- 2.00 % for 15.0 ppb of AFB1. The method
was applied for determination of the AFB1 in ground nut samples after extraction and clean-up steps. The
results were compared with that obtained by high performance liquid chromatography (HPLC) technique.
Abstrak Satu kaedah elektroanalisis telah dibangunkan untuk mengesan dan menentukan 2,3,6a,9a-
tetrahydro-4-methoxycyclo penta[c] furo[3’,2’:4,5] furo [2,3-h][l] benzopyran-1,11-dione (aflatoxin B1,
AFB1) menggunakan teknik voltammetri perlucutan katodik denyut pembeza di atas elektrod titisan raksa
tergantung (HMDE) di dalam larutan akuas dengan larutan penimbal Britton-Robinson (BRB) pada pH
9.0 bertindak sebagai larutan penyokong. Kesan parameter peralatan seperti keupayaan pengumpulan
(Eacc), masa pengumpulan (tacc), kadar imbasan (v), frekuensi gelombang bersegi, kenaikan keupayaan dan
amplitud denyut telah dikaji. Keadaan terbaik yang diperolehi adalah Eacc; -0.8 V, tacc; 100 s, v; 3750
mV/s, frekuensi; 125 Hz, kenaikan keupayaan; 30 mV dan amplitud denyut; 50 mV. Keluk kalibrasi
adalah linear pada julat di antara 0.01 ke 0.15 uM dengan had pengesan pada 0.125 x 10-8 M. Sisihan
piawai relatif untuk 5 kali pengukuran AFB1 dengan kepekatan 0.01uM ialah 0.83 %. Nilai perolehan
semula di dalam larutan elusi sampel kacang yang disuntik dengan 3.0 ppb, 9 ppb dan 15.0 ppb AFB1
adalah 94.00 +/- 0.67 %, 91.22 +/- 1.56 % dan 92.56 +/- 2.00 % masing-masingnya. Kaedah ini telah
digunakan untuk menentukan kandungan AFB1 di dalam sampel kacang tanah selepas proses
pengekstraksian dan pembersihan dijalankan. Keputusan yang diperolehi telah dibanding dengan
keputusan dari kaedah kromatografi cecair berprestasi tinggi.
Keywords: square wave stripping voltammetry, HMDE, aflatoxin B1, ground nut
Introduction
Aflatoxins (AF), the mycotoxin produced mainly by Aspergillus flavus and parasiticus and
display strong carcinogenicity [1]. They are dangerous food and contaminants and represent a worldwide
threat to public health. AFB1, B2, G1 and G2 and their metabolites M1 and M2 are the most common,
1
Corresponding address:
Chemistry Dept, Faculty of Science, UTM,
81310 Skudai, Johor, Malaysia
tel: 07-553 4492
2
and of these, AFB1 and AFG1 are observed most frequently in food [2]. Of these, researches have shown
AFB1 (Fig 1) exhibits most toxic [3] with the order of toxicity is AFB1 > AFG1 > AFB2 > AFG2
indicates that the terminal furan moiety of AFB1 is a critical point for determining the degree of
biological activity of this group of mycotoxins [4]. Many countries including Malaysia have stringent
regulatory demands on the level of aflatoxins permitted in imported and traded commodities.
O
O
O O
Figure 1 Chemical structure of AFB1
One of the foodstuffs which is most occurrence of AFB1 is ground nut. In Malaysia, the AFB1
level in peanut is regulated with maximum level that cannot be greater than 15 ppb [5]. Several analytical
techniques for quantitative determination of the AFB1 in ground nut have been proposed such as thin
layer chromatograhpy [6], high performance liquid chromatography[ 7,8,9 ]and an enzyme-linked
immunosorbent assay, ELISA [10], All these methods, however, require specialist equipment operated
by skilled personel and expensive instruments and high maintenance cost [11].
Due to all these reasons, a voltammetric technique which is fast, accurate and require low cost
equipment [12,13] is proposed. A square wave cathodic stripping which is presented in this paper is one
of the voltammetric technique that in particularly, has a few advantages compared to other voltammetric
technique such as high speed, increased analytical sensitivity and relative insensitivity to the presence of
dissolved oxygen [14]. Previous experiment using cyclic voltammetric technique showed that AFB1
reduced at mercury electrode and the reaction is totally irreversible [15]. This work aimed to study and
develop a SWSV method for determination of AFB1 at trace levels and to determine this aflatoxin in
ground nut samples. No such report has been published regarding this experiment until now.
Experiment
Apparatus
Square-wave voltammograms were obtained with Metrohm 693 VA Processor coupled with a
Metrohm 694 VA stand. Three electrode system consisted of a hanging mercury drop electrode (HMDE)
was used as the working electrode, Ag/AgCl/3 M KCl reference electrode and a platinum wire auxiliary
electrode. A 20 ml capacity measuring cell was used for placing supporting electrolyte and sample
analytes. All measurements were carried out at room temperature. All pH measurements were made
with Cyberscan pH meter, calibrated with standard buffers at room temperature.
Reagents
AFB1 standard (1mg per bottle) was purchased from Sigma Co. and was used without further
purification. Stock solution (10 ppm or 3.21 x 10-5 M) in benzene:acetonitrile (98:2) were prepared and
stored in the dark at 14 0 C. The diluted solution were prepared daily by using certain volume of stock
solution, degassed by nitrogen until dryness and redissolved in Britton-Robinson buffer (BRB) solution at
3
pH 9.0. Britton-Robinson buffer solutions (prepared from a stock solution 0.04 M phosphoric (Merck),
boric (Merck) and acetic (Merck) acids; and by adding sodium hydroxide (Merck) 1.0 M up to pH of 9.0.
All solutions were prepared in double distilled dionised water (~ 18M Ω cm). All chemicals were of
analytical grade reagents.
Procedure
For voltammetric experiments, 10 ml of Britton-Robinson buffer solution with pH 9.0 was placed
in a voltammetric cell, through which a nitrogen stream was passed for 600 s before recording the
voltammogram. The selected Eacc = - 800 mV was applied during the tacc = 100 s while the solution was
kept under stirring. After the accumulation time had elapsed, stirring was stopped and the selected
accumulation potential was kept on mercury drop for a rest time (tr = 10 s), after which a potential scan
was performed between -1.00 as initial potential (Ei) and finished at -1.400 V as final potential (Ef ) by
SWSV technique.
Procedure for the determination in ground nut samples
AFB1 was extracted according to the standard procedure developed by Chemistry Department,
Penang Branch, Ministry Of Science, Technology and Innovation, Malaysia [16]. 1ml of the final
solution from extraction and clean-up steps in chloroform was pipette into amber bottle, degassed with
nitrogen and redissolved in 1ml of BRB solution. 200 ul of this solution was spiked into 10 ml supporting
electrolyte in volumetric cell. After that, the general procedure was applied and voltammogram of sample
was recorded. This experiment was followed by a standard addition of 10 ppb of AFB1 standard addition
and voltammogram of sample with AFB1 standard was recorded.
Result and discussion
Using previous differential pulse stripping voltammetry (DPCSV) optimum parameters [17],
SWSV was run to determine 0.1 uM AFB1 in BRB pH 9.0. It gave a single reduction signal with peak
height (Ip) of 250 nA at peak potential (Ep) of -1.26 V (against Ag/AgCl). The SWS voltammograms
shows improved Ip compared to that obtained bt DPCSV where the Ip was increased almost 4 times as
shown in Figure 2.
Cyclic voltammogram of AFB1 in BRB pH 9.0 is shown in Figure 3 which only produced a
cathodic peak that indicates the non-reversibility of the electrode process [18].
Figure 2 Voltammograms of 0.1 uM AFB1 obtained by (a) DPCSV and (b) SWSV techniques.
Experimental condition; for DPCSV: Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, υ = 50 mV/s and
4
pulse amplitude = 80 mV and for SWSV: Ei = -1.0 V, Ef = -1.4 V, Eacc = -0.6 V, tacc = 80 s, frequency =
50 Hz, voltage step = 0.02, amplitude = 50 mV and υ = 1000 mV/s.
Figure 3 Cyclic voltammogram of AFB1 in BRB pH 9.0. Experimental conditions: Ei = 0, Elow = -1.5
V, Ehigh = 0 and scan rate = 200 mV/s.
The effect of the pH of the BRB on the stripping was studied in a pH range of 6 – 13 (Figure 4).
The Ip increased slowly with increasing pH up to 8.0 followed with sharply increased for pH 8.0 to 9.0,
then decreased in pH 10.0 and continuously decreased in a range of 10 – 13. Thus pH 9.0 was chosen for
the analysis. This result is not contradicted with that found by Smyth et al. (1979) when they performed
polarographic study of AFB1 [19].
300
250
200
I p (nA)
150
100
50
0
5 6 7 8 9 10 11 12 13 14
pH of BRB
Figure 4 Influence of pH of BRB on the Ip of 0.10 uM AFB1 using SWSV technique. The instrumental
parameters are the same as in Figure 2.0.
Figure 5 shows a dependence of the Ep on pH. Shifting of the Ep towards the negative direction at
higher pH implies that the reduction process takes up hydrogen ions [20]. A double bond in aromatic ring
conjugates with ketone group, in general, undergoes a reduction at mercury electrode. The suggested
mechanism of this reaction in BRB pH 9.0 is illustrated in Figure 6 as reported by Smyth et al. (1979)
[21].
5
1.34
1.32
1.3
1.28
Ep (-V)
1.26
1.24
1.22
1.2
1.18
5 6 7 8 9 10 11
pH
Figure 5 Relationship between Ep of AFB1 with pH of BRB
O O
O O
O
O
2e-
2 2H + 2
CH3
CH3
2 H 2O
D im e r + 2 OH-
Figure 6 Mechanism for reduction of AFB1 at mercury electrode in BRB pH 9.0
Optimisation of condition for the stripping analysis
The voltammetric determination of analytes at trace levels normally involves very small current
response. For that reason it is important to optimise all those parameters which may have an influence on
the measured current. The effect of Ei , Eacc , tacc, frequency, voltage step and amplitude were studied.
Square-wave voltammetric technique was used with stirring. For this study, 1.0 x 10-7 M of AFB1 was
spiked into supporting electrolyte.
A study of the influence of Ei showing a peak height (Ip) was obtained for Ei = -1.0 V (Figure 7).
The Ip was slowly decreased for Ei more negative than -1.0 V. This value was chosen for subsequent
studies further optimisation step. Influence of Eacc to Ip of AFB1 was investigated where the Eacc was
varied between 0 to -1.4 V. The maximum value of Ip obtained at -0.8 V (366 nA) as shown in Figure 8.
This value was selected for subsequent experiments.
6
The dependence of Ip on tacc was studied. The effect of tacc on the Ip was studied where tacc was
varied from 0 to 160 s. The result is shown in Figure 9 which reveals that there is linearly up to 100s
according to equation y = 3.8557x + 21.383 (n=6) with R2 = 0.9936, then it increased rather slowly
leveling off at about 140 s. At 160 s, Ip decreased which may be due to the electrode saturation [21].
Thus, 100 s was appeared to be an optimum tacc for the pre-concentration prior to stripping.
300
250
200
Ip (nA)
150
100
50
0
0.2 0.4 0.6 0.8 1 1.2 1.4
Ei (-V)
Figure 7 Effect of Ei on the Ip of 0.1 uM AFB1 in BRB pH 9.0
400
350
300
250
Ip (nA)
200
150
100
50
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
Eacc (-V)
Figure 8 Effect of Eacc on the Ip of 0.1 uM AFB1 in BRB pH 9.0
450
400
350
300
Ip (nA)
250
200
150
100
50
0
0 40 80 120 160 200
T acc (s)
Figure 9 Effect of tacc on the Ip of 0.1 uM AFB1 in BRB pH 9.0
7
For other instrumental condition such as square wave frequency, step potential and pulse
amplitude were examined, varying one of them and maintaining constant others. The variable ranges
were: 25 to 125 Hz for the frequency, 0.01 to 0.04 for the voltage step and 25 – 100 mV for pulse
amplitude. Generally, the Ip increase by increasing all of these instrumental parameters [22]. At higher
potential values the peak width increase; at higher frequency values the current background increases.
Finally, the condition selected were: frequency = 125 Hz, voltage step = 0.03 V and pulse amplitude = 50
mV (Figures 10 to 12). Under optimised parameters, Ip of AFB1 was 956 nA which is 16 times higher
compared to that obtained by DPCSV. Figure 13 shows voltammograms of 0.1 uM AFB1 obtained by
SWSV and DPCSV techniques.
1000
800
600
Ip (nA)
400
200
0
0 25 50 75 100 125 150
Fre que ncy (Hz)
Figure 10 Dependence of the Ip of AFB1 on SWSV frequency
1200
1000
800
Ip (nA)
600
400
200
0
0 0.01 0.02 0.03 0.04 0.05
Voltage step (V)
Figure 11 Dependence of the Ip of AFB1 on SWSV voltage step
8
1200
1000
800
Ip (nA) 600
400
200
0
0 0.025 0.05 0.075 0.1 0.125
Amplitude (V)
Figure 12 Dependence of the Ip of AFB1 on SWSV pulse amplitude
Figure 13 Voltammograms of AFB1 obtained by (a) DPCSV and (b) SWSV techniques in BRB pH 9.0.
Voltammetric determination of AFB1 and analytical characteristics of the method
Using the selected conditions already mentioned, a study was made of the relationship between Ip
and concentration. There is a linear relationship in the concentration range 0.01 to 0.15 uM as shown in
Figure 14. Limit of detection (LOD) was 0.389 ppb (0.125 x 10-8 M) which was determined by standard
addition of low concentration of AFB1 until obtaining the sample response that is significantly difference
from blank [23]. Relative standard deviation (RSD) of the analytical signals at several measurement
(n=5) of 0.10 uM was 0.83%.
1400
1200
1000
Ip(nA)
800
600
y = 62.373x + 246.15
400
R2 = 0.9956
200
0
0 5 10 15 20
[AFB1] / 10-8 M
Figure 14 Calibration plot of AFB1 in BRB pH 9.0 obtained by SWSV technique.
9
Determination of AFB1 in ground nut samples
The proposed method was applied to the analysis of the AFB1 in ground nut samples. Recovery
studies were performed by spiked with difference concentration levels of AFB1 standard into eluate of
ground nut sample. In this case the concentrations used were 3 ppb (0.963 x 10-8 M), 9 ppb (2.889 x 10-8
M) and 15 ppb (4.815 x 10-8 M). The results of these studies are shown in Table 1. For analysis of AFB1
in ground nut samples, the standard addition method was used in order to eliminate the matrix effects.
Figure 15 show voltammograms of real sample together with spiked AFB1 standard. Table 2 listed the
content of AFB1 in 6 samples obtained by proposed technique compared with that obtained by HPLC.
The results show that there are no significant different of AFB1 content obtained by both techniques.
Table 1 Percent recovery of AFB1 spiked in real samples (n=3)
Amount added (ppb) Amount found (ppb) Recovery (%) n=3
3.00 2.82 +/- 0.02 94.00 +/- 0.67
9.00 8.21 +/- 0.14 91.22 +/- 1.56
15.00 13.88 +/- 0.30 92.53 +/- 2.00
Figure 15 SWS voltammograms of real sample (b) and spiked AFB1 (c) obtained in BRB pH 9.0 as the
supporting electrolyte (a)
10
Table 2 AFB1 content in ground nut samples by proposed technique compared with those obtained by
HPLC.
No of sample AFB1 content in real sample
By SWSV By HPLC
1 ND ND
2 ND ND
3 ND ND
4 9.21 8.25
5 13.92 14.34
6 36.30 36.00
Conclusion
A SWSV technique was successfully developed for the determination of AFB1 in ground nut as
an alternative method for determination of AFB1 which is sensitive, accurate and fast technique. The
results are not significant different with that obtained by accepted technique used for routine analysis of
AFB1.
Acknowledgement
One of the author would like to thank University Science Malaysia for approving his study leave
and financial support to carry out his further study at Chemistry Dept., UTM. We also indebt to UTM for
short term grant (Vot No 75152/2004) and to Chemistry Department, Penang Branch, Ministry of
Science, Technology and Innovation (MOSTI) for their help in analysis of aflatoxin in ground nut
samples using HPLC technique.
References
1. Akiyama, H. Goda, Y. Tanaka, T and Toyoda, M. (2001). “Determination of aflatoxinsB1, B2,

G1 and G2 in spices using a multifunctional column clean-up” J. Of Chromatography A, 932.
153-157.
2. Yoruglu, T, Oruc, H.H. and Tayar, M. (2005) “Aflatooxin M1 levels in cheese samples from
some provinces of Turkey” Food Control. 16(10): 883-895.
3. World Health Organisation in: IARC Monograph on the evaluation of carcinogenic risk to
human, WHO, Lyon, 1987.
11
4. Hall, A.J., Wild, C.P in: Eaton, D.L., Groopman, J.D. (Eds). “The toxicology of aflatoxins:
Human Health, Veterrinary and Agricultural Significance” Academic, New York, 1994.
5. Malaysian Food Act (1983) Act No 281, Reviewed 2002, Food Quality Control Dept, Ministry of
Health, Malaysia.
6. Bicking,M.K.L., Kniseley, R.N and Svec, H.J (1983) “Coupled-Column for quantitating low
levels of aflatoxins” J . Assoc. Off. Anal. Chem. 66, 905-908.
7. Beebe, B.M. (1978). “Reverse phase high pressure liquid chromatographic determination of
aflatoxins in foods” J. Assoc. Off. Anal. Chem. 81. 1347-1352.
8. Cepeda, A., Franco, C.M., Fente, C.A., Vazquez, B.I., Rodriguez, J.L., Prognon, P. and
Mahuzier, G. (1996) “Postcolumn excitation of aflatoxins using cyclodextrins in liquid
chromatography for food analysis” J Of Chrom A. 721. 69-74.
9. Garner, R.C., Whattam, M.M. , Taylor, P.J.L. and Stow, M.W ( 1993) “Analysis of United
Kingdom purchased spices for aflatoxins using immunoaffinity column clean up procedure
followed up by high-performance liquid chromatographic analysis and post-column derivatization
with pyridium bromide perbromide” J. Chromatography, 648. 485-490
10. Beaver, R.W., James, M.A. and Lin ,T.Y. (1991). “ELISA-based screening test with liquid
chromatography for the determination of aflatoxin in corn” J. Assoc.Off. Anal. Chem , 74: 827 –
829
11. Garden, S.R. and Strachan, N.J.C.(2001). “Novel colorimetric immunoassay for the detection of
aflatoxin B1” Anal Chim Acta, 444. 187-191.
12. Braitina, Kh. Z., Malakhova, N.A. and Stojko, Y. (2000) “Stripping voltammetry in
environmental and food analysis” Fresenius J Anal Chem 368. 307-325
13. Skrzypek, S., Ciesielski, W., Sokolowski, A., Yilmaz, S. and Kazmierczak, D. (2005) “Square
wave adsorption stripping voltammetric determination of famotidine in urine”Talanta. Article in
press.
14. Economou, A., Bolis, S.D., Efstathiou, C.E. and Volikakis, G.J (2002) “A virtual electrochemical
instrument for square wave voltammetry”.Anal. Chim. Act. 467. 179-188.
15. Yaacob, M.H., Mohd Yusoff, A.R. and Ahmad, R. (2003). “Cyclic voltammetry of AFB2 at the
mercury electrode”. Paper presented at Simposium Kimia Malaysia ke 13 (SKAM-13), 9 – 11th
September 2003, Kucing, Sarawak, Malaysia.
16. Standard procedure for determination of aflatoxins (2000). Chemistry Dept. Penang Branch,
Ministry of Science, Technology and Innovation (MOSTI), Malaysia – unpublished paper.
17. Yaacob, M.H., Mohd Yusoff, A.R. and Ahamad, R. (2005). “Determination of the aflatoxin B1 in
ground nut by differential pulse cathodic stripping voltammetry technique”. Paper presented at
2nd National Seminar On Chemistry, 14th April 2005, Universiti Sumatera Utara, Medan,
Indonesia.
12
18. Rodriguez, J., Berzas, J.J., Casteneda, G. and Rodriguez, N. (2005). “Voltammetric determination
of Imatinib (Gleevec) and its main metabolite using square-wave and adsorptive stripping square-
wave techniques in urine samples”. Talanta. 66. 202-209.
19. Smyth, M. R., Lawellin, D. W. and Osteryoung, J.G. (1979). “Polarographic study of Aflatoxin
B1, B2, G1 and G2: Application of differential pulse polarography to the determination of
Aflatoxin B1 in various foodstuffs” Analyst. 104. 73-78
20. Sun, N., Mo, W-M., Shen, Z-L. and Hu, B-X. (2005). “Adsorptive stripping voltammetric
technique for the rapid determination of tobramycin on the hanging mercury electrode”. J Pharm.
Biomed. Anal. Article in press.
21. Wang J. (1994) “Analytical Electrochemistry” VCH Publisher, USA.
22. Komorsky-Lovric, S., Lovric, M. and Branica, M. (1992). “Peak current-frequency relationship in
adsorptive stripping square-wave voltammetry”. J of Electroanal. Chem. 335(1-2). 297-308.
23. Barek, J. , Fogg, A. G., Muck, A. and Zima, J. (2001). “Polarography and Voltammetry at
Mercury Electrodes”, Critical Reviews In Analytical Chemistry, 31. 291-309
13
PAPER SENT TO PROF BAREK
TO BE PUBLISHED IN
J. COLLECT. CZECH. CHEM. COMMON
( IN PRESS )
1
DEVELOPMENT OF DIFFERENTIAL PULSE STRIPPING VOLTAMMETRIC
(DPSV) TECHNIQUE FOR DETERMINATION OF AFLATOXIN G1 AT THE
MERCURY ELECTRODE
Mohamad Hadzri Yaacob1, Abdull Rahim Hj. Mohd. Yusoff2 and Rahmalan
Ahamad2
1
School of Health Sciences, USM, 16150 Kubang Krian, Kelantan
2
Chemistry Dept., Faculty Of Sciences, UTM, 81310 Skudai, Johor
Abstract
DPSV technique was developed for determination of aflatoxin G1 (AFG1) in
Britton-Robinson Buffer (BRB). Controlled Growth Mercury Electrode (CGME)
and Ag/AgCl have been used as working and reference electrodes respectively.
In this experiment, the analyte was initially scanned from -200 mV to -1500 mV in
BRB from pH 2.0 to 9.0. The effect of pH of supporting electrolyte, scan rate,
accumulation potential, accumulation time and pulse amplitude on the peak
potential and peak current of the analyte were studied. The results showed that
AFG1 compound undergoes reduction at potential -1175 mV ( versus Ag / AgCl ).
Maximum peak height was observed in BRB pH 9.0 with the optimised
parameters such as scan rate; 50 mV/s, accumulation potential; -600 mV,
accumulation time; 80s and pulse amplitude; 80 mV . Under the optimum
condition, the peak height of AFG1 was proportional to the concentrations of the
AFG1 in the range of 0.02 to 0.26 uM with a limit of detection of 0.015 uM.
Relative standard deviation for a replicate measurements of AFG1 ( n= 8) with
the concentrations of 0.06 and 0.20 uM were 3.52% and 1.54% respectively.
Recoveries of the different AFG1 concentrations in prepared standard solutions
were found to be 85% to 95% with the precision around 0.4% to 1.0%.
1
Correspondence address;
Chemistry Dept, Faculty Of Science,
UTM 81310 Skudai Johor
2
Introduction
Aflatoxin G1 ( 3,4,7a,10a-tetrahydro-5-methoxy-1H, 12H furo[3’,2’:4,5]furo[2,3-

h]pyrano[3,4-c][1]-benzopyran-1,12-dione), structural formula is shown in Figure
1.0, is one of the type of aflatoxin which is naturally occurance (1). It is one of the
compound in mycotoxin group (2) that is produced by Aspergillus flavus and
Aspergillus parasticus fungi (3,4). It is found in various contaminated food such
as peanuts and peanut products, barley, maize, cottonseed, coffee beans and
others (5). It produces green color under ultraviolet light (6,7). In health aspect, it
is considered as human carcinogen by the International Agency for Research on
Cancer (IARC) as reported in World Health Organisation (WHO)’s monograph
(8). It has been reported to be teratogenic, tremorogenic, and haemorrhagic to
wide range of organisms and to cause hepatic carcinoma in man (9)
Consequently, AFG1 level in animal feed and various human food is now
monitored and tightly regulated by various countries. In this country, regulatory
level for AFG1 and total aflatoxins is 15 ppb in raw peanuts and 5 ppb for others
(10). Due to all these reasons, selecting appropriate and accurate method of
analysis of AFG1 is absolutely necessary to determine AFG1 at the part-of-billion
(ppb) level.
O O
O O
O
O
Figure 1.0 Chemical structure of AFG1

There have been several reports for the determination of AFG1 including thin
layer chromatography (11,12), high performance liquid chromatography with
various type of detectors ( 13,14,15, 16 ) and polarographic technique (17) but
3
no report has been published concerning the use of stripping voltammetric
technique. The development of new method capable of determining AFG1 in
food sample is important. Stripping voltammetric technique was selected for this
study due to its simple, easy to use, sensitive and accurate technique (18). This
technique applies pre-concentration step that will increase sensitivity of the
method (19). This paper will describe the development of DPSV technique for
determination of AFG1 using CGME as the working electrode.
Materials and Methods

All reagents employed were of analytical grade. Water purified from a Nano Pure
Ultrapure water system ( Branstead / Thermolyne) was used for all dilution and
sample preparation. AFG1 was purchased from SIGMA: 1 mg was dissolved in
hexane: acetonitrile (98:2) to produce 10 ppm solution. A stock of Britton-
Robinson buffer (BRB) solution 0.04 M was prepared as follows: 2.47 g boric
acid (Fluka), 2.30 ml acetic acid (MERCK) and 2.70 ml orthophosphoric acid (
Ashland Chemical ) were dissolved in 1 l deionised water. The pH of the solution
was adjusted to the desired values by adding 1 M sodium hydroxide (BDH) or 1
M hydrochloric acid ( MERCK). Hexadistilled mercury, grade 9N, used for
working electrode was purchased from MERCK.
All voltammograms were recorded with a BAS CV-50W Voltammetric Analyser
in connection with a Controlled Growth Mercury Electrode (CGME) stand
equipped with a three-electrode system consisting of an Ag / AgCl reference
electrode, a platinum wire counter electrode and the CGME as working
electrode. A 20 ml capacity BAS MR-1208 cell was used. In all voltammetric
analysis, the supporting electrolyte solution was deaerated by a stream of
nitrogen for at least 10 min. A pH meter Cyber-scan model equipped with a
glass electrode combined with an Ag / AgCl reference electrode, was employed
for the pH measurements.
Differential pulse stripping voltammograms (DPSVs) of AFG1 were run after
purging the test solution for at least 5 min. The DPSVs were scanned from -200
to -1500 mV with scan rate of 40 mV/s, accumulation potential ( Eacc ) -800 mV,
4
accumulation time ( tacc ) 40 s, pulse amplitude 50 mV, pulse width 50-ms and
pulse period 200-ms.
Results and Discussion

From our previous studies on the cyclic voltammograms of AFG1 in BRB pH 9.0,
a reduction peak was observed at -1245 mV ( against Ag / AgCl) and no anodic
peak at oxidation wave. The results of this CVs showed that AFG1 underwent
irreversible reduction reaction at the mercury electrode (20). Due to this
electroanalytical properties, differential pulse cathodic stripping voltammetric
technique was applied in this study using the same supporting electrolyte. By
using initial parameters as mentioned before, the peak potential ( Ep ) of 0.15 uM
AFG1 was obtained at -1175 mV with peak current ( Ip ) of 34.73 nA ( Figure
2.0 ). Several parameters have been optimised so as to obtain a more
symmetrical and higher reduction peak.
Figure 2.0 Voltammograms of 0.15 uM AFG1 (b) in BRb pH 9.0 (a) . Condition;
scan rate; 40 mV/s, accumulation potential ( Eacc ); -800 mV, accumulation time
( tacc ); 40 s, pulse amplitude; 50 mV, pulse width; 50-ms and pulse period; 200-
ms.
Effect of pH of BRB
The effect of different pH medium of BRB have been studied. The results
showed that a reduction peak was first observed at pH 5.0 which increases in
5
peak height as the pH increases. It reaches its optimum height at pH 9.0. At pH
more than 9.0, the peak height decreases rapidly and no peak was observed at
pH more than 12.0 ( Fig 3.0 ). This results suggested that proton ion played a
significant role in the reduction of AFG1 on mercury electrode. Hence, BRB with
pH 9.0 was applied in this experiment..
50
40
Ip(nA)
30
20
10
0
4 6 8 10 12
pH of BRb
Figure 3.0 The effect of pH of BRB on peak current of AFG1. [AFG1] = 0.15 uM
Condition: scan rate; 40 mV/s, accumulation potential ( Eacc ); -800 mV,
accumulation time ( tacc ); 40 s, pulse amplitude; 50 mV, pulse width; 50-ms and
pulse period; 200-ms.
Effect of potential windows, accumulation potential ( Eacc ) and

accumulation time ( tacc) )
The influence of potential windows on the cathodic peak current of AFG1 was
studied by varying the potential windows from -200 to -1000 mV and from -1400
to -1500 mV for initial ( Ei ) and final potential ( Ef ) respectively. The results
showed that with Ei = -950 mV and Ef = -1400 mV, the peak current at Ep = -
1160 mV was 41.50 nA which is higher than using initial potential windows as
described in previous experiment. Therefore, this potential window was used for
all subsequent measurements. The dependence of the cathodic peak current on
Eacc was also studied. The highest peak current was found when Eacc was -600
mV and when Eacc changed to more negative than -600 mV, the Ip decreases
6
( Figure 4.0 ) which indicated that maximum accumulation of AFG1 at mercury
electrode occurred when potential of -600 mV was applied on it in BRB pH 9.0.
The effect of tacc on Ip of AFG1 also was studied. The Ip of AFG1 was found to
increase with increasing tacc ( Figure 5.0 ) indicating continueous accumulation
of AFG1 at the electrode surface with increasing accumulation time. Normally,
the increase in the response current continued until a maximum signal level
presumable corresponding to a saturation electrode surface was attained (21).
The results thus obtained indicated that the optimum tacc for AFG1 was 80 s.
Hence 80 s accumulation time was employed throughout this experiment.
60
40
I p (n A )
20
0
0 200 400 600 800 1000 1200 1400 1600
Eacc (-mV)
Figure 4.0 Effect of accumulation potential on Ip of AFG1
80
60
ip (n A )
40
20
0
0 50 100 150 200
tacc (s)
Figure 5.0 Effect of accumulation time on Ip of AFG1
7
Effect of instrumental parameters
As the scan rate was varied from 20 to 100 mV s-1, the Ip increases and reached
it maximum value at scan rate of 50 mV s-1 , after which the peaks became
broader with undesired tail at their end. A similar pattern for Ip was obtained with
increasing pulse amplitude ( from 30 to 120 mV ). A pulse amplitude of 80 mV
with scan rate of 50 mV s-1 were chosen for further studies. Using all optimised
parameters ( Ei = -950 mV, Ef = 1400 mV, Eacc = -600 mV, tacc = 80 s, scan rate =
50 mV s-1, pulse amplitude = 80 mV ) the Ip of 0.15 uM AFG1 was 75.10 nA
with Ep at -1150 mV was obtained
Calibration graph, detection limit, precision and recovery
The differential pulse cathodic stripping voltammograms at different

concentrations of AFG1 under optimum conditions are shown in Fig. 6.0. The Ip
of AFG1 increased linearly with increasing concentration up to 0.26 uM. A linear
calibration graph was obtained in the concentration range 0.02 to 0.26 uM AFG1
( n = 10, r = 0.9980). At higher concentrations Ip tend to level off which may be
due to electrode surface saturation (20). The detection limit ( three times signal-
to-noise ) was found to be 0.015 uM AFG1. For eight successive determinations
of 0.06 and 0.20 uM AFG1, relative standard deviations were 3.52 and 1.54 %
respectively which indicated that the precision of the method was good.
Recovery of the different AFG1 concentrations in prepared standard solutions
were found to be 85 to 95% with precision around 0.4 to 1.0 % as listed in Table
1.0.
8
160
140
120
100
Ip (n A )
80
60
40 y = 5.5958x + 3.7725
20 R2 = 0.998
0
0 0.05 0.1 0.15 0.2 0.25 0.3
[AFG1] / uM
(A) (B)
Figure 6.0 (A) : Differential pulse cathodic stripping voltammograms of AFG1 at
mercury electrode. AFG1 concentrations in (a) BRB pH 9.0; (b) 0.02 uM, (c)
0.04 uM, (d) 0.06 uM, (e) 0.08 uM, (f) 0.1 uM, (g) 0.12 uM, (h) 0.14 uM, (i) 0.18
uM, (j) 0.22 uM, (k) 0.26 uM. (B) The related calibration graph. Conditions: Ei = -
950 mV, Ef = -1400 mV, Eacc = -600 mV, tacc = 80 s, scan rate = 50 mV s-1, pulse
amplitude = 80 mV .
9
Table 1.0 Recovery of spiked AFG1 standard in BRB pH 9.0
[AFG1] Ip (nA) Ep (-mV) [AFG1] % recovered

injected obtained
0.10 uM 56.35 1150 0.0940 uM 94.00

56.84 1150 0.0948 uM 94.80
56.62 1150 0.0942 uM 94.20
x = 94.50 +/- 0.41
(RSD = 0.43%)
0.15 uM 78.21 1140 0.1330 uM 88.68

77.19 1140 0.1313 uM 87.51
77.20 1140 0.1313 uM 87.51
x = 87.91 +/- 0.58
(RSD = 0.77%)
0.20 uM 98.42 1140 0.1691 uM 84.57

99.23 1140 0.1705 uM 85.75
98.14 1140 0.1686 uM 84.30
x = 84.87 +/- 0.77
(RSD = 0.91%
10
Conclusion
A differential pulse cathodic stripping voltammetric technique using mercury

electrode as the working electrode for determination of AFG1 has been
developed. The method presented here is fast, reliable and sensitive technique
for analysis of AFG1 with good precision and recovery. Further study is currently
been carried out for analytical application of this method for quantitation of AFG1
in food sample.
Acknowledgement
The author gratefully acknowledge the University Science Malaysia for giving me
a study leave together with a scholarship under the Academic Staff For Higher
Education Scheme (ASHES). Also we would like to thank all Electroanalytical
group and Chemistry Department staff, UTM for their fully cooperation.
References
[1] Goldblatt L.A. 1969. Aflatoxin:Scientific background, control and implication.
Academic Press, New York, pp 5-10
[2] Salleh B. 1998. Mikotoksin: Implikasinya terhadap kesihatan manusia dan
haiwan, USM, Penang, pp 32 - 49
[3] Begum F and Samajpathi N. 2000. Mycotoxin production in rice, pulses and
oilseeds, Naturwissenschaften, 87: 275-277.
[4] Ordaz J.J., Fente C.C., Vazquez B.I., Franco C.M. and Cepeda A. 2003.
Development of a method for direct visual determination of aflatoxin production
by colonies of the Asperfillus flavus group, Int. J. Food Microbiology, 83, 219-
225.
[5] Batista L. R., Chalfoun S.M., Prado G. Schwan R.F. and Wheals A. E. 2003.
Toxigenic fungi associated with processed (green) coffee beans ( Coffea arabica
L.) . Int. J. Food Microbiology, 85, 293-300.
11
[6] Akiyama H. Goda Y. Tanaka T and Toyoda M. 2001. Determination of
aflatoxins B1, B2, G1 and G2 in spices using a multifunctional column clean-up,
J. Of Chromatography A, 932. 153-157
[7] Aziz N.H., Youseff Y.A., El-Fouly M.Z. and Mousse L.A. 1998. Contamination
of some common medicinal plant samples and spices by fungi and their
mycotoxins. Bot. Bull. Acad. Sin 39, 279-285.
[8] World Health Organisation 1987 in: IARC Monograph on the Evaluation of
Carcinogenic Risk to Human, WHO, Lyon , pp 82 Supl. I
[9] Refai M.K. 1988. Aflatoxins and aflatoxicosis. J. Egypt Vet. Med. Ass. 48, 1-
19
[10] Ministry of Health, Malaysia, Food Act No 231. 1983. Reviewed 2003
[11] Shotwell O.L and Goulden M.L. 1977. Aflatoxins: Comparison of analysis of
corn by various methods, J. Assoc. Off. Anal. Chem 60: 83-88
[12] Gimeno A. and Martins M. L. 1983 Rapid thin layer chromatographic
determination of patulin, citrinin and aflatoxin in apples and pears and their juices
and jams, J. Assoc. Anal. Chem, 66, 85-91.
[13] Scholten J.M and Spanjer M.C. 1996. Determination of aflatoxin B1, B2, G1
and G2 in pastachio kernels and shells, , J. Assoc. Off. Anal. Chem 79: 1360-
1364
[14] Jeffery H.W., Lenorich L.M and Martin R. Jr. 1982. Liquid chromatography
determination of aflatoxins in artificially contaminated cocoa beans. , J. Assoc.
Off. Anal. Chem 80: 1215-1219
[15] Joshua, H. 1993 Determination of aflatoxin by reversed-phase high
performance liquid chromatography with post-column in-line photochemical
derivatization and fluorescence detection, J. Chromatography A, 654: 247-254
[16] Gonzalez M.P.E., Mattusch J. and Wennrich R 1998. Stability and
determination of aflatoxins by high-performance liquid chromatography with
amperometric detection, J. Chromatography A, 828: 439-444
[17] Smyth M. R., Lawellin D. W. and Osteryoung J.G 1979. Polarographic
Study of Aflatoxin B1, B2, G1 and G2: Application of Differential-pulse
12
Polarography to the Determination of Aflatoxin B1 in Various Foodstuffs, Analyst,
104: 73-78
[18] Braitina Kh. Z., Malakhova N.A. and Stojko Y. 2000 Stripping voltammetry
in environmental and food analysis, Fresenius J Anal Chem 368: 307-325
[19] Fifield, F.W. and Haines, P.J. 2000. .Environmental Analytical Chemistry
2nd Ed, Balckwell Science UK, pp 241-242
[20] Yaacob M.H., Mohd Yusof A.R., Ahamad R. and Yakob A.R. 2004. Cyclic
Voltammetry of AFG1 at mercury electrode, Paper will be presented at 3rd
Annual Seminar on Sustainable Science and Management, KUSTEM,
Terengganu, 4 – 5 May 2004.
[21] Wang. 2000. Analytical Electrochemistry. Wiley-VCH, USA.
13
PAPER SENT TO PROF BAREK
TO BE PUBLISHED IN
CHEMICAL ANALYSIS (WARSAW)
( IN PRESS)
DIFFERENTIAL PULSE STRIPPING VOLTAMMETRIC TECHNIQUE FOR
THE DETERMINATION OF AFLATOXIN B2 AT THE MERCURY
ELECTRODE
Mohamad Hadzri Yaacob, Abdull Rahim Hj. Mohd. Yusoff and Rahmalan Ahamad
ABSTRACT
Differential pulse voltammetric technique was developed for determination of aflatoxin
B2 standard (AFB2) in Britton-Robinson Buffer (BRB). Hanging mercury drop electrode
and Ag/AgCl have been used for working and reference electrodes respectively. In this
experiment, the analyte was initially scanned from -1000 mV to -1500 mV in BRB pH
9.0. The effect of pH of supporting electrolyte, scan rate, accumulation potential,
accumulation time and pulse amplitude on the peak potential and peak current of the
analyte were observed. The results showed that AFB2 compound undergoes reduction
reaction at a potential of -1230 mV ( versus Ag/AgCl ). BRB pH 9.0 was the best
supporting electrolyte with the optimised parameters such as scan rate; 50 mV/s,
accumulation potential; -800 mV, accumulation time; 80s and pulse amplitude; 80 mV
which gave the maximum peak height. Under the optimum condition, the peak height of
AFB2 was proportional to the concentrations of the AFB2 in the range of 0.02 to 0.32 uM
with a limit of detection was 0.0159 uM. Relative standard deviation for a eplicate
measurements of AFB2 ( n= 8) with the concentrations of 0.06 and 0.20 uM were 2.80%
and 2.53% respectively. Recoveries of the different AFB2 concentrations in prepared
standard solutions were found to be 85% to 95% with the precision around 0.7% to 2.0%.
Keywords: aflatoxin B2 compound, differential pulse stripping voltammetry, Britton-Robinson buffer and
hanging mercury drop electrode (HMDE)
1. INTRODUCTION
Aflatoxins are mycotoxins produced by certain fungi, especially, Aspergillus flavus and
Aspergillus parasiticus which are found on crops and foodstuffs under certain conditions
(1). They display strong carcinogenicity. Therefore, they are dangerous food
contaminants. They were first identified as the causative agent of the severe outbreak of
“Turkey X” disease, a toxicosis that killed more than 100,000 turkey poults in England in
1960 (2) There are a major concern as human hepatocarcinogens and are considered to
play an important role in the high incidence of human hepatocellular carcinoma in certain
areas of the world. Due to this reason, many countries including Malaysia have set
regulatory demands on the level of aflatoxins permitted in imported and trade
commodities.
2
There are six main aflatoxin compounds such as Aflatoxin B1, B2, G1 and G2 (
designated as AFB1, AFB2, AFG1 and AFG2 ) and aflatoxin M1 and M2 ( designated as
AFM1 and AFM2). The former four compounds are naturally prevalent and the others are
hydroxylated metabolite of AFB1 and AFB2 and were found in the milk of lactating
animals following ingestion of B1 and B2- contaminated feeds (3)
Among them AFB1 exhibits potent carcinogenic and mutagenic characteristics in a

numbers of human and animals. AFB2 has a similar chemical structure with AFB1
whereby the presence of an 8,9-double bond in the form of a vinyl ether at the terminal
furan ring in AFB1, but not in AFB2. The chemical structure of AFB2 is shown in Figure
1.0. This small difference in structure is associated with very significant change in
activity; whereby AFB1 is more toxic than AFB2 (4).
O
O
O O
Figure 1.0: Chemical structure of AFB2
Due to their toxicity, it is important that the level of these contaminants be controlled in
food, soils and water to prevent toxic effect. The way to achieve this is by monitor
samples so that any problem can be identified and dealt with, by doing analysis of
aflatoxins using the analytical techniques at the parts-per-billon (ppb) level. Current
analysis is done by various methods including thin-layer chromatography (TLC) (5),
liquid chromatography (LC) and high-performance liquid chromatography (6,7) and
microtitre plate enzyme-linked immunosorbant assay (ELISA) (8). All these methods
require special equipments operated by skilled personnel and time consume. With the aim
of rapid, simple, accurate and relative low cost analysis, the voltammetric technique is
proposed for this purposes (9).
Voltammetric technique generally refer to a system that combines a triple electrodes

system with a potentiostate for controlling applied potential. It measures the current
response generated by mass transport of electroactive species in the solution to the
working electrode while potential is scanned. The total current response is proportional
to the amount of analyte present in the solution (10). This paper reports the differential
pulse voltammetric determination of AFB2 at a mercury electrode using a cathodic
stripping voltammetric technique.
3
2. EXPERIMENT
2.1 Reagents
Ultrapure water system ( Barnstead / Thermolyne) was used for all dilution and sample
preparation. AFB2 was purchased from SIGMA ( 1 mg per bottle ) and dissolved in
hexane:acetonitrile (98:2) produced 10 ppm standard solution. The stock solutions were
placed in small amber glass tubes and kept in a freezer at -4.0oC.
A stock solution of 0.04 M Britton-Robinson Buffer (BRB) solution was prepared as

follows: 2.47 g boric acid (Fluka), 2.30 ml acetic acid (MERCK) and 2.70 ml
orthoposphoric acid ( Ashland Chemical ) were diluted to 1 l with deionised water.
Britton-Robinson Buffer of pH 2.0 to 13.0 were prepared by adding sodium hydroxide
1.0 M (MERCK) into the stock solution and was used as the supporting electrolyte.
Hexadistilled mercury, grade 9 N, used in a control growth mercury electrode ( CGME )
stand, was purchased from MERCK. The purging was carried out with 99.99% nitrogen.
2.2 Instruments
A BAS CV-50W voltammetric analyser in connection with a CGME stand equipped with
a three-electrode system and a 20 ml capacity BAS MR-1208 cell were used for all the
voltammetric determination. The working electrode was a hanging mercury drop
electrode ( HMDE ). A silver-silver chloride (Ag / AgCl ) and a platinum wire were used
as the reference and counter-electrode respectively. BAS CV-50W was connected to a
computer for data processing. A pH meter Cyber-scan model equipped with a glass
electrode combined with an Ag/AgCl reference electrode, was used for all pH
measurements.
2.3 Procedure
2.3.1 General procedure
The general procedure used to obtain cathodic stripping voltammograms was following
this procedure. A 10 ml aliquot of supporting electrolyte solution was put in a
voltammographic cell. The stir was switched on and the solution was purged with
nitrogen gas for 25 minutes. After forming a new HMDE, accumulate was effected for
the required time at the appropriate potential while stirring the solution. At the end of the
accumulation time the stirring was switched off and after 10 s had elapsed to allow the
solution to become quiescent, the negative going potential was initiated. Ag/AgCl
saturated was used as the reference electrode throughout this study.
4
2.3.2 Procedure for determination of aflatoxin
For the aflatoxin stock standard solution, organic solvent was removed by flowing
nitrogen to dryness followed by addition of the BRB supporting electrolyte solution. The
solution was placed in small amber tube. This solution has been freshly prepared before
voltammetric experiment started. When further volume of aflatoxin were added using a
micropipette into the cell, the solution was redegassed with nitrogen for another 5
minutes before further voltammetric experiment was carried out.
3. RESULTS AND DISCUSSION
3.1 The effect of pH of supporting electrolyte
From our previous cyclic voltammetric studies of AFB2 compound, it was capable of
undergoing reduction process at the mercury electrode in BRB pH 9.0 (11). In the present
study, 2.0 uM AFB2 was cathodically scanned in BRB solution in the pH range of 3.0 to
13.0. This study was carried out with the aim to determine if protons are directly
involved in the reduction process of AFB2. The result showed that in the BRB with pH <
4.0 , no peak was observed. The single and characteristic AFB2 peak was initially
observed starting from pH 5.0 with Ip and Ep were 11.0 nA and -1119mV respectively.
The effect of the pH of supporting electrolyte on the Ip of the AFB2 peak is shown in
Figure 1.0
60
50
40
Ip (n A )
30
20
10
0
4 6 8 10 12
pH
Figure 1.0: Dependence of the peak height of AFB2 on the pH of 0.04 M BRB
solution. AFB2 concentration: 2.0 uM, Ei = 0, Ef = -1.5 V, Eacc = 0, tacc =
15 s and υ = 50 mV/s.
The Ip increased with increasing pH and the highest peak ( 52 nA at Ep of -1192 mV) was
obtained in pH 9.0. The Ip sharply decreased at pH 10.0 to 12.0 and no peak was
significantly appeared the pH 13.0. These results show that the reduction of AFB2 was
totally depends on the pH of supporting electrolyte. The Ep of AFB2 was shifted toward
5
more negative direction with increasing of the pH of the BRB solution which indicates
that in more alkali solution, the reduction of AFB2 was much difficult to take place.
3.2 Optimisation of conditions for the stripping analysis
The voltammetric determination of analyte at trace level normally involves very small
current response. For that reason it is important to optimise all the parameters which may
have influence on the measured current. The effect of initial potential ( Ei), final potential
(Ep) accumulation potential (Eacc ), accumulation time (tacc ), scan rate (υ ) and pulse
amplitude to the Ep and Ip of the AFB2 peak have been studied in BRB pH 9.0. In this
optimisation procedures, 0.06 uM AFB2 was used with initial parameters as follows; Ei
= -1000 mV, Ef = -1500 mV, Eacc = -800 mV, tacc = 80 s, υ = 40 mV/s and pulse
amplitude = 100 mV/s. Using these parameters, the Ip and Ep of 0.06 uM AFB2 were
found to be 13.90 nA and -1250 mV ( vs Ag/AgCl ) respectively.
Effect of Eacc, Ei and Ef
For Ei = -1000 mV and Ef = -1500 mV, the effect of Eacc on the Ip and Ep of AFB2 was
studied. The results revealed that the Ip of AFB2 obtained by using Eacc of -200 mV, -400
mV, -600 mV, -800 mV and -1000 mV were not significantly different which were about
14 nA. To get the maximum response, the Ef was changed from -1500 mV to -1400
mV. The result is shown in Figure 2.0
25
20 A B
Ip (nA)
15
10
0
200 400 600 800 1000
Accum ulation potential (-m V)
Figure 2.0: Effect of Eacc on the Ip of 0.06 uM AFB2 obtained using different
scan potential windows. (A) Ei = -1000 mV, Ef = -1500 mV and
(B) Ei = -1000 mV, Ef = -1400mV.
Using Ef = -1400 mV, the optimum Eacc was -600mV which gave the Ip of 16.84 nA.
The Ep of AFB2 was not changed with different Eacc used in this experiment.
Effect of tacc
6
The effect of the tacc to the Ip of AFB2 (Figure 3.0) shows that by increasing tacc , the Ip
linearly increases following this equation:-
Ip (nA) = 0.4038x ( x s ) - 0.2847 ( R2 = 0.9990, n = 5)
for the range of 20 to 80 s. The maximum peak current ( 31.86 nA) was obtained when
tacc = 80 s. When AFB2 was longer deposited at mercury electrode, the Ip was not
significantly increased, hence the tacc at 80s was chosen as the optimum tacc. The Ep of
AFB2 shifted toward less negative direction with increasing tacc.
50
40
Ip (nA )
30
20
10 [AFB2]=0.6X10-7M
0
0 30 60 90 120 150
Accumulation time (s)
Figure 3.0: Effect of tacc on the Ip of 0.06 uM, Ei = - 1000 mV, Ef = -1400 mV,
tacc = 80 s, υ = 40 mV/s and P.A = 100 mV.
Effect of υ
Relationship between υ and the Ip of the AFB2 also was investigated which revealed that
when the υ increased, the Ip increased as shown in Figure 4a. The Ip was not significantly
different when the υ was more than 60 mV/s. Therefore, 50 mV/s has been selected as
the best υ for determination of AFB2 in BRB pH 9.0. The peak potential of AFB2 has
linearly shifted toward more negative direction for the range of 20 to 50 mV/s and
became level off for the υ of 60 and 80 mV/s as shown in Figure 4b. For the υ of 50
mV/s, the pulse amplitude has to be changed to 80 mV instead of 100 mV otherwise, the
Ip cannot be calculated by software. The maximum Ip obtained from this optimisation was
38.18 nA which was about 200% higher than before being optimised. Figure 6.0
illustrates the voltammogramms of 0.06 uM AFB2 obtained under optimum and
unoptimum conditions.
50 1225
40 1220
E p (-m V )
Ip (nA)
30 1215
20 1210
10 [AFB2]=0.6X10-7M 1205 7
[AFB2]=0.6X10-7M
0 1200
0 20 40 60 80 100 0 20 40 60 80 100
Scan rate (mV/s) Scan rate (mV/s)
(a) (b)
Figure 4.0: Effect of υ on the Ip (a) and Ep (b) of AFB2. Ei = - 1000 mV,
Ef = -1400 mV, Eacc = -600 mV, tacc = 80s and P.A = 100 mV
Figure 5.0: Voltammogramms of 0.06 uM AFB2 obtained under

unoptimum (a) and optimum (b) parameters.
From this optimisation procedures, the selected optimum parameters for determination of
AFB2 compound in BRb pH 9.0 were Ei = -1000 mV, Ef = -1400 mV, Eacc = -600 mV,
tacc = 80 s, υ = 50 mV/s, pulse amplitude = 80 mV and quite time = 10 sec.
3.2.4 Calibration curve of AFB2

Using the optimum parameters, a calibration curve has been prepared by a series of
standard addition of AFB2 as shown in Figure 6.0. The range of linearity was found to be
from 0.02 u M to 0.32 u M with the limit of detection of 0.0158 uM. Figure 7.0 shows
the voltammograms of AFB2 obtained by this standard addition.
8
200
180
160
140
120
ip (nA)
100
80
y = 5.55x + 3.6435
60
40 R2 = 0.9978
20
0
0 10 20 30 40
[AFB2] X10-8M
Figure 6.0: Linear plot of Ip versus concentration of AFB2 in BRb pH=9.0
Figure 7.0: Cathodic stripping voltammograms for increasing concentration

of AFB2 (1) background (2) 0.02 (3) 0.06 (4) 0.10 (5) 0.14 (6) 0.18
(7) 0.22 (8) 0.26 and (9) 0.32 uM. E acc = -600 mV, t acc = 80 s, υ = 50
mV/s, P.A = 80 mV and BRB of pH 9.0
For determination of accuracy of the proposed method, the reproducibility was calculated
from 8 independent runs of 0.06 uM and 0.20 uM of AFB2 solution, obtaining the
relative standard deviation (RSD) of 2.80 % and 2.53 % respectively. In order to check
the precision of the method, a recovery study has also been carried out by adding of the
different concentration of AFB2 (0.10 uM and 0.25 uM) into the voltammetric cell and
measuring the peak currents of the respective concentrations. The actual amount of AFB2
found in the cell were calculated using a regression equation obtained from prepared
calibration curve. The recovery obtained were 86% to 95% with RSD of 1.68% and
0.86% ( n = 3) respectively as shown in Table 3.1. The recovery study using real sample
will be performed in future work..
9
Table 1.0: Mean values for the recovery of AFB2 (n =3) in prepared AFB2 standard
solution ( n = 3 )
No of experiment Amount added Peak current Amount found Recovery (%)

( uM) (nA) (uM)
1 0.10 51.85 8.683 86.83 +/- 1.46
2 0.25 134.43 23.56 94.26+/0.81
4. CONCLUSION
The use of the DPCSV technique has been proved to be a useful, simple, accurate and
efficient alternative in the quantitative assay of trace amounts of AFB2. The sensitivity of
this approach is much greater than obtained by differential pulse polarographic technique
(12). The optimised parameters will be applied to determine other aflatoxin compounds
such as AFB1, AFG1 and AFG2. Further experiment will be performed to increase it’s
sensitivity in future work together with a recovery study of AFB2 using real samples such
as ground nut and nut products. An alternative approach will also be studied by using
different working electrode such as screen print electrode.
5. ACKNOWLEDGEMENTS
The author gratefully acknowledge the Universiti Sains Malaysia (USM) for granting a
study leave together with a scholarship under the Academic Staff For Higher Education
Scheme (ASHES). Also we would like to thank all Chemistry Department staff, UTM
for their fully cooperation.
6. REFERENCES
1. Eaton D.L and Groopman J.D. (Eds) (1994), The toxicity of aflatoxin. Academic
Press, United Kingdom.
2. Goldblatt L.A (1972) Aflatoxin: Scientific background, control and implication,

Academic Press, New York (1972)
3. Andeou V.G., Nikolelis D. and Tarus B. (1997). Electrochemistry investigation of

transduction of interactions of alfatoixin M1 with bilayer lipid membranes (BLMs),
Anal. Chim. Acta, 350. 121-127.
10
4. Jaimez J., Fente C.A., Vazquez B.I., Franco C.M., Cepeda A., Mahuzier G. and
Prognon P (2000). Application of the assay of aflatoxins by liquid chromatography
with fluorescence detection in food analysis. J. Chromatography A.” 882: 1-10
5. Gimeno A. and Martins M. L (1983) Rapid thin layer chromatographic determination

of patulin, citrinin and aflatoxin in apples and pears and their juices and jams, J.
Assoc. Anal. Chem, 66, 85-91.
6. Vahl M. and Jorgensen (1998). Determination of aflatoxins in food using LC/MS/MS,

Z Lebensm Unters Forsch A , 206. 243-245
7. Kussak A., Anderson B and Anderson K. (1995). Determination of aflatoxins in

airborne dust from feed factories by automated immunoaffinity column clean-up and
liquid chromatography, J Of Chromatography A, 708. 55-60
8. Pesavento M., Domagala S., Baldini E. and Cucca L. (1997). Characterisation of

enzyme linked immunosorbent assay for aflatoxin B1 based on commercial reagents,
Talanta, 45. 91-104.
9. Braitina Kh.Z Malakhova N.A. and Stojko Y. (2000) Stripping voltammetry in

environmental and food analysis, Fresenius J Anal Chem 368: 307-325
10 Fifield F.W. and D. Kealey (2000), Principle and practice of analytical chemistry 3rd
Edition, Blackwell science, UK
11.Yaacob M.H, Yusoff A.R, Ahamad R and Yaakob A.R (2003), Cyclic voltammetric
study of aflatoxin B2 at the mercury electrode, Paper presented at 16th Symposium Of
Chemical Analysis, 9-11th September 2003, Kucing, Sarawak, Malaysia.
12. Smyth M. R., Lawellin D. W. and Osteryoung J.G (1979) Polarographic Study of
Aflatoxin B1, B2, G1 and G2: Application of Differential-pulse Polarography to the
Determination of Aflatoxin B1 in Various Foodstuffs, Analyst, 104: 73-78
11
Paper will be published in
Malaysian Journal of Analytical Science

CYCLIC VOLTAMMETRIC STUDY OF AFLATOXIN B2 AT THE MERCURY
ELECTRODE
Mohamad Hadzri Yaacoba, Abdull Rahim Hj. Mohd. Yusoffb and Rahmalan
Ahamadb
a
School Of Health Sciences, USM, 16150 Kubang Krian, Kelantan
b
ABSTRACT
A cyclic voltammetry (CV) study of aflatoxin B2 (AFB2) in Britton-Robinson

buffer using a HMDE is described. CV was carried out by anodic and cathodic potential
scan through within the range of 0 to -1500 mV with no accumulation time. The effect of
the different scan rate on the peak height and peak potential of the analyte were also
studied. The results from this study showed that the reduction process on the hanging
mercury electrode gives a single characteristic cathodic peak at -1315 mV ( versus
Ag/AgCl ) in pH of 9.0. Effect of the scan rate on both responses has proved that the
reduction of AFB2 is irreversible reaction and the limiting current is adsorption
controlled.
Keywords: aflatoxin B2 compound, cyclic voltammetry, Britton-Robinson buffer and hanging mercury
drop electrode (HMDE)
1. INTRODUCTION
Aflatoxins are mycotoxins produced by certain fungi, especially, Aspergillus

flavus and Aspergillus parasiticus which are found on crops and foodstuffs under certain
conditions (1). They were first identified as the causative agent of the severe outbreak of
“Turkey X” disease, a toxicosis that killed more than 100,000 turkey poults in England in
1960 (2) They display strong carcinogenicity. Therefore, they are dangerous food
contaminants and many countries including Malaysia have set regulatory demands on the
level of aflatoxins permitted in imported and trade commodities.
Aflatoxin B2 (AFB2) is one of the naturally occurrence aflatoxins. The chemical

structure of this compound is shown in Figure 1.0.
a
Corresponding Address:
Chemistry Dept, Faculty of Science, UTM, 81300 Skudai, Johor
Tel: 07- 553 4540 e-mail: mhy02psk@yahoo.com
2
O
O
O O
Figure 1.0: Chemical structure of AFB2
Due to its toxicity, it is important that the level of this contaminant be controlled
in food, soils and water to prevent toxic effect. The way to achieve this is to monitor
samples so that any problem can be identified and dealt with by doing analysis of
aflatoxin using the analytical techniques that are necessary to determine aflatoxin at the
parts-per-billon (ppb) level. Currently, analysis is done by various methods including
thin-layer chromatography (TLC) (3), liquid chromatography (LC) and high-performance
liquid chromatography (4,5) and microtitre plate enzyme-linked immunosorbant assay
(ELISA) (6). All these methods are time consuming and require special equipments
operated by skilled personnel.
The proposed voltammetric technique generally refer to a system that combines a

triple electrodes system with potentiostat for controlling applied potential. This technique
is rapid, simple and relative low cost (7). It measures the current response generated by
mass transport of electroactive species in solution to the working electrode while
potential is scanned The total current response is proportional to the amount of analyte
present in the solution (8). This paper reports the results from the cyclic voltammetric
study of AFB2 at a mercury electrode.
2. EXPERIMENT
2.1 Reagents
Ultrapure water system ( Barnstead / Thermolyne) was used for all dilution and sample
preparation. AFB2 was purchased from SIGMA: 1 mg was dissolved in hexane:
acetonitrile (98:2) to produce 10 ppm each. The stock solutions were placed in small
glass tubes covered with an aluminum foil and kept in a freezer at -4.0 oC. A stock of
Britton-Robinson Buffer (BRB) solution 0.04 M was prepared as follows: 2.47 g boric
acid (Fluka), 2.30 ml acetic acid (MERCK) and 2.70 ml orthoposphoric acid ( Ashland
Chemical ) and were diluted to 1 l with deionised water. Hexadistilled mercury, grade
3
9N, used by the Controlled Growth Mercury Electrode (CGME) stand, was purchased
from MERCK. The purging was carried out with nitrogen gas with purity 99.99%.
2.2 Instruments
A BAS CV-50W voltammetric analyser in connection with a Control Growth

Mercury Electrode (CGME) stand equipped with a three-electrode system and a 20 ml
capacity BAS MR-1208 cell was used for all the voltammetric determination. The
working electrode was a Hanging Mercury Drop Electrode (HMDE). As reference and
counter-electrode, a silver-silver chloride (Ag / AgCl) and a platinum wire were used,
respectively. All potentials are quoted relative to this reference electrode. BAS CV-50W
was connected to a computer for data processing. A pH meter Cyber-scan model
equipped with a glass electrode combined with an Ag/AgCl reference electrode, was
employed for the pH measurements.
2.3 Procedure
Cyclic voltammetry was studied within the range of 0 to -1500 mV with different
scan rate from 20 to 500 mV/s in BRB pH 9.0 as the supporting electrolyte. The HMDE
and Ag/AgCl were used as the working and reference electrode respectively through out
this experiment . The general procedure used to obtain cyclic voltammograms was as
follows. A 10 ml aliquot of buffer solution was placed in a voltammographic cell using a
micropipette. The stir was switched on and the solution was purged with nitrogen gas for
10 minute. After forming a new HMDE, potential scan was effected using different scan
rate while stirring the solution. For the aflatoxin standard stock solution, organic solvent
was removed by flowing nitrogen gas to dryness followed by adding of the BRB
supporting electrolyte solution. When further volume of aflatoxin were added to the cell,
the solution was redeoxygenated with nitrogen gas for 5 minute before carried out further
voltammetry followed the mentioned procedures.
3. RESULTS AND DISCUSSION
Cathodic cyclic voltammogrammes of 1.3 X 10-6 M AFB2 in BRB solution at

pH=9.0 and a scan rate (υ) of 200 mV/s can be observed in Figure 2.0. A sharp and well
defined cathodic wave around -1.315 V ( against Ag/AgCl ) with a peak current of 30 nA
is observed due to the reduction of the AFB2. No oxidation wave appears in the anodic
branch, which indicates that the AFB2 reduction is irreversible. This was confirmed by
anodic cyclic voltammetric that gave the same results as shown in Figure 3.0
4
Figure 2.0: Cathodic cyclic voltammogram for 1.3 X10-6 M AFB2,
obtained at υ of 200 mV/s, Ei = 0, Elow = -1.5 V and Ehigh = 0
in BRB solution at pH=9.0. ( 1 ) cathodic and (2) anodic scans
Figure 3.0: Anodic cyclic voltammogram for 1.3 X10-6 M AFB2, obtained at υ
of 200 mV/s, Ei = -1.5, Elow = -1.5 V and Ehigh = 0 in BRB solution at
pH=9.0. ( 1 ) anodic and (2) cathodic scans
Standard additions of AFB2 was carried out to confirm that the observed peak is
related to the reduction of AFB2. Figure 4.0 shows that the Ep at around -1.315 V is
referred to the AFB2 peak since there are no significant change in location of the peak
and the Ip increases with increasing of the AFB2 concentration. There are no extra peak
apparently observed with this standard addition. The corresponding equation of this
5
dependence of Ip to concentration of AFB2 for cathodic cyclic voltammetric is at pH
=9.0,
Ip (nA) = 23.839 x ( x 10 -6 M) + 4.44 ( r = 0.9817, n = 4 )

The regression equation shows that the Ip of AFB2 does not very linearly with the
concentration of AFB2 suggesting formation of a compact film on the electrode surface
(9).
Figure 4.0 Effect of AFB2 concentration on the Ip of cathodic cyclic

voltammetric curve in BRB at pH =9.0. (a) 1.3 X10-6 M ,
(b) 2.0 X 10-6 M (c) 2.7 X10-6 M and (d) 3.4 X 10-6 M.
A result from repetitive cathodic stripping voltammetry experiment is shown in

Figure 5.0. The result showed that the reduction peak of AFB2 increases with number of
cycles indicating irreversible reduction with adsorption of AFB2 at the mercury electrode
surface. No other cathodic peak was observed.
6
Figure 5.0: Repetitive cathodic cyclic voltammograms of 1.3 X 10-6 M AFB2 in
BRB solution at pH of 9.0.
The adsorption of AFB2 on the electrode surface is expected due to the AFB2
chemical structure, that consists of functional groups such as ketone, ester and ether. The
presence of these groups increases the polarity and enhance absorption of molecules at
the electrode surface (10). Ip increases with the number of cycle. However, a further
increases in the repetitive cycle tend to slow down the increment of the Ip due to the
deformation of double layer at the mercury electrode surface (11).
The effect of increasing scan rate from 20 to 500 mV/s to the Ep and Ip of AFB2
cathodic peak were observed under the same experiment conditions. Linear relationship
was observed between the log Ip versus log υ with a slope of 0.5097 ( R2 = 0.995, n = 9 )
as shown in Figure 6.0. The slope of more than 0.5 indicates that the diffusion current is
influenced by an adsorption on the electrochemical process at the electrode surface (12,
13). The plot of Ep versus log scan rate which is a linear graph as shown in Figure 7.0
( R2 = 0.9936, n = 9 ) confirmed that the reduction of AFB2 on the electrode surface is
totally irreversible. This irreversible reaction is also confirmed by observing the shifted
of Ep towards more negative direction when the υ is increased, according to the following
equation:
Ep (-mV) = 40.065log υ + 1223.2 ( R = 0.9936, n = 9)
1.9
1.7
1.5
log Ep
1.3
1.1
0.9
y = 0.5097x + 0.4115
R2 = 0.995
0.7
0.5
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8
log V
Figure 6.0: Plot of log Ip versus log υ for 1.3 X10-6 M AFB2 in BRB solution at pH 9.0
7
1340
1330
1320
Ep (-mV)
1310
1300
1290 y = 40.065x + 1223.3
1280 R2 = 0.9936
1270
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8
log V
Figure 7.0: Plot of Ep versus log υ for 1.3 X10-6 M AFB2 in BRB solution at pH 9.0
4. CONCLUSION
From the CV study the AFB2 reduced at the mercury electrode produced a single
and well characteristic cathodic peak at -1.315 V ( versus Ag/AgCl) . The reduction is
totally irreversible reaction and the diffusion current is controlled by adsorption process.
Further work on stripping voltammetric study of this compound will be carried out.
5. ACKNOWLEDGEMENTS
The author gratefully acknowledge the University Science Malaysia for giving me a
study leave together with a scholarship under the Academic Staff For Higher Education
Scheme (ASHES). Also we would like to thank all Chemistry Department staff, UTM for
their fully cooperation.
6. REFERENCES
1. Eaton D.L and Groopman J.D. (Eds) (1994), The toxicity of aflatoxin. Academic
Press, United Kingdom.
2. Goldblatt L.A (1972) Aflatoxin: Scientific background, control and implication,

Academic Press, New York (1972)
8
3. Gimeno A. and Martins M. L (1983) Rapid thin layer chromatographic determination
of patulin, citrinin and aflatoxin in apples and pears and their juices and jams, J.
Assoc. Anal. Chem, 66, 85-91.
4. Vahl M. and Jorgensen (1998). Determination of aflatoxins in food using LC/MS/MS,

Z Lebensm Unters Forsch A , 206. 243-245
5. Kussak A., Anderson B and Anderson K. (1995). Determination of aflatoxins in

airborne dust from feed factories by automated immunoaffinity column clean-up and
liquid chromatography, J Chromatography A, 708. 55-60
6. Pesavento M., Domagala S., Baldini E. and Cucca L. (1997). Characterisation of

enzyme linked immunosorbent assay for aflatoxin B1 based on commercial reagents,
Talanta, 45. 91-104.
7. Braitina Kh.Z Malakhova N.A. and Stojko Y. (2000) Stripping voltammetry in

environmental and food analysis, Fresenius J Anal Chem, 368. 307-325
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York .
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Verlag, New York.
11. Fifield F.W. and Kealey D (2000) Principle and Practice of Analytical Chemistry 3rd
Edition, Balckwell Science, U.K
12. Gosser D.K (1993) Cyclic voltammetry, Wiley-VCH, New York
13. Yaridimer C. and Ozaltin N. (2001) Electrochemical studies and differential pulse
polarographic analysis of lansoprazole in pharmaceuticals, Analyst, 126. 361-366
9
PAPER WAS PUBLISHED IN
J. Kimia Analisis (Universiti Sumatera Utara, Medan, Indonesia)
Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. (2005). Application of Differential
Pulse Cathodic Stripping voltammetry (DPCSV) Technique in Studying Stability of
Aflatoxins. J. Sains Kimia. 9(3): 64-70.
1
APPLICATION OF DIFFERENTIAL PULSE CATHODIC
STRIPPING VOLTAMMETRIC TECHNIQUE IN STUDYING
STABILITY OF AFLATOXINS
Mohamad Hadzri Yaacob1, Abdull Rahim Hj. Mohd. Yusoff2 and Rahmalan Ahamad2
1
School Of Health Sciences, USM, 16150 Kubang Krian, Kelantan, Malaysia
2
Dept of Chemistry , Faculty of Sciences, UTM, 81310 UTM, Skudai, Johor, Malaysia
Abstract A stability studies of aflatoxins (AFB1, AFB2, AFG1 and AFG2 ) in Britton-Robinson buffer (BRb) using a
Differential Pulse Cathodic Stripping Voltammetric (DPCSV) technique is described. The DPCSV was performed by
cathodic potential scan through within the range of -950 to -1400 mV with 80s accumulation time using a BRb at pH
9.0 as the supporting electrolyte. The samples were exposed for 0 to 8 hrs to normal laboratory condition before being
scanned under optimised voltammetric parameters. Using the same procedure, aflatoxins in different pH of BRb were
also studied after 0 to 3 hours exposed to the same condition. Other stability study was performed by scanning
aflatoxins in BRb solution which were kept in freezer from first to six months of storage time. The results from these
studies showed that AFB1 and AFB2 in BRb pH 9.0 were stable within 8 hours exposure time while AFG1 and AFG2
were stable up to a few hours. All aflatoxins scanned in BRB pH 6.0, 7.0 and 9.0 were more stable as compared when
there were scanned in BRB pH 11.0 within 3 hours exposure time. The results also showed that the stability of the
samples which were prepared in BRB pH 9.0 was affected by longer storage time in freezer.
Abstrak Satu kajian terhadap kestabilan aflatoksin (AFB1, AFB2, AFG1 and AFG2 ) di dalam larutan penimbal
Britton-Robinson (BRb) menggunakan teknik Voltammetri Perlucutan Kethodik Denyut Pembeza (DPCSV) adalah
dilaporkan. Kaedah ini dijalankan dengan cara mengimbas keupayaan katodik di dalam julat di antara -950 ke -1400
mV dengan masa penjerapan 80 s menggunakan larutan penimbal pada pH 9.0 sebagai larutan penyokong. Sampel
didedahkan kepada persekitaran makmal dari 0 hingga 8 jam sebelum diimbas menggunakan parameter voltammetri
yang optimum. Menggunakan prosedur yang sama, aflatoksin yang disediakan di dalam larutan penimbal yang
berbeza pH juga dikaji kestabilan dari 0 hingga 3 jam terdedah kepada kondisi yang sama. Kajian kestabilan yang lain
yang dijalankan termasuk mengimbas aflatoksin yang disediakan di dalam larutan penimbal dan disimpan di dalam
penyejukbeku dari bulan pertama hingga ke bulan keenam. Keputusan dari kajian ini menunjukkan bahawa AFB1 dan
AFB2 di dalam larutan BRb stabil di dalam tempuh 8 jam terdedah kepada keadaan biasa makmal dan AFG1 serta
AFG2 hanya stabil di dalam tempuh beberapa jam sahaja. Semua aflatoksin yang diimbas di dalam pH 6.0, 7.0 dan 9.0
di dalam tempuh 3 jam masa pendedahan adalah lebih stabil berbanding jika ianya diimbas di dalam pH 11.0.
Keputusan juga menunjukkan bahawa kestabilan sampel yang disediakan di dalam BRb dengan pH 9.0 adalah
dipengaruhi oleh jangkamasa simpanan di dalam penyejukbeku.
Keywords: AFB1, AFB2, AFG1, AFG2 compounds, stability studies and differential pulse cathodic
stripping voltammetry
INTRODUCTION
Aflatoxins are a group of secondary metabolites produced by Aspergillus flavus and Aspergillus
parisiticus, which are found on crops and foodstuffs under certain conditions [[1]. Aflatoxins B1, B2, G1
and G2 have been found to be naturally and they display strong carcinogenicity. There are dangerous food
contaminants and have been classified as Type I toxic materials by International Agency for Research
1
Corresponding Address:
Chemistry Dept, Faculty of Science, UTM, 81300 Skudai, Johor
Tel: 07- 553 4540 or 019-939 2440 or e-mail: mhy02psk@yahoo.com
( Note: A part of this paper was presented in Simposium Kimia Analisis Malaysia ke 17 (SKAM-17), 24 –
26th August 2004 at Kuantan, Pahang, Malaysia)
2
Cancer (IARC) in 1984 [2]. Due to its toxicity, many countries have set stringent regulatory demands on
the level of aflatoxins permitted and traded commodities. In our country, the regulatory levels for total
aflatoxins in raw peanuts are 15 ppb and 5 ppb for other foodstuffs [3].Aflatoxins are not totally diminished
by normal and cooking temperatures [4,5]. The chemical structures of AFB1, AFB2, AFG1 and AFG2 are
shown in Figure 1.0 [6].
O
O O
O
O
O
O O
O O
O
O
(a) (b)
O O O O
O O O O
O
O
O
O
O
O
(c) (d)
Figure 1.0: Chemical structures of (a) AFB1, (b) AFB2, (c) AFG1 and (d) AFG2
Many methods have been used for quantitative determination of aflatoxins includes thin layer
chromatography (TLC) [7-9], high performance liquid chromatography (HPLC) with different type of
detectors such as ultra-violet [10-12], fluorescence [13-15] and amperometer detectors [ 16]
In this study, all aflatoxins have been investigated for its stability in BRB pH 9.0 using DPSV
technique. This technique was chosen rather than UV-VIS spectrophotometer because it uses the principle
of the measurement of peak height of electro active species in sample solution when potential was applied
into it [17]. It can avoid problem arise from weak fluorescence property of AFB1 and AFG1 [16]. This
technique also offers shorter time analysis compare to the HPLC technique [17].
In the present paper, we describe the results of stability studies on AFB1, AFB2, AFG1 and AFG2
which were prepared in BRB pH 9.0 and underwent hour to hour monitoring and also for shelf life of all
aflatoxins in BRB pH 9.0 which was kept in fridge for six months.
3
EXPERIMENTAL
Reagents
All reagents employed were of analytical grade. Water purified from a Nano Pure Ultrapure water
system ( Barnstead / Thermolyne) was used for all dilution and sample preparation. Aflatoxins were
purchased from SIGMA ( 1 mg per bottle ) and dissolved in benzene:acetonitrile (98:2) to produce 10 ppm
standard solution each. The stock solutions were placed in small amber glass tubes and kept in a freezer at -
4.0oC. The solution was measured under UV-VIS spectrometer for it absorbance at 365 +/- nm before
being used for voltammetric analysis.
A stock solution of 0.04 M Britton-Robinson Buffer (BRB) solution was prepared as follows: 2.47
g boric acid (Fluka), 2.30 ml acetic acid (MERCK) and 2.70 ml orthophosphoric acid ( Ashland Chemical )
were diluted to 1 l with deionised water. Britton-Robinson Buffer of pH 5, 7.0 and 11.0 were prepared by
adding sodium hydroxide 1.0 M (MERCK) into the stock solution.. Hexadistilled mercury, grade 9 N, used
in a control growth mercury electrode ( CGME ) stand, was purchased from MERCK. The purging was
carried out with 99.99% nitrogen.
Instruments
A BAS CV-50W voltammetric analyser in connection with a CGME stand equipped with a three-
electrode system and a 20 ml capacity BAS MR-1208 cell were used for all the voltammetric
determination. The working electrode was a hanging mercury drop electrode (HMDE). A silver-silver
chloride (Ag / AgCl) and a platinum wire were used as the reference and counter-electrode respectively.
BAS CV-50W was connected to a computer for data processing. A pH meter Cyber-scan model equipped
with a glass electrode combined with an Ag/AgCl reference electrode, was used for all pH measurements.
Procedure
General procedure
The general procedure used to obtain cathodic stripping voltammograms was as follows. A 10 ml
aliquot of supporting electrolyte solution was put in a voltammographic cell. The stir was switched on and
the solution was purged with nitrogen gas for at least 10 minutes. After forming a new HMDE, accumulate
was effected for the required time at the appropriate potential while stirring the solution. At the end of the
accumulation time the stirring was switched off and after 10 s had elapsed to allow the solution to become
quiescent, the negative going potential was initiated. Ag/AgCl saturated was used as the reference electrode
throughout this study.
Procedure for determination of aflatoxin
The aflatoxin standard solutions have been freshly prepared by removing of organic solvent in
aflatoxin stock solutions until dryness followed by the addition of the BRb supporting electrolyte solution.
The solution was placed in small amber bottles. When further volume of aflatoxin were added using a
micropipette into the voltammetric cell, the solution was purged with nitrogen for another 2 minutes before
further voltammetric experiment was carried out.
Procedure for stability studies
For hour to hour stability studies, 312 ul, 315 ul, 328 ul and 330 ul of 1 ppm AFB1, AFB2,
AFG1 and AFG2 standard solution in BRB pH 9.0 respectively were injected into 10 ml supporting
electrolyte in the voltammetric cell separately and were scanned following the above stated procedures.
4
Eight measurements have been made. The peak heights and peak potential of each aflatoxins were
recorded. After these measurements, the aflatoxins standard solutions in voltammetric cell were exposed to
normal laboratory condition without purging. Measurements were taken every hour up to 8 hours exposure
time.
For shelf life stability studies, all aflatoxin standard solutions which were kept in freezer were
subjected to the voltammetric measurement in BRb pH 9.0 every month up to 6 months keeping period.
For aflatoxins in different pH of BRB, the stability studies were performed by scanning aflatoxins
solution (prepared in BRB pH 9.0) in four difference pH of BRB as the supporting electrolytes which were
6.0, 7.0, 9.0 and 11.0. The voltammetric determination procedures were followed the same procedures as
for the hour to hour stability studies except the exposure time was limited up to 3 hours.
From our previous experiment using a cyclic voltammetry technique , AFB1, AFB2, AFG1 and
AFG2 show an irreversible reduction peak at -1235 +/- 10 mV, ( all against Ag/AgCl) respectively [18,
19]. Based on this electro active property, differential pulse cathodic stripping voltammetry (DPCSV)
technique was applied in this study.
Voltammograms of aflatoxins
The voltamograms of 0.1 uM AFB1, AFB2, AFG1 and AFG2 scanned in BRb pH = 9.0 are
shown in figures 2.0a -2.0d. Replicate measurements of peak height of respective aflatoxins give the
repeatability results of 63.19 nA, 65.67 nA, 60.76 nA and 61.79 nA respectively with coefficient variation
of 1.50 to 2.60 % for all aflatoxins. The results show that the DPSV technique give high consistent results
with very small error and could be applied in stability studies of aflatoxins.
(a) (b)
(c) (d)
Figure 2.0 :Voltammograms ( n = 8) of 0.1 uM AFB1 (a), AFB2 (b), AFG1 (c) and AFG2 (d) in BRb pH = 9.0.
5
Hour to hour stability studies;
The results of these studies using 0.10 uM AFB1, AFB2, AFG1 and AFG2 in BRB pH 9.0 are
represented in Figure 3.0.
70
60
Peak height (nA)
50 AFB1
40 AFB2
30 AFG1
20 AFG2
10
0
0 1 2 3 4 5 6 7 8 9
Exposure time (hrs)
Figure 3.0: Peak heights of AFB1, AFB2, AFG1 and AFG2 in BRb pH = 9.0 exposed to normal condition up to 8
hours.
The results show that AFB1 and AFB2 in BRb pH 9.0 were stable within 8 hours exposure time
where the peak heights for both aflatoxins were reduced only 15%. In contrast, AFG1 and AFG2 gave
significant reduced of peak heights within the same period of exposure time where the peak height of
AFG1 and AFG2 were reduced to 50% and 58% respectively. The results show that AFG1 and AFG1 were
less stable in BRb pH 9.0 as compared to AFB1 and AFB2. Based on their chemical structures, the most
reactive functional groups for ease of attack by chemical reagents are two lactones rings of AFB1 and
AFG1 and cyclopentanone rings in AFB2 and AFG2. These lactones can be readily opened by hydrolysis
with strong alkalis such as sodium hydroxide. Other functional group of this aflatoxin is less readily
attacked by chemical reagents. The methyl ether and furan ether groups would be cleaved only by very
strong acids such as hydriodic acid. The double bond of terminal furan rings of AFB1 and AFG1 are
susceptible to attack by electrophilic reagents and can be reduced or oxidised [20].
Shelf life stability studies
For shelf life stability studies, the results are shown in Figure 4.0
80
70
60
Peak height (nA)
AFB1
50
AFB2
AFG1
40
AFG2
30
20
10
0 1 2 3 4 5 6 7
Figure 4.0: Peak heights of aflatoxins in BRb pH 9.0 which were kept up to 6 months in freezer.
6
The results showed that after three months storage in freezer, the peak heights of AFG1 and AFG2
in BRB pH 9.0 were decreased to about 70% from original peak height measured at first day of preparation.
AFB2 was the most stable within 2 months keeping period. These results revealed that AFB1 and AFB2
were stable within 3 months kept in fridge but gradually decreased after that time. Peak heights of AFG1
and AFG1 were gradually decreased since first month keeping time up to 6 months.
Stability studies in different buffer solutions
The results for these studies are shown in Figure 5.0a to 5.0d. The results show that in acid and
neutral conditions, peak heights of all aflatoxins were increased when the exposure time were longer. In
BRb pH 9.0, the peak heights were not significantly affected by longer exposure time. This phenomenon
may be because of all aflatoxins need enough time to be completely dissolved in acid and neutral condition
compared in basic medium. In strong base, the peak height was significantly decreased where AFG2 has
not given any peak after 3 hours exposure time. These results indicate that in neutral and acid conditions,
aflatoxins take time to stabilise themselves and at strong base, it was slowly reacted while cyclopentanone
and lactone groups slowly damaged (21). AFG1 and AFG2 show decreasing in peak height faster than
aFB1 and AFB2. This was due to the easy of lactones group in both compounds attacked in strong basic
condition compared to acidic condition. Other chemical group was not affected in acid or neutral medium
as can be observed from the peak heights of all aflatoxins in BRB pH 6.0 and 7.0 within 3 hours standing
time. At 0 hrs standing time, the peak heights of all aflatoxins in BRB pH 6.0, 7.0 and 11.0 were lower as
compared in BRb pH 9.0 due to those pH were not an optimum pH for DPCSV analysis of aflatoxins as
found in our previous experiment [22].
70 70
60 60
50 50
AFB1 AFB1
40 40
Ip (nA)
Ip (nA)
AFB2 AFB2
30 AFG1 30 AFG1
AFG2 AFG2
20 20
10 10
0 0
0 1 2 3 0 1 2 3
Standing tim e (hrs ) Standing tim e (hrs )
(a) (b)
70
45
60 40
35
50
30 AFB1
AFB1
Ip (nA)
40 25 AFB2
Ip (nA)
AFB2
AFG1 20 AFG1
30
AFG2 15 AFG2
20
10
10 5
0
0
0 1 2 3
0 1 2 3
Standing tim e (hrs ) Standing tim e (hrs )
(c) (d)
Figure 4.0: Peak heights of 0.1 uM aflatoxins in BRb at pH (a) 6.0, (b) 7.0, (c) 9.0 and (d) 11.0 exposed to normal
laboratory condition up to 3 hrs standing time.
7
CONCLUSION
Using DPCSV technique, stability studies of aflatoxins have been successfully performed. It was
concluded that AFB1 and AFB2 in BRb pH 9.0 which were exposed to normal condition were more stable
as compared to AFG1 and AFG2 in the same medium. Shelf life of AFB1 and AFB2 keeping in freezer
were longer compared to AFG1 and AFG2. All aflatoxins in BRb pH 6.0, 7.0 and 9.0 which were exposed
to normal condition up to three hours were stable but in BRb pH 11.0, their peak heights were
significantly decreased.
ACKNOWLEDGEMENT
One of the author is gratefully acknowledge the University Science Malaysia for approved him a
study leave and financial support for his study at UTM. Also we would like to thank UTM for a
fundamental research grant (Vot No 75152) to carry out this study.
REFERENCES
1. Begum F and Samajpathi N (2000), Mycotoxin production in rice, pulses and oilseeds,Naturwissenschaften, 87:
275-277
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humans, vol. 56. World Health Organisation, Lyon.
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Malaysia
4. Creepy E.E (2002). Update of survey, regulation and toxic effect of mycotoxins Europe, Toxicology Letter, 127:
19-28
5 Samarajeewa U., Sen A.C., Cohen M.D. and Wei C.L (1990). Detoxification of Aflatoxins in foods and feeds by
physical and chemical methods Food Protection, 53, 489-501.
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7. Gimeno A. and Martins M. L (1983) Rapid thin layer chromatographic determination of patulin, citrinin and
aflatoxin in apples and pears and their juices and jams, J. Assoc. Anal. Chem, 66, 85-91.
8. Bicking M.K.L., Kniseley R.N and Svec H.J (1983), Coupled-Column for quantitating low levels of aflatoxins, J .
Assoc. Off. Anal. Chem. 66, 905-908.
9. Tutour L.B., Elaraki A. T. and Aboussalim A.,(1984). Simultaneous thin layer chromatographic determination of
aflatoxin B1 and ochratoxin A in black olives, J Assoc Off Anal Chem 67: 611-620.
10. Rastogi S., Das M. and Khanna S.K. (2001). Quantitative determination of aflatoxin B1-oxime by column liquid
chromatography with ultraviolet detection. J Of Chromatography A, 933: 91-97
11. Joshua, H (1993) Determination of aflatoxin by reversed-phase high performance liquid chromatography with
post-column in-line photochemical derivatization and fluorescence detection, J. Chromatography A, 654: 247-254
12 Miller N, Pretorius H.E. and Trinder D.W (1985). Determination of aflatoxins in vegetable oils, J. Assoc. Off. Anal.
Chem, 68 : 136-137
13. Scholten J.M and Spanjer M.C (1996) Determination of aflatoxin B1 in pistachio kernels and shells, , J. Assoc. Off.
Anal. Chem 79: 1360-1364
8
14. Brera C., Caputi R., Miraglia M., Iavicoli I., Salerno A. and Carelli G. (2002). Exposure assessment to mycotoxins
in workplace: aflatoxins and ochratoxin A occurrence in airborne dusts and human sera, Micro chemical J
(uncorrected proof).
15. Garcia-Villanova R.J., Cordon C., Paramas A.M.G., Aparicio P. and Rosales M.E.G., (2004). Simultaneous
immunoaffinity column clean up and HPLC analysis of aflatoxins and ochratoxin A in Spanish bee pollen, J
Agric. Food Chem.52; 7235-7239
16. Gonzalez M.P.E., Mattusch J. and Wennrich R ( 1998) Stability and determination of aflatoxins by high-
performance liquid chromatography with amperometric detection, J.Chromatography A, 828: 439-444
17. Fifield F.W. and Kealey D (2000) Principle and practice of analytical chemistry 3rd Edition, Blackwell science,
UK
18. Yaacob M.H., Mohd Yusof A.R., and Ahamad R (2003), Cyclic Voltammetry of AFB2 at mercury electrode,
Paper presented at SKAM –VI, Unimas, Kucing, Sarawak, Malaysia, 9-11 September 2003
19. Yaacob M.H., Mohd Yusof A.R., and Ahamad R. (2004), Cyclic Voltammetry of AFG1 at mercury electrode,
Paper presented at 3rd Annual Seminar on Sustainable Science and Management, KUSTEM, Terengganu,
Malaysia, 4 – 5 May 2004.
20. Jaimez J., Fente C.A., Vazquez B.I., Franco C.M., Cepeda A., Mahuzier G. and Prognon P (2000). “Application of
the assay of aflatoxins by liquid chromatography with fluorescence detection in food analysis.” J.
Chromatography A.” 882: 1-10
21. Miller K (1987) Toxicology aspects of food. Elsevier Applied Science, New York
2.2. Yaacob M.H., Mohd Yusof A.R. and Ahamad R (2005). Differential pulse stripping voltammetric (DPSV)
technique for determination of AFB2 at the mercury electrode. To be published in Collection of Czechoslovak
Chemical Communication. In process.
9
PAPER WAS PUBLISHED IN
J. Kimia Analisis (Universiti Sumatera Utara, Medan, Indonesia)
Yaacob, M.H., Mohd. Yusoff, A.R. and Ahamad, R. (2005). Development of Differential
Pulse Cathodic Stripping voltammetry (DPCSV) Technique for the Determination of
AFB1 in Ground Nut Samples. J. Sains Kimia. 9(3): 31-36.
1
DETERMINATION OF THE AFLATOXIN B1 IN GROUND NUT BY
DIFFERENTIAL PULSE CATHODIC STRIPPING VOLTAMMETRY (DPCSV)
TECHNIQUE
Mohamad Hadzri Yaacob1, Abdull Rahim Hj. Mohd. Yusoff2, Rahmalan Ahamad2
and Marpongahton Mismi3
1
School of Health Sciences, USM, 16150 Kubang Krian, Kelantan, Malaysia
2
Dept. of Chemistry, Faculty of Sciences, UTM, 81310 UTM, Skudai, Johor, Malaysia
3
Dept. of Chemistry, Faculty of Mathematic and Science, USU, Medan, Indonesia
ABSTRACT
An electro analytical method has been developed for the detection and
determination of the 2,3,6a,9a-tetrahydro-4-methoxycyclo penta[c] furo[3’,2’:4,5] furo
[2,3-h][l] benzopyran-1,11-dione (aflatoxin B1, AFB1) by a differential pulse cathodic
stripping voltammetry on a hanging mercury drop electrode (HMDE) in aqueous solution
with Britton-Robinson buffer (BRb) as supporting electrolyte. Effect of instrumental
parameters such as accumulation potential (Eacc), accumulation time (tacc) and scan rate
(v) were examined. The best condition were found to be pH 9.0, Eacc of -0.6 V, tacc of 80
s and v; 50 mV/s. Calibration curve is linear in the range of 0.02 to 0.32 uM with a
detection limit of 0.75 x 10-8 M (3SDblank/m). Relative standard deviation for a replicate
measurements of AFB1 ( n= 8) with the concentrations of 0.02 and 0.20 uM were 1.27%
and 0.83% respectively. The method is applied for determination of the AFB1 in ground
nut samples after extraction and clean-up steps. The recovery values obtained in spiked
ground nut sample are 90.93 +/- 2.53 % for 3.0 ppb and 86.15 +/- 2.03 % for 15.0 ppb of
AFB1.
ABSTRAK
Satu kaedah elektroanalisis telah dibangunkan untuk mengesan dan menentukan

2,3,6a,9a-tetrahydro-4-methoxycyclo penta[c] furo[3’,2’:4,5] furo [2,3-h][l] benzopyran-
1,11-dione (aflatoxin B1, AFB1) menggunakan teknik voltammetri perlucutan katodik
denyut pembeza di atas elektrod titisan raksa tergantung (HMDE) di dalam larutan akuas
dengan larutan penimbal Britton-Robinson bertindak sebagai larutan penyokong. Kesan
parameter peralatan seperti keupayaan pengumpulan (Eacc), masa pengumpulan (tacc) dan
kadar imbasan (v) telah dikaji. Keadaan terbaik yang diperolehi adalah pH 9.0, Eacc; -0.6
V, tacc; 80 s dan v; 50 mV/s. Keluk kalibrasi adalah linear pada julat di antara 0.02 ke
0.32 uM dengan had pengesan pada 0.75 x 10-8 M (3SDblank/ m). Sisihan piawai relatif
1
Corresponding address:
Chemistry Dept, Faculty of Science, UTM,
81300 Skudai, Johor, Malaysia
tel: 07-553 4492 or 016-926 0354
or e-mail address: mhy02psk@yahoo.com
2
untuk pengukuran AFB1 dengan kepekatan 0.02 dan 0.2 M sebanyak 8 kali ialah 1.27 %
dan 0.83 % masing-masingnya. Kaedah ini telah digunakan untuk menentukan
kandungan AFB1 di dalam sampel kacang tanah selepas proses pengekstraksian dan
pembersihan. Nilai perolehan semula di dalam sampel kacang yang disuntik dengan 3.0
ppb dan 15.0 ppb AFB1 adalah 90.93 +/- 2.53 % dan 86.15 +/- 2.03 % masing-
masingnya.
Keywords: Cathodic stripping voltammetry; aflatoxin B1; ground nut
INTRODUCTION
Aflatoxins are mycotoxin produced by certain fungi especially, Aspergillus flavus

and parasiticus , and they display strong carcinogenicity [1]. Therefore, they are
dangerous food contaminants and many countries have stringent regulatory demands on
the level of aflatoxins permitted in imported and traded commodities. Aflatoxin B1, B2,
G1 and G2 are the main toxins and may be present in many foodstuff or animal feed
such as maize, peanuts, nuts, copra and cottonseeds [2]. Of these, AFB1 (Fig 1) is the
most commonly occurring variety and one of the most toxic [3]. The order of toxicity ,
AFB1 > AFG1 > AFB2 > AFG2, indicates that the terminal furan moiety of AFB1 is a
critical point for determining the degree of biological activity of this group of mycotoxins
[4].
O
O
O O
Figure 1. Structural formula of AFB1
One of the foodstuff which is most occurrence of AFB1 is ground nut. In

Malaysia, the AFB1 level in peanut is regulated with maximum level that cannot be
greater than 15 ppb [5]. Several analytical techniques for quantitative determination of
the AFB1 in ground nut have been proposed such as thin layer chromatography [6], high
performance liquid chromatography, where a derivatisation is required either using
trifluoroacetic acid [7], iodine [8] or bromine [9], and an enzyme-linked immunosorbent
assay, ELISA [10], All these methods, however, require specialist equipment operated
by skilled personnel and expensive instruments and high maintenance cost [11].
Due to all these reasons, a voltammetric technique which is fast, accurate and
requires low cost equipment [12] is proposed. This technique uses the concept where the
3
peak height of reduction peak of AFB1 is measured when a certain potential is applied
[13]. Previous experiment using cyclic voltammetric technique showed that AFB1
reduced at mercury electrode and the reaction is totally irreversible [14]. This work aimed
to study and develop a DPCSV method for determination of AFB1 at trace levels and to
determine this aflatoxin in ground nut samples. No such report has been published
regarding the determination of AFB1 in ground nut using this technique until now.
EXPERIMENTAL
Apparatus
Voltammetric measurements were performed using BAS CV-50W voltammetric

analyzer with Control Growth Mercury Electrode (CGME) stand. The three-electrode
system consisted of a CGME as working electrode, Ag/AgCl2 reference electrode and
platinum wire as the auxiliary electrode. A 20 ml capacity measuring cell was used for
placing supporting electrolyte and sample analytes. All measurements were carried out at
room temperature. All pH measurements were made with Cyberscan pH meter,
calibrated with standard buffers (pH 4, 7 and 10) at room temperature.
Reagents
AFB1 standard. (1mg per bottle) was purchased from Sigma Co. and was used
without further purification. Stock solution ( 10 ppm) in hexane:acetonitrile (98:2) were
prepared and was stored in the dark at -4 0 C. The diluted solution were prepared daily
by using certain volume of stock solution, degassed by nitrogen until dryness and
redissolved in Britton-Robinson solution at pH 9.0.
Britton-Robinson buffer solution was prepared from a stock solution 0.04 M

phosphoric (Merck), boric (Merck) and acetic (Merck) acids. BRb solution with other
pH also was prepared by adding sodium hydroxide (Merck) 1.0 M up to pH value
required. All solutions were prepared in double distilled dionised water. All chemicals
were of analytical grade reagents.
General procedure
For voltammetric experiments, 10 ml of Britton-Robinson buffer solution was

placed in a voltammetric cell, through which a nitrogen stream was passed for 600 s
before recording the voltammogram. The selected Eacc = - 600 mV was applied during
the tacc = 80 s while the solution was kept under stirring. After the accumulation time had
elapsed, stirring was stopped and the selected accumulation potential was kept on
mercury drop for a rest time (tr = 10 s), after which a potential scan was performed
between -0.950 and -1.500 V by DPCSV.
4
Procedure for the determination in ground nut samples
Four batches of ground nut included an artificial contaminated have been

analysed for AFB1 content. AFB1was extracted from ground nut samples according to
the standard procedure developed by Chemistry Department, Penang Branch, Ministry
Of Science, Technology and Innovation (MOSTI), Malaysia [15]. 1ml of the final
solution from extraction and clean-up steps in chloroform was pipette into an amber
bottle, degassed with nitrogen and re-dissolved in 1ml of Britton-Robinson. 200 ul of
this solution was spiked into 10 ml supporting electrolyte in volumetric cell. After that,
the general procedure was applied and voltammogram of sample was recorded. A 10 ppb
of AFB1 standard solution was then spiked into the sample solution before general
procedure for voltammetric analysis The content of AFB1 in ground nut sample was
calculated based on standard addition method to minimize any matrix effect.
RESULT AND DISCUSSION
Cyclic voltammetric determination of AFB1
Cyclic voltammogram of 1.3 uM AFB1 in Britton-Robinson buffer solution as

shown in Figure 2.0 indicates the non-reversibility of the electrode process. It is also
confirmed by the study of the influence of the scan rate in this technique, where a linear
relationship between peak potential (Ep ) and log scan rate was found as Ep (-mV) =
61.41x + 1171.6 (R2 = 0.9967).
Figure 3.0 Typical repetitive cyclic voltammogram of 1.3 uM AFB1 in BRb

pH 9.0; scan rate = 200 mV/s.
5
Effect of pH
The influence of pH on the voltammetric behaviour of AFB1 has been studied

using DPCSV. AFB1 gave a single reduction signal in the pH range of 5.0 to 12.0 where
at pH 9.0, the highest peak height has been obtained as shown in Figure 3.0. This can be
assigned to the reduction of the ketone group to give the corresponding alcohol [19].
60
50
40
Ip (nA)
30
20
10
0
4 6 8 10 12
pH
Figure 3.0 Effect of pH of BRb on peak height of 1.0 uM AFB1
Optimisation of condition for the stripping analysis
The voltammetric determination of analytes at trace levels normally involves very

small current response. For that reason it is important to optimise all those parameters
which may have an influence on the measured peak height. The effect of various
parameters such as accumulation potential (Eacc ), accumulation time (tacc) and scan rate
on the peak height of AFB1 have been studied. The results of these optimization were
Eacc; -0.6 V, tacc ; 80 s and scan rate 50 mV/s. Other parameters are initial potential (Ei )
= -0.1 V, final potential (Ef ) = -1.4 V and pulse amplitude = 80 mV. Using these
optimized parameters, 0.1 uM of AFB1 has produced a single and sharp peak at Ep of -
1.21 V with Ip of 60 +/- 5 nA ( n=5) as shown in Figure 4.0.
Figure 4.0 Voltammogram of 0.1 uM AFB1 in BRb pH 9.0 (n=5). Condition;

Eacc = -0.6 V, tacc = 80 s and scan rate = 50 mV/s
6
Effect of standard addition of AFB1 and analytical characteristics of the method
Using the optimized conditions that already mentioned, a study was made of the
relationship between peak current and concentration. There is a linear relationship in the
concentration range 0.02 uM and 0.32 uM with a regression equation of y = 5.4363x +
3.7245 ( R2 = 0.9989) as shown in Figure 5.0a and 5.0b. The relative standard deviations
(RSD) of the analytical signals at several concentration values were 1.27% and 0.83% for
AFB1 at concentration of 0.02 uM and 0.20 uM respectively. The analytical sensitivity
has been calculated as 3SDblank / m (16) and was found as 0.75 x 10-8 M. SDblank is the
standard deviation from 8 measurements of blank and m is slope from calibration curve.
200
150
ip (n A )
100
y = 5.4363x + 3.7245
50 2
R = 0.9989
0
0 10 20 30 40
[AFB1] x 10-8M
(i) (ii)
Figure 5.0 (i) Voltammograms of AFB1 with increasing concentration (b) 0.02 uM ,
(c) 0.04 uM, (d) 0.06 uM, (e) 0.10 uM, (f) 0.14 uM, (g) 0.18 uM (h) 0.22 uM, (i) 0.26
uM, (j) 0.30 uM and (k) 0.32 uM in (a) BRb pH 9.0; (ii) it’s calibration curve.
Conditions: Eacc= -0.6 V, tacc = 80 s, scan rate = 50 mV/s.
Determination of AFB1 in ground nut samples
The proposed method was applied to the analysis of the AFB1 in eluate of ground
nut samples, spiked with different concentration of AFB1 for recovery studies. In this
7
study, the concentrations used were 3.0 ppb and 15.0 ppb The results of this studies are
represented in Table 1.0. The results show that the accuracy of the method is good.
Table 1.0 Recovery study of AFB1 spiked in eluate of ground nut

samples (n = 3)
No Amount added Amount found % of recovery

(ppb) (ppb)
1 3.00 2.73 +/- 0.08 90.93 +/- 2.53
2 15.00 12.92 +/- 0.30 88.15 +/- 2.03
Analyses of AFB1 content in ground nut samples were determined by standard

addition in order to eliminate the matrix effects. Voltammograms of ground nut samples
are shown in Figures 6(i) to 6(iv). AFB1 was not detected in three samples while in
artificial contaminated sample, 333 ppb of AFB1 was found. The results were compared
to that obtained by HPLC technique as stated in Table 2.0 which show a good agreement
between them. It was concluded that the proposed technique is good, accurate and
reliable for determination of AFB1 in ground nut samples.
Table 2.0 The results of DPCSV analysis of AFB1 in ground nut samples
compared with HPLC technique.
Sample By DPCSV technique By HPLC technique
1 ND ND
2 ND ND
3 ND ND
4 333 ppb 337 ppb
Note: ND = not detected
8
(i) (ii)
(iii) (iv)
Figure 6.0 Voltammograms of ground nut sample No. 1 (i), sample No. 2 (ii),
sample No. 3 (iii) and sample No. 4 with standard addition of 10 ppb AFB1 (iv) in
BRb pH 9.0 as the blank. Condition; Eacc = -0.6 V, tacc = 80 s and scan rate = 50
mV/s.
ACKNOWLEDGEMENT
One of the author would like to thank University Science Malaysia for approving
his study leave and financial support to carry out his further study at Dept of Chemistry,
UTM. We also indebt to UTM for fundamental short term grant (Vot No 75152) and to
Chemistry Department, Penang Branch, Ministry of Science, Technology and
Innovation(MOSTI), Malaysia for the procedure of extraction and clean-up of ground nut
samples.
9
REFERENCES
1. Akiyama H. Goda Y. Tanaka T and Toyoda M. (2001). Determination of

aflatoxinsB1, B2, G1 and G2 in spices using a multifunctional column clean-up,
J. Of Chromatography A, 932. 153-157.
2. Chiavaro E., Dall’Asta C., Galaverna G., Biancardi A., Gambarelli E., Dossena A.
and Marchelli R. (2001). New reversed-phase liquid chromatographic method to
detect aflatoxins in food and feed with cyclodextrins as fluorescence enhancers
added to the eluent, J Of Chromatography A, 937. 31-40.
3. World Health Organisation in: IARC Monograph on the evaluation of

carcinogenic risk to human, WHO, Lyon, 1987.
4. Hall A.J., Wild C.P in: Eaton D.L., Groopman J.D. (Eds). The toxicology of
aflatoxins: Human Health, Veterinary and Agricultural Significance, Academic
New York, 1994.
5. Malaysian Food Act (1983) Act No 281, Reviewed 2002, Food Quality Control
Dept, Ministry of Health, Malaysia.
6. Bicking M.K.L., Kniseley R.N and Svec H.J (1983), Coupled-Column for
quantization low levels of aflatoxins, J . Assoc. Off. Anal. Chem. 66, 905-908.
7. Beebe B.M. (1978). Reverse phase high pressure liquid chromatographic

determination of aflatoxins in foods, J. Assoc. Off. Anal. Chem. 81. 1347-1352.
8. Cepeda A., Franco C.M., Fente C.A., Vazquez B.I., Rodriguez J.L., Prognon P.
and Mahuzier G. (1996). Post column excitation of aflatoxins using cyclodextrins
in liquid chromatography for food analysis. J Of Chrom A. 721. 69-74.
9. Garner R.C., Whattam, M.M. , Taylor, P.J.L. and Stow M.W ( 1993) Analysis of
United Kingdom purchased spices for aflatoxins using immunoaffinity column
clean up procedure followed up by high-performance liquid chromatographic
analysis and post-column derivatization with pyridium bromide per bromide, J.
Chromatography, 648: 485-490
10. Blesa J., Soriano J.M., Molto J.C., Marin R. and Manes J. (2003). Determination
of aflatoxins in peanuts by matrix solid-phase dispersion and liquid
chromatography. J Of Chrom A. 1011. 49-54.
11. Garden S.R. and Strachan N.J.C.(2001). Novel colorimetric immunoassay for the
detection of aflatoxin B1, Anal Chim Acta, 444. 187-191.
10
12. Braitina Kh. Z., Malakhova N.A. and Stojko Y. (2000) Stripping voltammetry in
environmental and food analysis, Fresenius J Anal Chem 368: 307-325
13. Fifield F.W. and Kealey D (2000) Principle and practice of analytical chemistry
3rd Edition, Blackwell science, UK
14. Yaacob M.H., Mohd Yusoff A.R. and Ahmad R. (2003). Cyclic voltammetry of
AFB2 at the mercury electrode. Paper presented at Simposium Kimia Malaysia ke
13 (SKAM-13), 9 – 11th September 2003, Kucing, Sarawak, Malaysia.
15. Standard procedure for determination of aflatoxins (2000). Chemistry

Department, Penang Branch, Ministry of Science, Technology and Innovation
(MOSTI), Malaysia (unpublished paper).
16. Smyth M. R., Lawellin D. W. and Osteryoung J.G (1979) Polarographic Study of
Aflatoxin B1, B2, G1 and G2: Application of Differential-pulse Polarography to
the Determination of Aflatoxin B1 in Various Foodstuffs, Analyst, 104: 73-78
17. Sabry S.M. (1999). Adsorptive stripping voltammetric assay of phenazopyridine

hydrochloride in biological fluids and pharmaceutical preparation, Talanta 50:
133-140.
11

Stripping Voltammetric Methods For The Determination of Aflatoxin Compounds

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Stripping Voltammetric Methods For The Determination of Aflatoxin Compounds

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STRIPPING VOLTAMMETRIC METHODS FOR

THE DETERMINATION OF AFLATOXIN COMPOUNDS

MOHAMAD HADZRI BIN YAACOB

UNIVERSITI TEKNOLOGI MALAYSIA

kerjasama antara _____________________ dengan _________________________

Tandatangan : .......................................................... Tarikh : ..........................

* Jika penyediaan tesis/projek melibatkan kerjasama.

BAHAGIAN B – Untuk Kegunaan Pejabat Sekolah Pengajian Siswazah

Tesis ini telah diperiksa dan diakui oleh:

Nama dan Alamat

Nama dan Alamat

Nama Penyelia Lain

Disahkan oleh Penolong Pendaftar di Sekolah Pengajian Siswazah:

Tandatangan : .......................................................... Tarikh : ..........................

Nama : .GANESAN A/L ANDIMUTHU

MOHAMAD HADZRI BIN YAACOB

A thesis submitted in fulfilment of the

Specially dedicated to:

My mother, wife, sons, daughters and all families for

School Of Health Sciences, USM, Health Campus, Kubang Krian Kelantan

UTM for awarding Short Term Grant No: 75152 / 2004

Aflatoxin, which is produced by Aspergillus flavus and Aspergillus parasiticus

CHAPTER TITLE PAGE

1.3.2 Voltammetric measurement 31

2.1 Apparatus, material and reagents 70

2.1.3.4 Ascorbic acid 74

2.2 Analytical Technique 77

2.2.3.3 Method validation 80

3 RESULTS AND DISCUSSION 88

3.1 Cyclic voltammetric studies of aflatoxins 88

3.2 Differential pulse cathodic stripping voltammetry 102

3.3.2 SWSV determination of other aflatoxins 166

4 CONCLUSIONS AND RECOMMENDATIONS 204

4.1 Conclusions 204

TABLE NO. TITLE PAGE

1.0 Scientific name for aflatoxin compounds 7

1.1 Chemical and physical properties of aflatoxin 10

1.2 Summary of analysis methods used for 19

1.3 Working electrode and limit of detection for 32

1.4 The application range of various analytical 33

1.5 List of different type of working electrodes and 36

1.6 Electroreducible and electrooxidisable organic 50

1.7 The characteristics of different type of 53

1.8 Application of Square Wave Voltammetry 62

2.0 List of aflatoxins and their batch numbers 72

2.1 Injected volume of aflatoxins into eluate of 86

3.0 The dependence of current peaks of aflatoxins to 97

3.1 Effect of buffer constituents on the peak height 109

3.2 Compounds reduced at the mercury electrode 116

3.3 Optimum parameters for 0.06 µM and 2.0 µM 126

3.4 The peak height and peak potential of aflatoxins 127

3.6 Mean values for recovery of AFB2 standard 132

3.7 Influence of small variation in some of the 133

3.8 Results of ruggedness test for proposed method 134

3.10 Mean values for recovery of AFB1 standard 137

3.12 Mean values for recovery of AFG1 standard 139

3.14 Mean values for recovery of AFG2 standard 142

3.15 Peak height and peak potential of 0.10 µM 143

3.16 Analytical parameters for calibration curves 145

3.17 LOD values for determination of aflatoxins 148

3.18 LOQ values for determination of aflatoxins 149

3.20 Analytical parameters for calibration curves for 172

3.21 Result of reproducibility study (intra-day and inter- 173

kerjasama antara _ dengan _____