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Determination of Histamine in Some Foods

by Isotachophoretic Method with Simple Sample Preparation


Aneta Jastrzbska & Marzanna Kurzawa & Anna Piasta &
Edward Szyk
Received: 23 August 2011 / Accepted: 6 December 2011 / Published online: 24 December 2011
#Springer Science+Business Media, LLC 2011
Abstract A one-dimensional capillary isotachophoretic
method in cationic system of the separation has been
applied for histamine determination in food samples.
The proposed electrolyte system consisted of 0.01 M
potassium hydroxide with L-valine to pH09.9 as the
leading electrolyte and 0.02 M 2-amino-2-hydroxy-
methyl-propane-1,3-diol adjusted to pH08.3 with 0.1 M
hydrochloric acid as terminating electrolyte. Proposed
method was characterized by linearity range 5
50 mg L
1
and R
2
00.9982, accuracy (recoveries ranged
from 95% to 102%), detection (2.10 mg L
1
), and quan-
tification (7.01 mg L
1
) limits. The sample preparation
for proposed electrophoretic method included only simple
extraction with trichloroacetic acid with filtration and
derivatisation stage are avoided. The histamine concen-
tration was determined in meat (turkey, chicken, beef and
pork) and meat products (ripened sausage and dry-cured
ham), fish (smoked salmon and mackerel), and different
kind of mildew and mold ripened cheeses samples. The
histamine content ranged from not detected level for
fresh meat to 29.63 mg 100 g
1
for cheese samples.
The reversed phase HPLC was applied as reference
method and the F-Snedecor test and the t test were
employed to compare the precision and accuracy of the
both methods. Positive correlations were found between
the two analytical methods for histamine determination in
food products. The obtained results indicate that the
proposed electrophoretic method is simple, precise, accu-
rate, and convenient.
Keywords Histamine determination
.
Isotachophoretic
method
.
Food samples
.
HPLC
Introduction
Biogenic amines (BA) are present in living organisms at low
levels where they are responsible for many essential biolog-
ical functions (Bodmer et al. 1999; Saccani et al. 2005;
Hernndez-Jover et al. 1997). Their presence in food, espe-
cially in fish, cheese, and meat products, varies by a great
extent depending on technological processes and microbial
factors. In fact their formation mainly depends on the activity
of microbial decarboxylases and in a minor role on the
endogenous amino acid decarboxylase activities (Galgano
et al. 2009; Talon and Leroy 2011). For this reason, the
content of biogenic amines can be relatively high in fer-
mented food, especially in raw materials (meat and dairy
products) presenting a high content of proteins combined
with high proteolytic activity (Talon and Leroy 2011).
BA can be generated during storage or processing of food
rich in proteins by thermal or bacterial enzymatic decarbox-
ylation of amino acids. Hence, the composition and number of
biogenic amines gives information about the freshness of
food. Consequently, they are important indicators of food
quality and hygiene and high level of BA is usually consid-
ered as an index of poor manufacturing practices (Galgano et
al. 2009; Mazzoli et al. 2009; Sml et al. 2003).
Histamine (HIS), beta-phenylethylamine, tyramine, trypt-
amine, putrescine, cadaverine, spermine, and spermidine are
the most important biogenic amines identified in food and
beverages. The high concentration of these compounds in
food causes different effects in susceptible individuals
(Mazzoli et al. 2009; Kaniou et al. 2001; Vinci and
Antonelli 2002; Gosetti et al. 2007). Among BA, histamine
A. Jastrzbska (*)
:
M. Kurzawa
:
A. Piasta
:
E. Szyk
Faculty of Chemistry, Nicolaus Copernicus University,
7 Gagarin Str.,
87-100 Toru, Poland
e-mail: aj@chem.uni.torun.pl
Food Anal. Methods (2012) 5:10791087
DOI 10.1007/s12161-011-9345-7
is the most important substance responsible for intoxications
and allergies (Gosetti et al. 2007; Bomke et al. 2009;
Hungerford 2010). Moreover, HIS is neither volatile nor
destroyed by cooking; hence, it is difficult to remove it from
food, what diminishes food quality. This amine does not
affect organoleptic characteristics, but clearly induces food
intolerance in an increasing number of human populations
and caused the potential risk to health (Bodmer et al. 1999;
Akbari-adergani et al. 2010).
Therefore, a regulation level on histamine in food has
been set by many countries or organizations (Hwang et al.
2003; Peng et al. 2008). The Polish Standards (in agreement
with The European Union (EU) 2007/642/WE) (Polish
Monitor 2009) regulate the permissible content of histamine
in fish and fish products from the families Scombridae,
Clupeidae, Eugraulidae, and Coryphaenidae. These prod-
ucts must fulfill the following requirements: (1) the mean
value must not exceed 10 mg 100 g
1
; (2) two samples may
have a value of more than 10 mg 100 g
1
but less than
20 mg 100 g
1
; (3) no sample may have a value exceeding
20 mg 100 g
1
. However, fish belonging to these families
which have undergone enzyme ripening treatment in brine
may exhibit higher histamine levels but not more than twice
the above values (2040 mg 100 g
1
). According to Talon
and Leroy (Talon and Leroy 2011), this regulation is already
obligatory in the fish industry in EU and may be extended to
fermented food in the future.
Because of its potential risk to health, the research on the
simultaneous and rapid analysis of BAs in a variety of
biological matrices is of the widest interest. The number
and variety of methods developed for histamine and other
amines determination in food samples is impressive and
reviewed by nal (nal 2007) and Karoviov and Kohajdov
(Karoviov and Kohajdov 2005). According to Cinquina et
al. (Cinquina et al. 2004), methods proposed for amines
determination may be divided into two classes: simple
method with short time analysis and low sensitivity and
complicated method with pre- or post-column derivatization
that is accompanied by good sensitivity and specificity, long
analysis times, nonreproducibility, and problems of derivati-
zation products stability.
The most popular methods for biogenic amines determi-
nation are various chromatographic techniques, such as: gas
chromatography (GC) (Hwang et al. 2003), liquid chroma-
tography (high-performance liquid chromatography (HPLC)
and UHPLC) coupled with different detection (Galgano et
al. 2009; Sml et al. 2003; Kaniou et al. 2001; Gosetti et al.
2007; Bomke et al. 2009; Hungerford 2010; Peng et al.
2008; Dadkov et al. 2009; Aygn et al. 1999; Latorre-
Moratalla et al. 2009; Sun et al. 2011), ion chromatography
(Saccani et al. 2005; Favaro et al. 2007). Most of the
separation methods for histamine determination applied de-
tection schemes based on pre-column derivatization to
produce fluorescent products or strong chromophores, but
direct UV detection of imidazole ring has also been applied
(Hungerford 2010; Cinquina et al. 2004).
According to literature (Cinquina et al. 2004; Dadkov et
al. 2009; Steiner et al. 2009; Gallardo et al. 1997; Oguri et al.
1997), capillary electrophoresis is an excellent alternative to
HPLC methods for the analysis of BA in complex matrices.
The fluorimetric methods based on the formation of a fluores-
cent derivative with ortho-phthalaldehyde were widely used
for the analysis of histamine because of their great sensitivity
and selectivity (Adamou et al. 2005; Larionova et al. 2009;
Leszczyska et al. 2004).
As the chromatographic and fluorescence analysis of
biogenic amines is complicated, further analysis meth-
ods have been elaborated. The immunochemical method
(enzyme-linked immunosorbent assay (ELISA)) (Aygn
et al. 1999; Leszczyska et al. 2004), colorimetric (Patange et
al. 2005), electrochemical method (Akbari-adergani et al.
2010), or biosensors (Keow et al. 2007; Ito et al. 2009) for
rapid assessment of histamine level were studied for this
purpose.
Although the histamine content of food has been the sub-
ject of considerable analysis in the past years, the problem of
searching new simple methods for the HIS determination is
still fundamental and interesting. It is related to the fact, that
most of methods required complicated and expensive instru-
mentation, toxic reagents, time consuming operations, cum-
bersome sample preparation and the derivatization reaction
products have a short life time. Moreover, in the complex
matrix samples, due to the high contents of protein and fat,
the presence of potentially interfering compounds, and the
occurrence of several BA simultaneously imposed problems
in the analysis.
In the literature, we have found only few reports on
biogenic amines in different food samples determination
by capillary isotachophoresis (Karoviov et al. 2003;
Rubach et al. 1981). This electrophoretic method has many
advantages (simplicity and short time of analysis) for deter-
minations of many ions in complex matrices and is known
as an analytical method with high reproducibility and can be
competitive to other separation techniques (Kvasnika
2000; Jastrzbska 2011). Moreover, conductometric detec-
tion permitted on biogenic amines determination based on
their electrophoretic properties.
For this reason, we applied capillary isotachophoretic
(ITP) method with conductometric detection for determina-
tion of histamine in food samples after simple sample prep-
aration. The proposed method was validated in terms of
linearity, sensitivity, precision, and recovery. To prove the
versatility of the method, the analysis was carried out on
three groups of food widely consumed all over the world
(meat and meat products, fishes, and cheeses). Furthermore,
these products belong to the usually studied ones in terms of
1080 Food Anal. Methods (2012) 5:10791087
the biogenic amines content. Our results from isotachopho-
resis were compared with those obtained by reversed-phase
(RP)-HPLC method.
Because, amines are strong organic bases it is very
useful to take advantage of this feature for their sepa-
ration from sample matrix. The extraction of amines
represents the critical step of the process and it influ-
ences negatively the analytical recoveries. The aim of
the extraction is to remove interfering compounds from
the matrix, but during this step losses of BA must be as
little as possible. Many different solvents have been
used for the extraction of amines from the matrix, such
as hydrochloric acid, trichloroacetic acid, perchloric ac-
id, methanol, and other organic solvents (Vinci and
Antonelli 2002). For this reason, the optimization of
extraction procedures was described.
Experimental
Reagents
All reagents were of analytical or HPLC grade. Histamine
dihydrochloride (HIS), L-valine, methanol, 2-amino-2-
hydroxymethyl-propane-1,3-diol (TRIS), and acetonitrile
were purchased from Sigma-Aldrich (Pozna, Poland). Am-
monium acetate, potassium hydroxide, hydrochloric acid,
and trichloroacetic acid (TCA) were obtained from Alchem
(Toru, Poland). Redistilled water was used in all solutions
preparation (specific conductivity, <10 S).
Apparatus
Isotachophoretic separations were performed using a Villa
Labeco EA 100/101 isotachophoretic analyzer equipped
with a conductometric detector. The PTFE pre-separation
capillary (900.8 mm, I.D.) was connected with PTFE
analytical capillary (1600.3 mm, I.D.). Samples of 30-l
fixed volume were injected via a sample valve by internal
sample loop. The isotachopherograms were evaluated with
the software supplied with analyzer (KasComp Ltd.,
Slovakia).
The HPLC system, equipped with an autosampler SIL-
20AC HT and a photodiode multi-wavelength detector
(SPD-M20A Prominence Diode Array Detector), SHI-
MADZU (Kyoto, Japan) was applied. Analyses were carried
out on Discovery C18 Supelco column (5-m particle size,
1504.8 mm), maintained at 25 C.
Food products samples were centrifuged by a laboratory
centrifuge (max speed, 9,000 rpm; RFC, 8693g; angle,
30; and falcon tubes, 50 mL; MPW, Warsaw, Poland).
Food samples
All food samples were purchased from different local mar-
kets and collected in Table 1.
Prior to analysis, purchased samples were cut up, homog-
enized in household food grinder with a plate of 3-mm
diameter holes.
Sample Preparation for ITP and HPLC Methods
Depending on the complexity of food matrix and the
natural amount of free amino acids, extraction and pu-
rification steps can be necessary prior to analytical
determination. Because proposed ITP method with con-
ductometric detection required ionic form of histamine,
the sample extraction was performed in acidic medium
as reported (Hernndez-Jover et al. 1997; Vinci and
Antonelli 2002; Innocente et al. 2007) for food matrices
with relatively low free amino acids content such as
meat, fish and vegetables. According to Moret and
Conte (Moret and Conte 1996), the choice of acid has
to be related to the characteristics of the analyzed ma-
trix. On the basis of their experience, 0.1 M HCI
appears to be a good choice for the analysis of cheese,
however, it is not a suitable for fish or meat products.
For these samples, TCA represented a better choice
because of its capacity to precipitate proteins. For this
reason, in our study we tested TCA as all-purpose
extraction solution in concentration from 1% to 5%.
The best precision and recovery for ITP and HPLC
methods were obtained for 2% of TCA.
Possible differences between extracted analytes and in-
terfering species were checked for the range 525 g of
Table 1 Food samples analyzed by ITP and HPLC methods
Sample no Type of sample
Sample 1 Minced turkey breast
Sample 2 Minced chicken breast
Sample 3 Minced pork shoulder
Sample 4 Minced beef shoulder
Sample 5 Minced pork ham
Sample 6 Raw beef
Sample 7 Ripened sausage
Sample 8 Dry-cured ham
Sample 9 Smoked salmon
Sample 10 Smoked mackerel
Sample 11, 11a; 11b;
11c; 11d
Different kind of mildew and mold
ripened cheeses
Sample 12 Fresh poultry meat
Sample 13 Fresh pork
Sample 14 Fresh beef
Food Anal. Methods (2012) 5:10791087 1081
processed food samples. For ITP and HPLC methods the
highest analytes/interferents ratio was noted for 15 g and
consequently this amount was used in all successive experi-
ments. The sample preparation procedure with all details is
presented below.
Procedure of Sample Preparation
The food products samples (150.0001 g purchased
products) were extracted with 25 mL of TCA (2%) using
an orbital shaker for 30 min. The extracts were separated
using centrifuge at 9000 rpm for 30 min, followed by
double filtration. All extracts were transferred into 50 mL
volumetric flasks, made up to the mark and analyzed
with ITP and HPLC methods. In the case of HPLC,
the obtained extracts were filtered through a 0.4-mm
membrane prior the analyses. Additionally, for several
samples, dilution with redistilled water was applied. It
should be noted, that applied sample preparation proce-
dure avoids a derivatization stage in the case of both
method. The sample preparation procedure is simple,
short and uncomplicated in comparison to the methods
requiring clean solution or derivatization procedure.
Determination of Histamine by ITP and HPLC Methods
The ITP analysis of histamine was performed with LE
(leading electrolyte): 10 mM KOH+L-valine (pH09.9)
and terminating electrolyte: 0.02 M TRIS+0.1 M HCl
(pH08.3). A driving current of the preseparation capil-
lary was 150 A, while of analytical capillary 100 A
for all samples in five repetitions. The histamine in
standard solution and food samples was identified by
the relative step height parameter (RSH
histamine
00.15).
The time of analysis was short and located between
510 min for standard solutions and 1525 min for
food extracts determination, respectively.
Precision of analysis was evaluated as the within-day
and between-days coefficient of variation (CV) (Miller
and Miller 2000). Within-day analyses were determined
by injection of the histamine standard solution
(20 mg L
1
) six times per day. The intralaboratory re-
producibility was determined by analysis of the standard
solution during five consecutive days. The CV values for
within-day and between-days determination varied from
0.98% to 4.12% and 2.544.89%, respectively, that con-
firmed stability of the proposed electrolyte system.
HPLC measurements were carried out using the
mixture of 0.1 M ammonium acetate + acetonitrile +
methanol (90:5:5) as a mobile phase (isocratic elution)
and detection at 0224 nm. The chromatographic data
were recorded and processed by the LC solution ver-
sion 1.23 SP.
Table 2 Linear regression calibration parameters of histamine deter-
mination by ITP and HPLC methods (n05)
Parameter ITP HPLC
Linearity range (mg L
-1
) 550 10100
Slope (b) 0.5863 16082
Standard deviation of slope (S
b
) 0.0874 149
Intercept (a) ()0.7422 134040
Standard deviation of intercept (S
a
) 0.2810 9220
Standard deviation of regression (S
r
) 0.7011 0.6818
Detection limit (DL; mg L
1
) 2.10 2.05
Quantification limit (QL; mg L
1
) 7.01 6.82
Coefficient of determination R
2
0.9982 0.9997
Where: DL0(y+3s
y/x
)b
1
, QL010s
y/x
b
1
A
histamine
B
histamine
C
histamine
D
histamine
Fig. 1 The isotachopherograms of food samples: a dry-cured ham
sample, b minced pork ham sample, c cheese sample, and d smoked
salmon sample
1082 Food Anal. Methods (2012) 5:10791087
Results and Discussion
Calibration Curves for Histamine Determination by ITP
and HPLC
Calibration curves were constructed using nine calibration
solutions of histamine and results were calculated as an aver-
age of five replicates. Calibration points were established by
measuring the zone length (L) or peak areas (A) versus stan-
dard concentration (c). The regression parameters of calibra-
tion curves are listed in Table 2.
The linearity for histamine determination by ITP and
HPLC methods was satisfactory and comparable with
values reported by other authors (Akbari-adergani et al.
2010; Latorre-Moratalla et al. 2009; Steiner et al. 2009;
Oguri et al. 1997; Karoviov et al. 2003). The
Table 3 Results of histamine
determination (mg 100 g
1
purchased products (pp)) in food
samples by ITP (n05)
Where: X is the average value
(mg 100g
1
pp), confidence
limit (0(t
n1
s/n
1/2
), p095%);
CV coefficient of variation (%),
nd not detected, n number of
samples
Samples X (mg 100 g
1
) CV (%)
Fresh poultry meat nd
Fresh pork nd
Fresh beef nd
Minced turkey breast 1.110.035 1.38
Minced chicken breast nd
Minced pork shoulder 1.180.052 3.57
Minced beef shoulder 1.650.11 2.89
Minced pork ham 6.200.29 1.22
Raw beef nd
Ripened sausage 19.200.83 1.88
Dry-cured ham 6.410.38 2.55
Smoked salmon 19.830.75 1.64
Smoked mackerel 18.250.72 2.46
Different kind of mildew and mold ripened cheeses 25.600.55 0.94
15.240.55 2.95
9.180.27 2.39
29.631.04 2.83
3.900.18 3.59
Table 4 Results of histamine
determination (mg 100 g
1
pur-
chased products (pp)) in food
samples by HPLC (n05)
For abbreviations, see Table 3
Samples X (mg 100g
1
) CV (%)
Fresh poultry meat nd
Fresh pork nd
Fresh beef nd
Minced turkey breast 2.510.14 2.47
Minced chicken breast nd
Minced pork shoulder 2.570.20 6.42
Minced beef shoulder 3.320.28 7.11
Minced pork ham 14.200.72 2.21
Raw beef nd
Ripened sausage 21.821.39 2.75
Dry-cured ham 13.140.22 0.73
Smoked salmon 20.830.95 3.67
Smoked mackerel 20.201.24 2.68
Different kind of mildew and mold ripened cheeses 27.341.51 2.40
17.410.72 3.31
11.510.62 4.33
31.801.56 3.97
5.030.31 5.03
Food Anal. Methods (2012) 5:10791087 1083
detection limits of the studied and HPLC methods were
sufficiently low for the determination of histamine in
food samples. In the case of quantification limit, values
were slightly higher than the lowest concentration of the
calibration curve. The last value indicates that the low-
est concentration of histamine solution cannot be deter-
mined with required precision.
The Determination of Histamine in Food Samples
by Isotachophoretic Method
The isotachopherograms of food samples are presented on
Fig. 1 and results are listed in Table 3.
Meat and Meat Products
As reported in Table 3 fresh meat (pork, poultry, and beef),
raw beef, and minced chicken breast samples revealed level
of histamine below the detection limit. In the case of remain-
ing meat samples, the amount of histamine ranged from
1.11 mg 100 g
1
for minced turkey breast (sample 1) to
6.20 mg 100 g
1
for minced pork ham (sample 5).
In our study, the maturing meat products (dry-cured
sausages and ham) revealed higher amount of histamine
than fresh or minced meat. The different concentration of
BA in fresh and meat products can be explained by the
properties of meat substrates and by microbial flora with
different biochemical potentiality for amino acids metabo-
lism (Galgano et al. 2009). On the other hand, the obtained
contents of HIS in minced meat suggested decrease of
quality or long time of storage before sale.
There are several studies on the determination of
biogenic amines in meat samples but usually level of
putrescine, cadaverine, tyramine, and spermine was dis-
cussed as coefficients for storage time and temperature
as well as for the microbiological quality of meat
(Kaniou et al. 2001; Vinci and Antonelli 2002; Rokka
et al. 2004). Fresh and processed pork contains high
levels of adrenalin, spermidine and spermine but low
levels of noradrenalin, putrescine, histamine, cadaverine,
and tyramine (Karoviov and Kohajdov 2005).
According to authors (Kaniou et al. 2001; Vinci and
Antonelli 2002; Rokka et al. 2004), some of amines can
be formed during the storage of fresh meat. Further-
more, during manufacturing of dry-cured and cooked
meat products several technological factors, such as
pH, temperature, and salt concentration are key factors
in the onset and the rate of amino enzymatic reactions
and their synergic effect (Saccani et al. 2005). Favaro et
al. (Favaro et al. 2007) found HIS in dry fermented
sausages 5. 7 mg 100 g
1
and dry-cured bel l y
11.4 mg 100 g
1
with CV values 10% and 6%, respec-
tively. The histamine in different food samples deter-
mined by Karoviov et al. (Karoviov et al. 2003)
was detected in four samples, while the highest level
was in frankfurters (14.67 mg 100 g
1
). Hernndez-
Jover et al. (Hernndez-Jover et al. 1997) applied HPLC
method for determination of BA in fresh pork and beef
meat and only spermidine and spermine were found in
both types of meat. Authors suggested that these amines
are naturally occurring in fresh pork and beef meat, and
0
50
100
150
200
250
0 1 2 3 4 5 6 7 8 9
min
A
1
2
0
40
80
120
160
0 1 2 3 4 5 6 7 8 9
min
min
B
C
2
1
1
2
m
A
U
m
A
U
m
A
U
Fig. 2 The chromatograms of food samples: a cheese sample, b
smoked salmon sample, and c minced pork ham sample; 1 histamine
and 2 histidine
Table 5 Results of standard addition method (n015)
parameter ITP HPLC
Precision (CV %) 1.103.47 1.989.17
Accuracy (recovery %) 95102 101110
For abbreviations, see Table 3
1084 Food Anal. Methods (2012) 5:10791087
their formation is not due to food spoilage or fermen-
tation processes. The range of HIS level in dry-cured
ham and ripened products was from below detection
limit to 15 mg 100 g
1
and to 35.7 mg 100 g
1
, re-
spectively. The levels of histamine in meat products
determined by Saccani et al. (Saccani et al. 2005) were
as follows: 060 mg 100 g
1
(fresh pork meat), 10
40 mg 100 g
1
(dry-cured sausage), 070 mg 100 g
1
(dry-cured ham), and 0110 mg 100 g
1
(cooked ham).
Samples of Cheeses
In contrast to the comparable contents of HIS in meat
samples, the observed level of this amine in cheese samples
demonstrated wide fluctuations from 3.90 mg 100 g
1
to
29.63 mg 100 g
1
. It should be noted that long ripened
cheeses are major sources of dietary biogenic amines. The
formation and presence of amines depend on a variety of
factors including the presence of substrate and microbial
enzymes, temperature, pH, salt and water content, presence
of enhancing substances and catabolism of amines. Accord-
ing to Bodmer et al. (Bodmer et al. 1999), in cheese pro-
duction the increase of histamine content was observed with
maximum level at 2,500 ppm in aged cheese. It is known
that dairy products are good examples to demonstrate the
undesired increase of histamine content during non-proper
food processing (Bodmer et al. 1999). A competitive direct
enzyme-linked immunosorbent assay for HIS determination
in cheese was proposed by Aygn et al. (Aygn et al. 1999)
and obtained mean values were as follows: 32.2 mg 100 g
1
for hard cheese, 3.3 mg 100 g
1
for semi-hard cheese, and
7.3 mg 100 g
1
for soft cheese.
Samples of Fishes
The histamine level has also been proposed as a chemical
index of freshness of fish, poor hygienic quality of raw materi-
als used and/or poor manufacturing conditions (Hwang et al.
2003). It should be noted, that fresh fish contains very low
level of histamine, but the content increases with the progress
of fish decomposition and processing. In this study the smoked
fish samples were analyzed and observed level of HIS (Table 3)
does not exceed the permitted level for fish products (Polish
Monitor 2009). According to Keow et al. (Keow et al. 2007),
histamine induced slightly poisoning at 840 mg 100 g
1
raw
fish, moderate poisoning at >40 mg 100 g
1
, and severe at
>100 mg 100 g
1
.
The Determination of Histamine in Food Samples
by RP-HPLC Method
As a reference method RP-HPLC was applied and obtained
results are listed in Table 4. The typical chromatograms of
food samples are presented on Fig. 2.
It is evident that obtained amounts of HIS by HPLC meth-
od were higher than by ITP. The differences ranged from
1.13 mg 100 g
1
(mold ripened cheese) to 8.00 mg 100 g
1
(minced pork ham). However, the correlation between meth-
ods was satisfactory. Only for two samples (minced pork ham
and dry-cured ham) the difference was higher than
3.00 mg 100 g
1
. Generally, chromatographic method requires
chemical derivatization of most BA due to the lack of a
suitable chromophore or fluorophore group for direct detec-
tion. Except that, the elution program usually consisted of the
gradient system and specific columns for amines separation
are often used. Also likely, the matrix interferences in complex
samples cannot be eliminated efficiently applying simple
sample preparation (single extraction). For this reason, the
pretreatment steps (for example, solid-phase extraction clean-
up) are necessary to eliminate the interferences with chro-
matographic separation. In our research simple elution
program without derivative and pretreatment steps was suffi-
cient for good separation.
Results of Statistical Analysis
Precision of ITP and HPLC Methods
Precision of methods for food samples was evaluated as the
within-day coefficient of variations values (Miller and Miller
2000). In the case of ITP method, the CV values for HIS
determination varied from 0.94% to 3.59% (Table 3), what
indicated satisfactory precision of the discussed method. The
relative standard deviations (RSD) at ITP determination of
amines obtained by Karoviov et al. (Karoviov et al. 2003)
ranged from 0.52% to 5.77% for all concentration levels.
Hwang et al. (Hwang et al. 2003) proposed GC method for
HIS determination in tuna and shrimp with reported precision
Table 6 Comparison between
ITP and HPLC methods by
F and t tests (F
crit
06.39
and t
crit
02.31)
For name of samples, see Table 1
No of samples
1 3 4 5 7 8 9 10 11 11a 11b 11c 11d
F 3.20 3.23 6.05 3.28 2.13 12.2 5.00 1.19 6.50 1.26 3.28 1.97 1.96
t 1.11 0.42 0.49 7.07 1.75 0.56 1.23 1.11 1.05 1.01 0.41
Food Anal. Methods (2012) 5:10791087 1085
of 2.77.8% and 2.78.9% (for standard addition method),
respectively. The RSD values obtained for cation exchange
chromatographic method were as follows: 10% for dry fer-
mented sausages and 6% for dry-cured belly (Favaro et al.
2007). The coefficient of variations for HPLC determination
ranged from 0.73% to 7.11% (Table 4) that indicated better
precision for ITP method. Comparing to the precision de-
scribed by other authors (Sml et al. 2003; Kaniou et al.
2001; Aygn et al. 1999; Latorre-Moratalla et al. 2009; Steiner
et al. 2009; Gallardo et al. 1997; Larionova et al. 2009;
Kvasnika 2000) and obtained with reference (RP-HPLC)
method one can conclude, that the proposed ITP method
reveals satisfactory repeatability.
Recovery Test (Standard Addition Method)
In order to evaluate the accuracy of the analytical method,
the food samples were spiked at two different concentrations
(5.00 and 10.00 mg 100 g
1
) of histamine to food samples,
analyzed by ITP and HPLC methods and obtained results
are listed in Table 5. Samples were homogenized and
extracted as previously described.
The obtained results clearly indicate that the proposed
method shows satisfactory accuracy and precision. Compar-
ing both methods (ITP and RP-HPLC) it is evident that
accuracy of isotachophoresis is better. Furthermore, the
recoveries obtained by ITP method were in agreement with
those reported in the literature (Saccani et al. 2005; Hwang
et al. 2003; Cinquina et al. 2004; Latorre-Moratalla et al.
2009; Steiner et al. 2009; Gallardo et al. 1997). Recovery of
histamine added to different marine products obtained by
Gallardo et al. (Gallardo et al. 1997) ranged from 97.8% to
101.8%. Cinquina et al. (Cinquina et al. 2004) tested two
techniques (HPLC-DAD and CE-DAD) for histamine deter-
mination and recoveries obtained by spiking tuna fish samples
at three different concentrations (50, 100, and 200 mg/kg)
resulting in average recoveries higher than 92% and RSD<
4%for HPLC, whereas for CE the recoveries were above 85%
and RSD below 3%. Similar recoveries (89115%) and RSD
from 0.5% to 3.6% were reported by Steiner et al. (Steiner et
al. 2009). Recovery determination of histamine from tuna by
GC method ranged from 97.5% to 110.9% and for shrimp
meat between 98.5% and 102.4% (Hwang et al. 2003). Re-
coveries of histamine determination in fish, cheese and dry
sausage samples obtained by Latorre-Moratalla et al. (Latorre-
Moratalla et al. 2009) ranged from 88.80% to 95.83%. A
method for simultaneous determination of underivatized bio-
genic amines based on the separation by cation-exchange
chromatography and suppressed conductivity coupled with
mass spectrometry detection was discussed by Saccani et al.
(Saccani et al. 2005), and average recoveries from meat sam-
ples ranged from 85 to 97% and coefficients of variation
ranged from 4.5 to 9.7%.
Comparison of Results for Proposed ITP and RP-HPLC
Methods by F and t Tests
The F-Snedecor and the t tests were employed to compare
the precision and accuracy of the used methods (Miller and
Miller 2000), and the results are listed in Table 6.
The calculated F test indicated that there was not a
significant difference in the precision of the proposed
and HPLC methods because calculated values were
above the theoretical values only in two cases (samples
8 and 11). The t values calculated for the both methods
were lower than the critical ones (except sample 5). The
results collected in Table 6 indicated that there are no
significant differences between the precision and average
concentrations of HIS in the samples assayed by both
analytical methods.
Conclusions
The proposed capillary ITP method with the conductometric
detection for determination of histamine is very simple,
relatively selective, highly precise and accurate. Moreover,
in comparison to HPLC discussed method is relatively in-
expensive, less laborious and is characterized by better
statistical parameters. The time of analysis is short and
located between 1525 min for food extracts determination.
The sample preparation method is simple (only extraction
and filtration), short and omits derivatisation stage. Further-
more, fat content and non-protein nitrogenous substances
(low weight peptides and free amino acids) does not inter-
fere in histamine determination. The presented results reveal
that proposed method can be successfully used for histamine
determination in variety of samples. Therefore, it can be
considered that the proposed ITP method can be usefully
employed by the food industry in assessing quality of dif-
ferent food products.
Acknowledgment The authors acknowledge the grant from the Polish
Ministry of Science and Higher Education: grant no. NN 312 465640
(4656/B/P01/2011/40)
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