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Food Additives & Contaminants: Part A

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Simplified RP-HPLC method for multi-residue analysis

of abamectin, emamectin benzoate and ivermectin in
rice
a b a a c
Xianchuan Xie , Shu Gong , Xiaorong Wang , Yinxing Wu & Li Zhao
a
State Key Laboratory of Pollution Control and Resource Reuse , Center for Hydrosciences
Research, Nanjing University, School of the Environment , Nanjing 210093, China
b
Zhejiang Economic & Trade Polytechnic , Hangzhou 310018, China
c
Shanghai Agricultural Extension and Service Center , Shanghai 201103, China
Published online: 04 Dec 2010.

To cite this article: Xianchuan Xie , Shu Gong , Xiaorong Wang , Yinxing Wu & Li Zhao (2011) Simplified RP-HPLC method for
multi-residue analysis of abamectin, emamectin benzoate and ivermectin in rice, Food Additives & Contaminants: Part A,
28:1, 19-25, DOI: 10.1080/19440049.2010.527377

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 Food Additives and Contaminants
Vol. 28, No. 1, January 2011, 19–25

Simplified RP-HPLC method for multi-residue analysis of abamectin, emamectin benzoate

and ivermectin in rice
Xianchuan Xiea*, Shu Gongb, Xiaorong Wanga, Yinxing Wua and Li Zhaoc
a
State Key Laboratory of Pollution Control and Resource Reuse, Center for Hydrosciences Research, Nanjing University,
School of the Environment, Nanjing 210093, China; bZhejiang Economic & Trade Polytechnic, Hangzhou 310018, China;
c
Shanghai Agricultural Extension and Service Center, Shanghai 201103, China
(Received 6 July 2010; final version received 22 September 2010)
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A rapid, reliable and sensitive reverse-phase high-performance liquid chromatography method with fluorescence
detection (RP-FLD-HPLC) was developed and validated for simultaneous analysis of the abamectin (ABA),
emamectin (EMA) benzoate and ivermectin (IVM) residues in rice. After extraction with acetonitrile/water (2 : 1)
with sonication, the avermectin (AVMs) residues were directly derivatised by N-methylimidazole (N-NMIM) and
trifluoroacetic anhydride (TFAA) and then analysed on RP-FLD-HPLC. A good linear relationship (r2 4 0.99)
was obtained for three AVMs ranging from 0.01 to 5 mg ml1, i.e. 0.01–5.0 mg g1 in rice matrix. The limit of
detection (LOD) and the limit of quantification (LOQ) were between 0.001 and 0.002 mg g1 and between 0.004
and 0.006 mg g1, respectively. Recoveries were from 81.9% to 105.4% and precision less than 12.4%. The
proposed method was successfully applied to routine analysis of the AVMs residues in rice.
Keywords: abamectin (ABA); emamectin (EMA) benzoate; ivermectin (IVM); residue

Introduction Kolar et al. 2004; Hernández-Borges et al. 2007) or

Avermectins (AVMs) are a group of fermentation
products from a strain of Streptomyces avermitilis and
are 16-membered macrocyclic lactones. They comprise
several derivatives, including abamectin (ABA), ema-
mectin (EMA) benzoate and ivermectin (IVM) (Brug
et al. 1979; Campbell et al. 1983; Yoshii et al. 2000)
(Figure 1), which have been widely applied as insec-
ticides on rice in China since 2008 because of their
efficiency and broad-spectrum effectiveness. Although
belonging to the biopesticide group, AVMs have a
potential risk to human health. Accidental death has
occurred through the ingestion of AVM (Chung et al.
1999). The Joint FAO/WHO Expert Committee has set
the acceptable daily intake (ADI) of abamectin at
0.1 mg kg1 body weight (Food and Agricultural
Organization/World Health Organization (FAO/
WHO) 1997). The European Union has established
the maximum residue levels (MRLs) of AVMs in food
and foodstuff at 10–50 mg kg1 (New Zealand Food
Safety Authority (NZ-FSA) 2003). There is, therefore,
a need for analytical methods capable of determining
multi-residues of AVMs in rice.
Various high-performance liquid chromatography
(HPLC) methods with ultraviolet detection (UV) or
fluorescence detection (FLD) (Roudaut 1998; Ali et al.
2000; Valenzuela et al. 2001; Wei and Li 2001;
mass spectrometry (MS) (Turnipseed et al. 2005;
Stubbings and Bigwood 2009; Thompsona et al.
2009) have been used for the determination of
AVMs. As HPLC-FLD has good sensitivity and
selectivity, it is the current method of choice for
analysis of AVMs residues, and selected as a residue
analytical method by the US Environmental Protection
Agency (USEPA) (2010).
As the main components of rice are hydrophilic
materials, such as starch, sugar and protein, the
lipophilic pesticides can be separated easily from
them using acetonitrile or acetone as an extraction
solvent (Bienvenida et al. 2009). The present study
aimed to develop a simple and sensitive HPLC-FLD
method without clean-up for simultaneous determina-
tion of multi-residues of ABA, EMA benzoate and
IVM in rice.
Materials and methods
Chemicals and reagents
Abamectin (ABA), emamectin (EMA) benzoate, iver-
mectin (IVM), trifluoroacetic anhydride (TFAA),
N-methylimidazole (N-NMIM) and triethylamine
(TEA) of analytical grade were purchased from
Sigma-Aldrich Co. (Shanghai, China) and used

*Corresponding author. Email: xchxie@yahoo.com.cn

ISSN 0265–203X print/ISSN 1464–5122 online

ß 2011 Taylor & Francis
DOI: 10.1080/19440049.2010.527377
http://www.informaworld.com
 20 X. Xie et al.
> 80% B1a: R=C2H5
< 20% B1b: R=CH3
1 Abamectin 2 Emamectin benzoate
Figure 1. Chemical structures of three AVMs.
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without further purification. The stock standard solu-
tions of ABA, EMA benzoate and IVM were sepa-
rately prepared in acetonitrile at 5000 mg ml1. A series
of working standard solutions were prepared by
diluting the stock solution with acetonitrile. All solu-
tions were stored at 20 C in the dark.
HPLC-grade acetonitrile, methanol and acetone
were purchased from Merck Co. (Shanghai, China).
All other chemicals and reagents were of analytical
grade and purchased from Sinopharm Chemical
Reagent Co. Ltd (SCRC) (Shanghai, China). All
aqueous solutions were prepared using reagent water
from a Milli-Q Gradient system (Millipore Co.,
Bedford, MA, USA).
Instrumentation
Chromatographic analysis was performed on a Waters
2696 Alliance system equipped with an autosampler
and Waters 474 Scanning Fluorescence Detector
(Massachusetts, USA). Chromatographic separation
was carried out in a Waters Xbridge C18 column
(250  4.6 mm i.d., 5 mm) with a guard column
(20  4.6 mm i.d., 5 mm) of the same material. The
Millennium Software of Waters Co. and a personal
computer were employed for data processing. A
sonication bath (SB-5200DTN, Ningbo Ultrasound
Co. Ltd, Ningbo City, China) was used as the
extraction and degassing device.
Sample collection and preparation
The rice samples were purchased from the local market
of Shanghai City, China. After being pulverised in an
electric grinder, the rice powders were sieved through
2 mm and stored at 20 C. A total of 1.0 g sample of
the rice powders was spiked by adding a standard
solution to give fortification levels of 0.01, 0.1 and
1 mg g1, respectively. To ensure good contact of
analytes with the whole sample, the spiked powders
were equilibrated by shaking overnight (8–12 h) in
the dark.
3 Ivermectin
Extraction
A total of 1.0 g sample of the powders was extracted
in an ultrasonic bath (40 kHz, 200 W) for 30 min with
15 ml acetonitrile/water (10 : 5, v/v) and then centri-
fuged for 5 min at 5000 g. The extraction was
transferred into a glass tube with a plug in which
0.1 g NaCl was pre-added, and then emphatically
stirred by hand for 5 min. After standing for 20 min,
5 ml of the supernatant were transferred into an amber
vial and then evaporated to dryness with a stream of
nitrogen at 60 C.
Fluorescence derivatisation
The evaporated residue in a tube reacted with
derivatisation reagents according to the following
processes: 200 ml of N-NMIM/acetonitrile (1 : 1, v/v)
were added to the tube and vortexed briefly, followed
by the addition of 300 ml TFAA/acetonitrile (1 : 2, v/v).
The reaction mixtures were stored in the dark for at
least 0.5 h. The derivatisation reagents had to be made
fresh every day. The derivatisation of standards was
carried out in the same way. To avoid the decrease of
fluorescent signal, the analysis of derivates was com-
pleted within 80 h.
Chromatographic conditions
A gradient programme of mobile phase with acetoni-
trile (solvent A), methanol (solvent B) and water
(solvent C) was carried out at a flow rate of 1.0 ml
min1 and at a column temperature of 40 C. The
gradient elution programme started with acetonitrile/
methanol/water (10 : 80 : 10, v/v/v) followed by a linear
adjustment for acetonitrile/methanol (20/80, v/v) in
10 min; the last condition was kept constant for 5 min.
Fluorescence detection was set with lex ¼ 254 nm and
lem ¼ 400 nm. The injection volume was 20 ml.
Quantification was performed by external calibration
and measured with peak areas.
 Food Additives and Contaminants 21

Table 1. Influence of solvents on recovery.

Solvents

Analyte Acetonitrile/water (2 : 1, v/v) Acetone/water (2 : 1, v/v) n-Hexane

ABA 103.4  6.0 66.8  20.1 50.6  15.3

EMA benzoate 86.7  5.5 70.4  17.6 42.3  7.5
IVM 94.5  4.9 77.2  13.2 68.2  10.7

Note: Values are given as the percentage mean  the percentage RSD (n ¼ 3).

Table 2. Influence of sonication extraction time on recovery.

Sonication extraction time (min)

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Analyte 10 20 30 40 50 60

ABA 78.2  11.3 85.9  9.7 103.6  6.2 96.2  6.5 107.2  8.3 98.5  7.3
EMA benzoate 75.1  12.6 81.2  4.7 89.7  3.7 87.6  4.8 90.4  6.3 87.8  6.2
IVM 73.8  14.0 85.5  8.9 94.6  4.6 90.1  5.2 88.6  8.4 96.1  4.9

Note: Values are given as the percentage mean  the percentage RSD (n ¼ 3).

Method validation To test for remaining interferences, the acetonitrile

The method was validated by evaluating system extract of rice, which was concentrated from 5 to
suitability, linearity, sensitivity, precision, accuracy, 0.5 ml, was directly analysed by HPLC-FLD without
selectivity and stability according to US Food and fluorescence derivatisation. Results showed that only a
Drug Administration (USFDA) bioanalytical method few interferences in the extract gave a fluorescence
validation guidance (US Department of Health and response (Figure 2). As shown, the derivatisation
Human Services, USFDA 2001). reaction could further eliminate the interference in
extract. Thus, no further clean-up step was needed
after the extraction process.
Results and discussion
Optimisation of extraction procedure
Optimisation of fluorescence derivatisation
In published methods, acetonitrile (Hernández-Borges
To optimise the parameters of the derivatisation
et al. 2007), acetone (Yoshii et al. 2000), n-hexane
reaction, the main factors, including reaction time,
(Prabhu et al. 1992) and other solvents have already
temperature, light, etc., were studied in a previous
been used for extracting AVM residues from a variety
study (Xie et al. 2006). It showed that the derivatisa-
of foods, including animal tissue (Ali et al. 2000;
tion of AVMs was entirely completed by at least 0.5 h
Stubbings and Bigwood 2009), milk (Turnipseed et al.
and its derivatives were stable within 80 h in the
2005), plasma (Wei and Li 2001), and vegetables and
dark, which was consistent with the results of
fruit (Valenzuela et al. 2001; Hernández-Borges et al.
Hernández-Borges et al. (2007). The presence of
2007). In preliminary experiments, acetonitrile/water
hydroxyl compound, including water, methanol, etha-
(2 : 1, v/v), acetone/water (2 : 1, v/v) and n-hexane were
nol, etc., could completely inhibit the derivatisation
tested as extraction solvents for spiked rice at 0.1 mg
reaction of AVMs. The derivatisation reaction was
g1. Results in Table 1 show that the best recoveries
sensitive to light because derivatives could be easily
were achieved by a mixture of acetonitrile/water, while
photo-degraded. The reaction temperature had no
acetone/water had unsatisfactory and variable recov-
influence on the fluorescence signal.
eries because of its high volatility, and there was no
sufficient extraction for AVMs provided by n-hexane.
As the sonication extraction time was increased from
10 to 30 min, the recoveries obviously increased. Optimisation of chromatographic conditions
However, the extended extraction time from 30 to The chromatographic conditions were optimised on
60 min did not result in significant increasing of optimal wavelengths of detection, type of columns,
recoveries (Table 2). Thirty minutes of extraction varying mobile phase and column temperature.
time, therefore, were recommended in this assay. Optimal excitation and emission wavelengths for
 22 X. Xie et al.

(a)
LU
3.4 Abamectin (ABA)
3.2 Ivermectin (IVM)
Emamectin (EMA) benzoate
3
2.8
2.6
2.4
2.2
2
1.8

2 4 6 8 10 12 14 m

(b)
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LU
3

2.8

2.6

2.4

2.2

1.8

1.6

2 4 6 8 10 12 14 m

(c)
LU
3.25 Abamectin (ABA)

3 Ivermectin (IVM)
Emamectin (EMA) benzoate
2.75

2.50
2.25

1.75

1.5
2 4 6 8 10 12 14 m

Figure 2. HPLC-FLD chromatogram: (a) for three AVMs standard solutions at 0.1 mg ml1; (b) for the bank rice sample; and
(c) for the spiked rice sample at 0.1 mg g1 of AVMs.

AVMs derivates were at 365 and 470 nm according to
the literature (Berendsen et al. 2007). To achieve good
peak shape and improve the separation efficiency,
three reverse HPLC columns, including an Xbridge C18
column (250  4.6 mm i.d., 5 mm), an SB-C8 column
(150  4.6 mm i.d., 5 mm) and an Inertsil ODS-3
column (150  4.6 mm i.d., 5 mm), were tested in the
preliminary study. Results showed that the sharp and
symmetrical peaks and complete separation for ABA,
EMA benzoate and IVM were achieved on an Xbridge
C18 column analysis (Figure 2a).
Acetonitrile and water along with methanol and
water systems are often used as the mobile phase in
reverse chromatography. Initially, several isocratic
elutions of acetonitrile–water and methanol–water
had been used as a mobile phase, but no success was
obtained for complete separation of ABA,
EMA benzoate and IVM. As a gradient elution
programme and reasonable column temperature
(described above) were used, three AVMs had
satisfactory peak separation and reasonable retention
times (Figure 2a).
 Food Additives and Contaminants 23

Table 3. Calibration results, LOD and LOQ values of three AVMs (n ¼ 5).

Line range LOD LOQ

Reference standards Slope  SD Intercept  SD r2 (mg ml1) (mg kg1) (mg kg1)

ABA 293.05  1.89 9.10  0.11 0.9968  0.0008 0.01–5.0 1.1 3.6
EMA benzoate 217.24  2.20 7.26  0.16 0.9952  0.0012 0.01–5.0 1.7 5.7
IVM 311.71  2.16 10.45  0.12 0.9977  0.0009 0.01–5.0 1.3 4.3

Table 4. Intra- and inter-day recovery and precision of three AVMs in rice.

Intra-day (n ¼ 5) Inter-day (n ¼ 3)

Mean  SD Recovery RSD Mean  SD Recovery RSD

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Spiked concentration (mg g1) calculated (mg g1) (%) (%) calculated (mg g1) (%) (%)

ABA 0.01 0.0105  0.0006 105.2 5.8 0.009  0.0008 89.6 8.6
0.1 0.107  0.0045 106.7 4.2 0.092  0.0067 92.4 7.3
1 0.92  0.08 92.2 8.7 0.88  0.076 87.8 8.6
EMA benzoate 0.01 0.0085  0.001 85.2 11.4 0.0082  0.001 81.9 12.2
0.1 0.093  0.003 87.8 3.6 0.087  0.0075 86.7 8.6
1 0.93  0.048 92.6 5.2 0.85  0.065 85.2 7.6
IVM 0.01 0.0096  0.0007 96.4 7.2 0.0104  0.0013 103.7 12.4
0.1 0.104  0.007 104.3 9.5 0.105  0.0069 105.4 6.5
1 0.90  0.028 90.1 3.1 0.92  0.074 92.3 8.0

Method validation their inter-day recoveries and precisions were within

System suitability
Standard stock solutions containing ABA, EMA
benzoate and IVM were prepared and diluted to
eight concentrations ranging from 0.01 to 5.0 mg ml1.
Each concentration was analysed in five replicates, and
the calibration curves were constructed by plotting the
peak areas versus the corresponding concentration.
The limit of detection (LOD) and the limit of quan-
tification (LOQ) were determined at signal-to-noise
ratios of 3 and 10, respectively. Results in Table 3
show that good linear relationships were achieved
(r2 4 0.99); the LOD and LOQ of three AVMs were
0.001–0.002 and 0.004–0.006 mg g1, respectively.
These results demonstrated good linearity and sensi-
tivity of the proposed method.
81.9–105.4% and less than 12.4%, respectively. These
results demonstrated that the assay had acceptable
precision and recovery.
Selectivity
Selectivity was assessed by comparing chromatograms
of five different types of blank rice with the corre-
sponding spiked sample. Figure 2b and c shows the
typical chromatograms of a blank rice sample and the
corresponding sample spiked with ABA, EMA benzo-
ate and IVM at 0.1 mg g1. No significant interference
from endogenous substances with three AVMs was
detected. Typical retention times for ABA, EMA
benzoate and IVM were 10.47, 13.57 and 14.80 min,
respectively. Thus, interference from rice matrix was
negligible in this method.

Precision and recovery Stability

Precision and recoveries were determined by repeated
analysis (n ¼ 5) of the spiked rice samples at 0.01, 0.1
and 1.0 mg g1 for 3 consecutive days. The concentra-
tion of each sample was determined using the calibra-
tion curve obtained in the same batch. Table 4
summarises the intra- and inter-day precision and
recoveries for ABA, EMA benzoate and IVM, which
were evaluated by assaying the spiked samples. As
shown, the intra-day recoveries and precisions for three
AVMs were within 85.5–105.2% and less than 11.4%;
The stability of ABA, EMA benzoate and IVM in rice
was evaluated by analysing replicates (n ¼ 5) of spiked
samples at 0.01, 0.1 and 1.0 g g1, which were exposed
to different conditions including 24-h storage at room
temperature (25 C), three freeze/thaw cycles, 30 days of
storage at 20 C, and 24-h storage on an autosampler
rack at room temperature (25 C). Results show that the
mean and RSD of recoveries were respectively within
87.6–105.7% and 2.6–12.6% (Table 5), indicating that
three AVMs were stable in rice.
 24 X. Xie et al.

Table 5. Stability of three AVMs in rice under different storage condition (n ¼ 5).

Nominal Short-term Long-term Three Autosampler

Analyte concentration (mg g1) (24 h at 25 C) (30 days at 20 C) freeze/thaw cycles for 24 h (25 C)

ABA 0.01 96.7  5.4 92.1  9.4 104.7  12.6 104.2  3.2
0.1 93.4  2.6 89.2  7.4 95.6  4.0 102.6  2.6
1 102.3  3.2 94.2  6.0 92.7  7.9 97.2  3.8
EMA benzoate 0.01 91.3  6.5 95.2  10.3 90.2  8.6 97.3  2.7
0.1 94.1  3.4 90.8  3.9 93.1  6.2 104.3  3.0
1 87.6  4.8 96.9  5.0 96.8  4.5 96.9  4.2
IVM 0.01 105.7  5.0 92.6  9.9 105.2  8.6 104.0  3.7
0.1 102.1  4.8 91.3  8.1 96.4  6.7 96.9  4.6
1 97.0  6.4 96.7  6.8 94.0  7.2 101.8  3.3
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Application of the method
The method was subsequently used to analyse the
ABA, EMA benzoate and IVM residues in 120 rice
samples on which the AVMs had been use during the
planting season. Results showed that the detectable
number for ABA was three and its contents were 0.009,
0.012 and 0.012 mg g1, respectively; the detectable
number for EMA benzoate is only one and its contents
was 0.017 mg g1; and no IVM residue was detected in
rice samples.
Conclusions
A rapid, reliable and sensitive HPLC method for
simultaneous analysis of the ABA, EMA benzoate and
IVM residues in rice was successfully developed and
validated. All validated parameters of the method were
sufficient for USFDA guidelines. The method was
successfully applied to routine analysis of the AVMs
residues in rice. Because of no required clean-up step,
the proposed method is rapid, has a lower cost and
is easy to perform. Furthermore, a small volume of
organic solvent in this method is beneficial to analysts
and the environment.
Acknowledgements
This study was supported by the key project of developing
agriculture (No. 2003-10-6) from the Shanghai Agriculture
Committee and the standard-technology programme (No.
08GWQ068) from the Shanghai Science & Technology
Committee.
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