Food Chemistry 126 (2011) 756–761

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Development and validation of an HPLC-FLD method for rapid determination

of histamine in skipjack tuna fish (Katsuwonus pelamis)
Saeed Tahmouzi a,b, Ramin Khaksar b,⇑, Mehran Ghasemlou a
a
Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, Campus of Agriculture and Natural Resources,
University of Tehran, P.O. Box 4111, Karaj 31587-77871, Iran
b
Department of Food Science and Technology, National Nutrition and Food Technology Research Institute, Faculty of Nutrition Science and Food Technology,
Shahid Beheshti University of Medical Sciences, Tehran, Iran

a r t i c l e i n f o a b s t r a c t

Article history: This study compared and validated two methods of high-performance liquid chromatography: fluores-
Received 18 May 2010 cence detection (HPLC-FLD) and UV–Vis detection. Recoveries greater than 55% at all spiking levels using
Received in revised form 25 October 2010 UV–Vis detection could not be obtained. However, derivatization with pre-column o-phthalaldehyde
Accepted 10 November 2010
(OPA) was found to efficiently enhance the method sensitivity. The analytical method was validated
based on the following criteria: linearity, sensitivity, accuracy, precision, repeatability, and recovery.
For fluorescence detection, excellent linear correlation with R2 = 0.9977 was observed over the range
Keywords:
of 5.0 to 100.0 mg/L. The minimum detection limit was calculated to be 1.5 mg/kg, while the limit of
Biogenic amine
Validation
quantification (LOQ) value of the validated method was determined to be 4.5 mg/kg. Good recoveries
Tuna fish (RSD% = 1.35) were obtained at all spiking levels ranging between 0 and 200 mg/kg. The proposed method
High-performance liquid chromatography was found to be suitable, selective, and rapid for the determination of histamine.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Biogenic amines are basic nitrogenous compounds formed as a
result of microbial decarboxylation of amino acids (Silla Santos,
1996). Large amounts of the biogenic amine can cause allergy-like
food poisoning, known as scombroid poisoning, which can lead to
death in very sensitive subjects (Hwang, Chang, Shiau, & Cheng,
1995). In previous studies, fish safety has been assessed purely
on the basis of histamine content, with most studies considering
cadaverine and putrescine as histamine toxicity potentiators
(Shakila, Vijayalakshmi, & Jeyasekaran, 2003). It was reported that
fish belonging to the Scomberesocidae family has often been impli-
cated in incidents of scombroid poisoning (Hwang et al., 1995).
Some studies have reported that histamine content increases with
the progress of fish decomposition at processing plants or even
immediately after catching (Lehane & Olley, 2000). Therefore, the
presence of histamine is of great importance when evaluating food
toxicity; it has also been proposed as an indicator of hygienic qual-
ity (Mah, Han, Oh, Kim, & Hwang, 2002). According to guidelines
issued by the US Food and Drug Administration (FDA), good-qual-
ity fish should contain less than 10 mg/kg of histamine, whereas a
level of 30 mg/kg indicates significant deterioration and 50 mg/kg
is considered to be a conclusive evidence of decomposition; fish
⇑ Corresponding author. Tel.: +98 21 22376426; fax: +98 21 22376470.
E-mail address: r.khaksar@sbmu.ac.ir (R. Khaksar).
with this level or greater should not be sold for human consump-
tion (Oguri, Enami, & Soga, 2007).
A sensitive and rapid method for monitoring histamines is ur-
gently needed to minimise the impact of biogenic amines, espe-
cially histamine, on human health, and also to practice hazard
analysis critical control point (HACCP) to ensure the safety of fish
products. This need is particularly great where decomposition is
suspected due to temperature abuse of fish (Önal, 2007).
Several methods to determine histamine levels in food have
been developed in recent decades, including oxygen-sensor based
assay using purified amine oxidase (Ohashi et al., 1994); copper
chelation assay (Bateman et al., 1994); enzyme-based screening
assay (Ben-Gigirey & Craven, 1998); enzyme-linked immunosor-
bent assay (ELISA) (Aygun et al., 1999); capillary electrophoretic
analysis (Mopper & Sciacchitano, 1994); and chromatographic
techniques such as gas chromatography (GC) (Laleye, Simatd,
Gosselin, Lee, & Giroux, 1987), thin-layer chromatography (Chin
& Koehler, 1983), and high-performance liquid chromatography
(HPLC) (Hwang, Chang, Shiau, & Chai, 1997; Yen & Hsieh, 1991).
Of these techniques, high-performance liquid chromatography is
most frequently used because of its high sensitivity and wide range
of linearity (Hwang et al., 1997). Generally, HPLC methods use a
pre-column or post-column derivatization step to facilitate detec-
tion, since the majority of biogenic amines do not possess chromo-
phoric or fluorophoric moieties (Lange, Thomas, & Wittmann,
2002). A number of reagents can be used for histamine
derivatization.

0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.11.060
 S. Tahmouzi et al. / Food Chemistry 126 (2011) 756–761
Dansyl chloride, fluorescamine, benzoyl chloride, and o-phthal-
aldehyde (OPA) are the four derivatization reagents most fre-
quently used by various researchers for histamine determination.
(Oguri & Yoneya, 2002).
 Dansyl chloride forms derivatives with limited stability and is
itself sensitive to light. Moreover, the reaction is time-consum-
ing even under heating.
 The reaction between fluorescamine and histamine is not rec-
ommended due to the instability and complexity of the prod-
uct; however, the reaction time is only a few minutes (Peng
et al., 2008).
 Short elution time, stability, relatively low cost and availability,
and the ability to react with both primary and secondary
amines are some advantages of benzoyl chloride (Özdestan &
Üren, 2009). Benzoyl derivatives of biogenic amines are not sen-
sitive to light (Özdestan & Üren, 2009).
o-Phthalaldehyde has been widely used as a fluorogenic reagent
in an alkaline medium for derivatization of biogenic amines such
as histamine (Yoshimura, Kaneuchi, Miura, & Kimura, 1987). Pre-
column derivatization with OPA is commonly used for HPLC anal-
ysis of histamine (Peng et al., 2008). One of the advantages of OPA
is that it reacts quickly with biogenic amines.
Among these derivatization procedures, the post- and on-col-
umn methods are most commonly chosen, due to broader peaks
of derivatives (Oguri & Yoneya, 2002).
Once a chromatographic method has been developed or modi-
fied, it should be validated by determining its linearity, sensitivity,
repeatability, and efficiency (Valls, Bello, & Kodaira, 1999).
This study was conducted to evaluate two HPLC methods for
quantitative determination of biogenic amines, particularly those
present in skipjack tuna fish samples. The objective was to estab-
lish a rapid, sensitive, and reproducible analysis method and to val-
idate it in terms of sensitivity, repeatability, linearity, accuracy, and
precision.
2. Materials and methods
2.1. Chemical reagents
Standard histamine dihydrochloride, putrescine dihydrochlo-
ride, cadaverine dihydrochloride, and tryptamine hydrochloride
were purchased from Sigma (St. Louis, USA). Acetonitrile of HPLC
grade was supplied from Merck (Darmstadt, Germany).
Analytical-grade reagents consisting of potassium dihydrogen
phosphate, trichloroacetic acid (TCA), perchloric acid, metformine
(internal standard, IS), 2-mercaptoethanol, diethyl ether, and
hydrochloric acid were obtained from Merck (Darmstadt,
Germany) and o-phthalaldehyde (chromatographic grade) from
Fluka (Buchs, Switzerland). The water used for eluent preparation
was purified by a Milli-Q System (Millipore, Billerica, MA, USA).
HPLC grade n-heptane was obtained from Mallinckrodt Baker
(Griesheim, Germany). Other chemicals and solvents used were
of analytical grade.
2.2. Apparatus and chromatographic conditions
Quantitative analysis was performed using the Waters HPLC
system (Waters Associates, USA), equipped with a Waters 616 gra-
dient pump (Waters-Millipore), a Waters 600S controller, and a
Waters 717 Plus autoinjector (Waters, Milford, MA). Separation
was performed on a Waters Nova Pak C18 HPLC column
(250 mm length, 4.6 mm internal diameter, and 5 lm particle size)
with a 4  4 mm security-guard cartridge column, and a Rheodyne
757
Model 7725i sample injector with an injection volume of 20 ll.
Solvents were vacuum filtered and degassed prior to use.
Two different elution conditions were used. In the first proce-
dure, a mobile phase with acetonitrile/potassium dihydrogen
(50:50), flow-rate of 2.5 ml/min and temperature of 20 °C were ap-
plied. Potassium dihydrogen concentration was increased gradu-
ally to 65% after 5 min, 75% after 10 min, and finally 80% after
15 min. Quantification was carried out by a Waters 486 variable
wavelength UV–Vis detector set at 254 nm. In the second method,
the separations were achieved with a simple mobile phase with
methanol/water (50:50) at a flow rate of 2.5 ml/min under iso-
cratic conditions. The detector was a Waters 470 scanning fluores-
cence detector at 320 nm (excitation wavelength, kex) and 523 nm
(emission wavelength, kem). A total separation time of 14 min was
required for each sample, and the column was equilibrated. A per-
sonal computer equipped with a Millenium 32 system (Waters,
Milford, MA) was used to analyse the chromatographic data.
2.3. Preparation of standard solution
A stock of standard solution as a histamine free base was pre-
pared by dissolving histamine dihydrochloride (16.55 mg) in a
50 ml volumetric flask and brought up to volume with 0.1 M HCl
to obtain a concentration of 1000 lg/ml. Similarly, cadaverine
dihydrochloride (17.14 mg), tryptamine hydrochloride (12.28 mg),
and putrescine dihydrochloride (18.29 mg) were prepared sepa-
rately as described above. The working standard solutions were
obtained by appreciate diluting the amine stock solutions in
0.1 M HCl to obtain aliquots of the following concentrations: 5,
10, 15, 20, 25, 50, 75, and 100 mg/l. The stock solutions were
prepared monthly and stored in brown glass bottles at 4 °C, while
working standard solutions were obtained daily.
2.4. Fish samples preparation for biogenic amines extraction
Frozen skipjack tuna samples (Katsuwonus pelamis), were pur-
chased from the Multifrost Fish Company (Karaj, Iran) and imme-
diately transferred in an icebox to the laboratory. Upon arrival,
frozen samples were thawed and exposed to room temperature.
A sharp knife was used to carefully remove the skins from the fil-
lets, and the samples were placed into sterile plastic bags prior to
any processing. Fifty grams of tuna fish were ground in a high-
speed Waring blender for 3 min and thoroughly homogenised with
20 ml trichloroacetic acid (TCA) 5% (w/v), 8 ml 0.1 M HCl and 16 ml
n-heptane (the solvent which we used for extraction, was chosen
after testing several solvents such as trichloroacetic acid (5% and
10%), perchloric acid, and methanol). Fifty millilitres of the result-
ing slurry was filtered into a centrifuge tube. The homogenate was
placed in an ultrasonic bath for 10 min, heated in a water bath at
60 °C, and cooled to room temperature. The supernatant was fil-
tered again through Whatman No. 1 filter paper into a clean plastic
tube after centrifugation at 4 °C for 15 min at 8500g (MSE model
GF6 centrifuge, Germany). One millilitre of the filtrate was used
for the analysis after derivatization with benzoyl chloride or o-
phthalaldehyde.
2.5. Derivatization with benzoyl chloride for UV detection
Derivatization with benzoyl chloride was performed according
to the method of Hwang et al. (1997) with minor modifications.
In brief, 1.0 ml of 2 M sodium hydroxide, 0.1 ml of internal stan-
dard solution (metformine), and 10 ll of benzoyl chloride were
added to 2.0 ml aliquot of the mixed biogenic amine solution.
The solution was mixed vigorously by using a vortex mixer (Cy-
clo-mixer) and allowed to stand for 30 min in a water bath at
40 °C. In order to stop the benzoylation reaction, 2.0 ml of
758 S. Tahmouzi et al. / Food Chemistry 126 (2011) 756–761
Table 1
Recoveries (%) of biogenic amines spiked to fish samples by using UV–Vis detection and banzoyl chloride derivation procedures were employed.
Analyte Concentration level (mg/kg)
50
Meana ± S.Db RSDc
Histamine 48.01 ± 4.05 8.45
Putrescine 37.76 ± 3.86 10.22
Cadaverine 39.37 ± 2.76 7.02
Tryptamine 37.7 ± 3.82 10.14
a
Means (%) of three replications.
b
Standard deviation.
c
Relative standard deviation (RSD% = (SD/mean)  100).
saturated NaCl solution was added. The solution was further mixed
by vortex mixer for 2 min and the resulting derivatives were ex-
tracted three times with 4 ml aliquots of diethyl ether. The solution
was then placed in an ultrasonic bath for 10 min, followed by cen-
trifugation at 8500g for 15 min. After centrifugation, the upper or-
ganic layer was decanted and transferred into a centrifugal vial and
evaporated on the rotary evaporator (Heidolph, Germany) until
completely dry. The residue was dissolved in 1.0 ml of methanol,
and 20-ll aliquots were injected for HPLC analysis.
2.6. OPA derivatization for fluorescence detection
Derivatization was performed by pre-column o-phthalaldehyde.
A 2.0 ml aliquot of the filtrate was transferred into a test tube,
1.0 ml of 1 M NaOH was added to make the solution alkaline,
and the solution was mixed well in a vortex mixer for exactly
1 min until it became yellow. After that, the tubes were allowed
to stand for 5 min at room temperature in the dark. One millilitre
OPA solution (previously prepared by dissolving 25.0 mg OPA,
1.0 ml methanol, and 100 lL 2-mercaptoethanol in 10.0 ml potas-
sium borate buffer) was added. Immediately after, 0.5 g of sodium
chloride was added. Finally, the o-phthalaldehyde derivatives were
extracted two times with 3.0 ml portions of ethyl acetate. For each
extraction, 3.0 ml of ethyl acetate was added to the extract, and the
tubes were vortexed for 1 min and then centrifuged for 15 min at
8500g. The ether layer was aspirated, and ether extracts were com-
bined and evaporated to dryness on the rotary evaporator. The res-
idue was then dissolved in 1.0 ml acetonitrile and 20 ll aliquot was
injected for HPLC analysis.
3. Results and discussions
3.1. Method validation of analysis by UV–Vis detector
As different authors report, detectors such as fluorescence, UV,
and electrochemical detectors may be used for determining the
presence of biogenic amines. It was reported that the major bio-
genic amines detected in tuna fish are putrescine, cadaverine,
and histamine. Because the literature revealed inconsistent results
using different detectors for determining biogenic amines, it was
necessary to improve analysis. HPLC–UV was used to determine
the suitability of a detector for identifying biogenic amines. Differ-
ent elution programs were evaluated to determine which achieved
good resolution in the shortest time. To validate the method, the
recovery and repeatability data were taken into consideration. Sur-
prisingly, we could not observe recoveries greater than 55% at all
spiking levels for biogenic amines. In an attempt to solve the prob-
lem, we applied liquid–liquid extraction and solid-phase extrac-
tion (SPE) to remove interfering compounds, but the recoveries
did not improve. We concluded that this detection technique can-
not be used owing to its limitations, and discontinued the use of
the UV detector. Recoveries of added biogenic amines are shown
100 200
Mean ± SD RSD Mean ± SD RSD
52.70 ± 3.22 6.11 55.04 ± 5.67 10.31
48.51 ± 3.21 6.63 50.75 ± 1.18 2.34
41.31 ± 3.15 7.63 45.70 ± 4.22 9.23
39.18 ± 3.51 8.97 41.04 ± 2.06 5.03
in Table 1. According to some studies, biogenic amines do not ex-
hibit satisfactory absorption at the visible or ultraviolet wave-
lengths, and fluorescence detection has been replaced in recent
decades by other methods (Proestos, Loukatos, & Komaitis, 2008).
The most probable reason for the low recovery was the com-
plexity of the sample matrix. In addition, due to the presence of
potentially interfering compounds in the sample matrix, simulta-
neous determination of several biogenic amines could have been
difficult and time-consuming. Therefore, only histamine as an
important biogenic amine in skipjack tuna fish samples was
evaluated.
3.2. Choosing the best conditions for extraction of samples
Extraction of biogenic amines from fish samples should be
undertaken prior to chromatographic analysis. The histamine sta-
bility was checked for different solvents; it is unstable in water,
while it is stable in acidic conditions (Cinquina et al., 2004). In a
preliminary test, different solvents were checked according to their
suitability for amine extraction. Two different concentrations (5%
and 10%) of trichloroacetic acid (TCA), perchloric acid, and metha-
nol (100%) were considered. Of these, a 5% TCA solution provided
rapid extraction and better chromatographic peak resolution. We
could not obtain good peak shape and effective extraction using
perchloric acid (Fig. 1A); this can probably be attributed to incom-
plete extraction and formation of additional peaks at the same
retention time as the eluted histamine. Earlier, Lange et al.
(2002) obtained similar results for this solvent for extraction of
histamine from fish muscle.
3.3. Method development by fluorescence detector
Fig. 1B shows a typical chromatogram of histamine standard
monitored at 320 nm (excitation wavelength, kem) by fluorescence
detector. Separation was completed in less than 14 min. This over-
all run time was very similar to those reported in the literature
(Hwang et al., 1997; Tsai et al., 2005). Fig. 1C shows a typical chro-
matogram of a blank tuna fish sample. Under the stated experi-
mental conditions, no interfering peaks were eluted in the region
of interest, and as seen in the blank sample chromatogram
(Fig. 1C), the interfering substances eluted much earlier than the
histamine peak. Fig. 1D shows a representative chromatogram of
a sample spiked with 100 mg/kg of histamine. Quantification was
based on the comparison of the analyte peak areas versus an exter-
nally generated calibration (Cinquina et al., 2004).
3.4. Method Validation of analysis by fluorescence detector
3.4.1. Linearity
Validation of analysis included linearity, limit of detection
(LOD), limit of quantification (LOQ), repeatability, accuracy, and
recovery. Five equispaced concentrations in the range of 5 to
 S. Tahmouzi et al. / Food Chemistry 126 (2011) 756–761 759

Fig. 1. Typical HPLC chromatograms: (A) after extraction with perchloric acid; (B) histamine standard monitored at 320 nm (excitation wavelength, kex) and 523 nm
(emission wavelength, kem) by fluorescence detection; (C) blank tuna fish sample after extraction with 5% TCA; (D) sample spiked with 100 mg/kg of histamine; conditions:
pre-column derivatization with OPA/2-ME, Nova Pak C18 column at 20 °C, isocratic condition, eluents A–B began (50:50, v/v) at a flow rate of 2.5 ml/min, injection loop 20 ll.
760 S. Tahmouzi et al. / Food Chemistry 126 (2011) 756–761

100 mg/l with three replications at each injection were chosen to 3.4.3. Precision and accuracy
generate an external calibration curve. A linear curve was found Intra-day repeatability was calculated by injecting each of the
between peak area and analyte concentration with a good correla- five different concentrations mixtures three times on the same
tion coefficient (R2 = 0.9977). day, while the inter-day reproducibility was assessed over six dif-
ferent days at the same concentration levels (Saaid, Saad, Hashim,
3.4.2. Sensitivity Mohamed Ali, & Saleh, 2009). The intra-day assay precision (ex-
The results obtained in the linearity test were analysed to calcu- pressed as% RSD) was in the range of 0.55% to 2.67%, while the in-
late LOD and LOQ. The LOD and LOQ values were calculated using ter-assay precision ranged from 1.13% to 1.94%. The accuracy of the
the following equation: method (trueness, %) was determined by assessing the agreement
between the observed and nominal concentrations of analysed
C ¼ SD  S=K samples (Cinquina et al., 2004). These values were considered very
where SD is the standard deviation of the Y-intercept values, and K satisfactory. The results of intra-day and inter-day assay precision
is the mean slope value from the five calibration curves constructed. and accuracy are shown in Tables 2 and 3 respectively.
The S values were 3 for LOD and 10 for LOQ, respectively (Numa-
noğlu, Hakki Boyaci, & Topcu, 2008). The LOD, defined as the small- 3.4.4. Recovery
est concentration from which it is possible to detect the presence of Recovery is an important step in the validation procedure, as it
the analyte with reasonable statistical certainty, was calculated to provides the analyst with knowledge about the efficiency of the
be 1.5 mg/kg; and the LOQ, defined as the lowest concentration of procedure (Valls et al., 1999). Recovery studies of the method were
the identified analyte in a sample that can be quantified with performed by spiking various known concentrations of histamine
acceptable precision and accuracy, was found to be 4.5 mg/kg for ranging from 0 to 200 mg/kg into fish samples. The test was per-
the proposed method (Cinquina et al., 2004). The sensitivity of the formed three times and recoveries were calculated by comparing
method as reflected by the LOD and LOQ values was similar to those the peak area of the fortified sample with that of the standard. Sat-
reported in the literature (Gosetti, Mazzucco, Gianotti, Polati, & isfactory recoveries were observed at all spiking levels of hista-
Gennaro, 2007; Oguri et al., 2007; Shakila, Vasundhara, & Kumuda- mine. Data from these experiments are reported in Table 4.
vally, 2001). Among the samples tested for recovery, two samples overesti-
mated the recovery by a margin that ranged from 3% to 5%. This
Table 2
can be attributed to non-uniform distribution of histamine origi-
Intra-day assay precision and accuracy data for histamine in tuna fish samples (n = 3). nally present in the fish muscle (Patange, Mukundan, & Kumar,
2005). A comparison of the current results with the literature on
Nominal Observed concentration Precisionb(%) Truenessc
concentration (mg/l, mean ± SDa) (%)
method validation revealed that the recoveries in the current study
(mg/l) were considerably higher than the range of those investigations.
25
For example, in a study conducted by Cinquina et al. (2004), an
(Day 1) 23.82 ± 0.23 0.99 4.70 average recovery of 92% was determined for the recovery of added
(Day 2) 24.11 ± 0.39 1.64 3.55 histamine at 5, 10, and 20 mg/100 g levels, which is below the re-
(Day 3) 23.83 ± 0.26 1.10 4.67 sults obtained in this research.
(Day 4) 23.86 ± 0.19 0.80 4.54
(Day 5) 23.93 ± 0.25 1.05 4.27
(Day 6) 24.09 ± 0.29 1.21 3.61
50 Table 3
(Day 1) 48.13 ± 1.10 2.28 3.72 Inter-day assay precision and accuracy data for histamine in tuna fish samples
(Day 2) 48.06 ± 0.88 1.83 3.87 (n = 18).
(Day 3) 47.43 ± 0.76 1.60 5.14
Nominal Observed concentrationa Precisionb Truenessc
(Day 4) 47.36 ± 0.37 0.79 5.26
concentration (mg/ (mg/l, mean ± SD) (%) (%)
(Day 5) 48.56 ± 0.64 1.31 2.86
l)
(Day 6) 47.64 ± 0.38 0.80 4.71
75 25 23.94 ± 0.27 1.13 4.22
(Day 1) 71.72 ± 1.11 1.55 4.37 50 47.86 ± 0.69 1.44 4.26
(Day 2) 72.53 ± 1.09 1.51 3.29 75 72.24 ± 1.26 1.75 3.67
(Day 3) 72.25 ± 1.29 1.79 3.66 100 100.94 ± 1.96 1.94 0.94
(Day 4) 72.08 ± 1.59 2.21 3.88 200 209.56 ± 2.62 1.25 4.78
(Day 5) 72.49 ± 1.93 2.67 3.34 a
n = 6 days with three replicates per day.
(Day 6) 72.36 ± 0.55 0.76 3.51 b
Expressed as RSD: RSD% = (SD/mean)  100).
c
100 Trueness % = (Observed concentration – nominal concentration)/nominal
(Day 1) 101.54 ± 2.30 2.27 1.54 concentration  100.
(Day 2) 100.85 ± 1.82 1.81 0.85
(Day 3) 99.77 ± 1.14 1.14 0.22
(Day 4) 100.46 ± 2.48 2.46 0.46
(Day 5) 101.47 ± 1.67 1.65 1.47 Table 4
(Day 6) 101.55 ± 2.37 2.33 1.55 Recovery (%) of histamine spiked to tuna fish samples by using HPLC-FLD.

200 Spiked level (mg/kg) Fish samples

(Day 1) 206.66 ± 4.11 1.99 3.33
Mean of recoverya (%) (n = 18) SDb RSD (%)c
(Day 2) 208.66 ± 5.32 2.55 4.33
(Day 3) 209.60 ± 1.87 0.89 4.80 25 95.77 1.09 1.13
(Day 4) 212.06 ± 1.22 0.57 6.03 50 95.73 1.38 1.44
(Day 5) 209.62 ± 1.98 0.94 4.81 75 96.32 0.99 1.03
(Day 6) 210.78 ± 1.21 0.57 5.39 100 100.94 1.96 1.94
200 104.78 1.31 1.25
a
SD: standard deviation.
b a
Expressed as RSD: RSD% = (SD/mean)  100). n = 6 days with three replicates per day.
c b
Trueness % = (Observed concentration – nominal concentration)/nominal Standard deviation.
c
concentration  100. Relative standard deviation (RSD% = (SD/mean)  100).
 S. Tahmouzi et al. / Food Chemistry 126 (2011) 756–761
4. Conclusion
The results of this survey confirmed that HPLC–UV was not a
good detector for determining biogenic amines in tuna fish. The
present study validated a rapid, sensitive, and reproducible meth-
od using fluorescence detection to determine histamine concentra-
tions in tuna fish samples. Two derivatization procedures were
applied and results were compared. The OPA derivatization proce-
dure is to be preferred to the benzoyl chloride method, as OPA re-
acts more quickly. Based on the results in this study, OPA
derivatization can be considered advantageous because it reduces
the time of analysis and allows excellent recoveries and repeatabil-
ity, and could be useful in fish-processing plants for routine
analysis.
Acknowledgements
The authors gratefully acknowledge the financial support ex-
tended for this research by the Nutrition and Food Science Faculty
of Shahid Beheshti Medical Science University of Iran. The first
author would also like to thank Dr Fariborz Shojaei for his technical
advice on HPLC analysis.
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