Food Control 22 (2011) 1154e1157

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

A simple and rapid method for histamine analysis in sh and shery products
by TLC determination
Zhihua Tao a, b, *, Minoru Sato b, Yali Han a, Zhujun Tan a, Toshiyasu Yamaguchi b, Toshiki Nakano b
a
b

Department of Food and Biology Technology, Guangdong University of Technology, Guangzhou University Town, 100, Guangzhou, China
Graduate School of Agricultural Science, Laboratory of Marine Biochemistry, Tohoku University, Sendai 981-8555, Japan

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 1 September 2010
Received in revised form
8 December 2010
Accepted 27 December 2010

A simple and rapid thin-layer chromatography (TLC)-densitometry method for histamine analysis in sh
and shery products was established. Histamine was extracted from seafood with 80% ethanol. The
sample extracts and standard solutions of histamine were applied to cellulose TLC plate and developed
by ascending chromatography with ammoniaeethanol 1:3 (v/v) as mobile phase. Histamine was visualized with paulys reagent, the intensity of the colored spots was digitized and calculated by image
processing method software, Lane & Spot Analyzer (ATTO, Tokyo, Japan). Successful separation of
histamine from other (imidazole compounds) paulys reagent-positive components in 80% ethanol sh
extracts was achieved. The linearity of the histamine estimation using this method was good within the
range from 30 ng to 1000 ng of histamine (r2 0.9997). Histamine can be satisfactorily detected and
completely separated from histidine, 4-methyl-imidazole and other paulys reagent-positive compounds.
This method does not need expensive instrument, any tedious pretreatment to eliminate potential
interference other imidazole compounds such as histidine or carnosine. It is also less reagents compared
with HPLC method. Moreover, it is a simple, rapid, sensitive, reproducible. Therefore this method is
suitable for monitor of histamine in multiple sh and shery products simultaneously that contain as
little as 20 ppm histamine (2.0 mg/100 g).
2010 Elsevier Ltd. All rights reserved.

Keywords:
TLC
Histamine
Fish
Paulys reagent development

1. Introduction
Histamine poisoning, an allergy-like food poisoning, was often
reported by ingesting seafood, mainly scombroid shes such as
mackerel, tuna, mahimahi, sardine and bluesh originating great
amount of histidine even though at present (Becker, Southwick,
Reardon, Berg, & MacCormack, 2001; CDC, 2000; Lehane & Olley,
2000; Lipp & Rose, 1997; Taylor, 1986; Ushiyama, Kan, Shindo, &
Saito, 2003). Since histamine is neither volatile nor destroyed by
cooking. Moreover, the cost is quite high for preventing scombroid
poisoning. Countries such as the USA through the Food and Drug
Administration (FDA), European Union, Canada have established
the regulatory guidelines for sh harvesting and processing.
In most cases histamine levels in illness-causing sh have been
above 200 ppm, often above 500 ppm (FDA, 1998a). Histamine was
often detected in various types of fresh and processed sh (Kan,
Ushiyama, Shindo, & Saito, 2005; Kanmuri, Kan, Hashimoto, Maki,
& Tamura, 1997; Kawabata, Ishizaka, & Miura, 1955; Lipp & Rose,
* Corresponding author. Graduate School of Agricultural Science, Laboratory of
Marine Biochemistry, Tohoku University, Sendai 981-8555, Japan.
E-mail address: tzh730@hotmail.com (Z. Tao).
0956-7135/$ e see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2010.12.014

1997). To prevent histamine poisoning because of ingesting seafood, the FDA established a hazards analysis and critical control
point (HACCP) program and set up guidelines for histamine at
5 mg/100 g (50 ppm) for scombroid sh (FDA, 1998b). Now, histamine monitoring has been globally accepted for safety conrmation
of sh and seafood products. A simple and rapid method for
histamine analysis is now required all over the world.
Different determination methods have been reported for the
analysis of histamine, including thin-layer chromatography (LapaGuimares & Pickova, 2004; Lieber & Taylor, 1978; Shakila,
Vasundhara, & Kumudavally, 2001), gas chromatography (Hwang,
Wang, & Choong, 2003), colorimetric assay (Hardy & Smith, 1976;
Kawabata, Uchida, & Akano, 1960), uorometric assay (AOAC,
2002), enzymaticassay (Lopez-Sabater, Rodriguez-Jerez, Roig-Sagues,
& Mora-Ventura, 1994), immunological assay (Serrar, Brebant,
Bruneau, & Denoyel, 1995) and high-performance liquid chromatography (Jensen & Marley, 1995; Jeyashakila, Vasundhara, &
Kumudavally, 2001; Sato et al., 1995; Yen & Hsieh, 1991). Concerning the determination of histamine, it is a very serious problem
to separate histamine completely from a very large amount of
interference compounds such as histidine or carnosine. Most
methods of analysis need a careful and tedious pretreatment to

Z. Tao et al. / Food Control 22 (2011) 1154e1157

eliminate potential interference substances, which is time consuming and prolongs the analytical process. We also developed
previously a simple and rapid method by paper electrophoresis
(Sato et al., 2006), but it also need electrophoresis equipment and
power supply.
A simple and rapid method for monitoring histamine levels in
sh and shery products is therefore needed to avoid delay in the
analysis in order to ensure safety of sh products. Our objective was
to adopt a simple and rapid TLC method for analysis of histamine to
monitor the suitability of sh products, according to regulatory
guidelines for histamine established by FDA. The TLC method with
paulys reagent visualization was studied because it does not
require tedious sample pretreatment. The TLC assay utilized the
reaction between the imidazole ring and sulfanilic acid as the basis
of a quantitative colorimetric method for estimation of histamine.
Sulfanilic acid is diazotized with sodium nitrite in HCl solution. And
diazotized sulfanilic acid reacts with imidazole ring compounds
under weak alkaline condition forming red azo dyes (Fig. 1). In
addition, 5e6 samples simultaneously can be analyzed on one plate
at a time, which results in low cost for the consumption of solvents.
2. Materials and methods
2.1. Chemical and supplies
L-Histamine, L-histidine, sodium nitrite, and sulfanilic acid were
purchased from Wako Pure Chemical Industries (Osaka, Japan).
L-carnosine, 2- and 4-methyl-imidazole, cadaverine, putrescine,
spermine, spermidine, tyramine were purchased from Sigma
(St. Louis, MO, U.S.A.), and all the reagents used were of analytical
grade. Paulys reagent (A and B) for the development of histamine
was prepared by the method of Sato et al. (2006). Reagent A also
consisted of two reagents, reagent A-1 and A-2. Reagent A-1 is
20 mM sulfanilic acid in a 1 M HCl solution and reagent A-2 is
a 200 mM sodium nitrite solution. Reagents A-1 and A-2 were
equally mixed just before spraying. Reagent B is l0% anhydrous
sodium carbonate in a 5% ethanol solution. Cellulose TLC plate was
purchased from Merck KGaA, Germany.

2.2. Histamine analysis

2.2.1. Preparation of standard solution
Stock standard solution of histamine (1000 mg/ml) was prepared
in 0.1 N HCl. Calibration standards were prepared by dilutions of the
stock histamine solutions (0e100 mg/ml) with 0.1 N HCl. Similarly,
the other standard samples such as histidine, 2- and 4-methylimidazole, L-carnosine and other amines were prepared separately
in 0.1 N HCl for each standard at 1000 mg/ml, then appropriate
dilutions were prepared for the assay.

1155

(200 mg) was homogenized in a sample tube with a vefold of 80%

ethanol using a Polytron homogenizer (KINEMATICA), and centrifuged at 12,000 rpm for 15 min, 5 ml of supernatant was applied to
cellulose TLC plate Paper electrophoresis assay, and HPLC assay
using a calibrated pipette (Drummond Scientic Company, USA).
Assay for the three methods were carried out in triplicate.
2.2.3. Thin-layer chromatography
Chromatography was performed on 10 cm  20 cm Cellulose
TLC plates (Merck KGaA Germany). Samples extract or standard
solution of histamine 5 ml was applied to the plates from the edge of
the plate. The cellulose TLC plate was developed in the mobile
phase ammoniaeethanol 3:1 (v/v). The plate was developed by
ascending chromatography over a distance of 60 mm. After development, the TLC plate was dried by dryer.
2.2.4. Color developing for histamine and quantication
The developed TLC plate was visualized by spraying with paulys
reagent A (equal mixture of A-1 and A-2) and then a reagent B. The
developed sample TLC plate with color density of histamine spot
was recorded using a digital camera CP-800 (EPSON, Tokyo, Japan).
Histamine spot was digitized and calculated by image processing
method software, Lane & Spot Analyzer (ATTO, Tokyo, Japan).
A standard histamine solution, 9 nmol (1.0 mg)/10 ml, was used as
the internal standard.
2.2.5. Recovery assay
Recovery assay was performed for examining the selectivity of
the method by adding the known concentration of histamine into
the extracts of sh sample. Each sample was rst analyzed separately with and without standard histamine solution at a concentration of 20, 40, 60 mg/100 g. A mixed sample was prepared from
equal parts of each sample to check the precision of the method.
3. Results and discussion
Histamine gave a red spot after pauly reagents development
and was completely separated from histidine (Fig. 2). It has been
conrmed that histamine also perfectly separate from other pauly
reagents positive compounds, such as histidine and carnosine, by
developing in mobile phase with ammoniaeethanol 1:3 (v/v). To
examine possible interference caused by reaction between the
reagent and other amines that can be extracted in the assay along

2.2.2. Extraction of histamine

To analyze histamine from sh samples freshly landed mackerel
were stored at 25  C for12 h, 24 h, 36 h. Each of the sh samples

Fig. 1. Pauly reaction equation.

Fig. 2. TLC separation pattern of standard histamine and histidine on the Cellulose TLC
plate (The spot of 4-methyl-imidazole was same as 2-methyl-imidazole, and the spot
of histidine was same as carnosine).

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Z. Tao et al. / Food Control 22 (2011) 1154e1157

Table 2
Recovery of added histamine in mackerel samples in TLC assay.

350000
y = 328568x + 619.56

300000

R = 0.9997

Parameter

Applied histamine level (mg/100 g)

0

20

40

60

Histamine range
Histamine recovered
SD
RSD (%)
Recovery (%)

16.1

34.0e35.7
18.53
0.93
5.02
93

53.4e55.7
38.3
1.17
3.05
96

72.9e76.6
58.67
1.85
3.15
98

Peak Area

250000
200000
150000
100000
50000
0
0

0.2

0.4

0.6
Histamine(g)

0.8

1.2

Fig. 3. Calibration curve of histamine, standard deviation (n 3) is expressed by error

bars.

with histamine, amines other than histamine were reacted with

pauly reagent in the same way. The other amines such as cadaverine, putrescine, spermine, spermidine or tyramine were negative
with the paulys reagent. Other amine compounds such as cadaverine, putrescine, spermine, spermidine or tyramine did not
interfere with this procedure.
Three different standard solutions of histamine were applied to
a TLC plate, leading to a calibration standard of 30, 500 and 1000 ng
histamine per spot. After TLC separation, the colored spot of
histamine was digitized and calculated by image processing
method soft lane spot analyzer. The linear calibration curve of
histamine estimation using this imaging processing method was
good within the range from 30 to 1000 ng (1 mg) of histamine
(r2 0.9997) (Fig. 3).
The histamine levels in mackerel incubated at 25  C were
compared using TLC method, paper electrophoresis and HPLC
method which we have developed (Sato et al., 1995). The histamine
contents in the mackerel esh samples determined by the three
methods agreed in the range of a relatively high histamine
concentration (Table 1).
In order to examine the sensitivity and accuracy of the TLC assay,
standard histamine solution (20, 40 and 60 mg/100 g) were added
into the mackerel esh samples, then the samples added with
histamine were analyzed for histamine recovery by the TLC assay.
The results are shown in Table 2. The histamine recoveries were 93,
96, 98 for mackerel sh samples. The precision of the assay was
determined by calculating the relative standard deviation for the
repeated measurements. The precision in the TLC assay ranged
from 3.05 to 5.02. The values in the result for the TLC assay were
regard as satisfactory.
Cellulose TLC method for histamine analysis by mobile phase
ammoniaeethanol was a simple and quicker than HPLC method. It
does not require tedious sample pretreatment such as sample
derivatization, clean-up step etc. The TLC assay only requires
applying sample solution to Cellulose TLC plate and dipping into
ammoniaeethanol solvent 3:1(v/v), after 30e40 min TLC development, histamine can be measured easily by not skilled person.
Table 1
Histamine contents in mackerel samples stored at 25  C by TLC, paper electrophoresis assay and HPLC assay.
Assay name

Histamine contents in mackerel samples

(mg/100 g) (Avg  SD)
Storage time (h)

TLC assay
Paper electrophoresis assay
HPLC assay
ND*: Not detected.

12

24

36

ND*
ND
ND

16.3  0.53
16.9  0.36
17.5  0.15

186.9  0.60
190.1  0.43
192.3  0.26

273.6  0.23
282.0  0.26
293.5  0.13

SD: Standard deviation.

RSD: Relative standard deviation.

Though this method is less sensitive than the HPLC method, the
differences in sensitive were not very signicant. Moreover, it can
simultaneously determine 5e6 samples on one plate at a time by
insert two plates simultaneously, 12 samples could be fractionated
about 30e40 min. It is noteworthy that entire estimation time of
one sample is quite short compared with the HPLC method and
other previously reported methods. The detection limit for histamine was 20 ppm (2.0 mg/100 g), which is more sensitive than the
guidance level of 50 ppm set by FDA (FDA, 1998b). It is well known
that histamine is generally not uniformly distributed in a decomposed sh. If 50 ppm is found in one section, there is the possibility
that other sections may exceed 500 ppm (FDA, 1998b). So, we
should check several sections simultaneously. From the aforementioned result, it is clear that the proposed TLC method has good
recovery, more precision and high sensitivity. Moreover, preparation of the samples is very simple and rapid and derivatization is
not needed before chromatography. This new method can be
therefore used in various scenes such as sh market, processing
plant, distribution, restaurants or at home.
Acknowledgments
This work was partly supported by Project 211 of Guangdong
province of China (No. 20091015).
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