Cogent Chemistry

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Validation of high-performance liquid

chromatography (HPLC) method for quantitative
analysis of histamine in fish and fishery products

B.K.K.K. Jinadasa, G.D.T.M. Jayasinghe & S.B.N. Ahmad

To cite this article: B.K.K.K. Jinadasa, G.D.T.M. Jayasinghe & S.B.N. Ahmad (2016) Validation of
high-performance liquid chromatography (HPLC) method for quantitative analysis of histamine in
fish and fishery products, Cogent Chemistry, 2:1, 1156806, DOI: 10.1080/23312009.2016.1156806

To link to this article: https://doi.org/10.1080/23312009.2016.1156806

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ANALYTICAL CHEMISTRY | RESEARCH ARTICLE

Validation of high-performance liquid

chromatography (HPLC) method for quantitative
analysis of histamine in fish and fishery products
B.K.K.K. Jinadasa, G.D.T.M. Jayasinghe and S.B.N. Ahmad

Cogent Chemistry (2016), 2: 1156806

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Jinadasa et al., Cogent Chemistry (2016), 2: 1156806
http://dx.doi.org/10.1080/23312009.2016.1156806

ANALYTICAL CHEMISTRY | RESEARCH ARTICLE

Received: 21 December 2015
Accepted: 17 February 2016
First Published: 22 February 2016
*Corresponding author: B.K.K.K.
Jinadasa, Analytical Chemistry
Laboratory (ACL), Institute of Post-
Harvest Technology (IPHT), National
Aquatic Resources Research &
Development Agency (NARA), Crow
Island, Colombo 15, Sri Lanka
E-mail: jinadasa76@gmail.com
Reviewing editor:
Federico Marini, Universita degli Studi di
Roma La Sapienza, Italy
Additional information is available at
the end of the article
Validation of high-performance liquid
chromatography (HPLC) method for quantitative
analysis of histamine in fish and fishery products
B.K.K.K. Jinadasa1*, G.D.T.M. Jayasinghe1 and S.B.N. Ahmad1
Abstract: A high-performance liquid chromatography method is described for
quantitative determination and validation of histamine in fish and fishery product
samples. Histamine is extracted from fish/fishery products by homogenizing with
tri-chloro acetic acid, separated with Amberlite CG-50 resin and C18-ODS Hypersil
reversed phase column at ambient temperature (25°C). Linear standard curves with
high correlation coefficients were obtained. An isocratic elution program was used;
the total elution time was 10 min. The method was validated by assessing the fol-
lowing aspects; specificity, repeatability, reproducibility, linearity, recovery, limits of
detection, limit of quantification and uncertainty. The validated parameters are in
good agreement with method and it is a useful tool for determining histamine in
fish and fishery products.

Subjects: Food Analysis; Meat & Poultry; Seafood

Keywords: high performance liquid chromatography (HPLC); histamine; method validation;

fish

ABOUT THE AUTHOR PUBLIC INTEREST STATEMENT

B.K.K.K. Jinadasa received the Bachelor of Science
(BSc-sp), Master of Science (MSc) degrees and
postgraduate diploma from the University of
Ruhuna, University of Sri Jayewardenapura, Sri
Lanka and United Nation University, Iceland,
respectively. During 2005-up to date, he stayed
in National Aquatic Resources Research and
Development Agency (NARA), Sri Lanka as a senior
scientist and technical manager of Analytical
Chemistry Laboratory (ACL).
B.K.K.K. Jinadasa
Scombrotoxin or histamine fish poisoning is a
seafood-related foodborne illness mainly found
in scombridae fish family (mainly tuna fish).
Scombrotoxin (histamine) forms as a result of time
and temperature abuse of certain species and
it may be due to consumption of inadequately
preserved and improperly refrigerated fish.
Histamine is a kind of biogenic amine produces
the results of enzyme histadine decarboxylase.
Generally the diagnosis is made on the skin
flushing, rash, gastrointestinal complaints and
throbbing headache, etc. There for analysis
of histamine content in the fish is very much
important and High Performance Liquid
Chromatography method (HPLC) is more sensitive
and accurate method used to quantify the
histamine content under the EU regulations. The
minimum detection limit is 1 mg/kg and it has to
range in-between 1 and 200 mg/kg. Validation of
this method should be very essential for enhancing
the method sensitivity, linearity, accuracy,
precision, recovery, repeatability and reproducibility.

© 2016 The Author(s). This open access article is distributed under a Creative Commons Attribution
(CC-BY) 4.0 license.

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1. Introduction
Histamine is a biogenic amine produced by decarboxylation of free histidine. Histamine is generally
present at low levels in the human body and can be present in a range of foods such as fish, cheese,
meat, wine and fermented foods (Rocco, Lina, Nicola, & Paolo, 2006). Scombroid fish (Teleostea,
Scombroidei), such as mackerel and tuna, or clupeid fish (Teleostea, Clupeoidei) such as sardines,
anchovies and herrings are often involved in histamine toxicity. Histamine remains one of the prob-
lems for exporting tuna and tuna-like species from the tropics and subtropics to international mar-
kets. The food and drug administrative (FDA, 1998) set the safety level of histamine 5/100 g to
ensure safety of the products. The European Union (EU/EC, 2005) has established that the average
content of histamine in fish should not exceed 100 mg/kg and no sample may contain more than
200 mg/kg and fishery products should not exceed 200 mg/kg and no sample may contain more
than 400 mg/kg out of nine samples. Mishandling coupled with high temperature abuse are com-
mon practices in handling fish in the tropic and subtropics, which significantly enhance histamine
formation (Nejib, Moza, Ismail, Ann, & Mohammad, 2005).

Several methods to analyse biogenic amines in food have been described so far, including thin-
layer chromatography, the use of the amino acid analyzer, liquid chromatography, gas chromatog-
raphy and seven several biochemical assays. The method routinely used for histamine involves an
extraction with methanol, subsequent ion-exchange chromatography and a chemical reaction with
O-Phtaldehyde (OPA) or dansyl chloride under defined conditions to measure the resulting fluores-
cent reaction products (Jana, Kathleen, & Christine, 2002).

The aim of this study was a modified high-performance liquid chromatography (HPLC) method on
the basis of an automated pre-column derivatization described by handouts of 5th regional training
course in fish quality assessment methods of seafood safety (2004), South Asian Fisheries
Development Center (SEAFDEC), Singapore. In addition, the method was optimized in terms of speci-
ficity, repeatability, reproducibility, linearity, recovery, limit of detection (LOD), limit of quantification
(LOQ) and uncertainty.

2. Methods

2.1. Apparatus
The HPLC model Shimadzu, SIL 20A (Kyoto, Japan) equipped with LC solution software, quaternary
pump and online degasser model LC20AD and injection valve with a loop capacity of 20 μL was used.
The detector used was a programmable fluorescence detector model RF10AXL with a 350-nm exci-
tation, 450-nm emission. The histamine compound was determined on reverse-phase ODS Hypersil
(150 × 4.6 mm), C18 column.

2.2. Reagents and standards

(a) Histamine standard solution: Weighed 82.9 mg of histamine dihydrochloride (C5H9N3.2HCl;

Fluka chemicals, Japan) to the nearest 0.1 mg on an analytical balance and dissolved in HPLC water
in a 50-mL one-mark volumetric flask and made up to the mark with HPLC grade water. Then, pre-
pared the 100 and 10 mg/L intermediate standards. Those are prepared fresh weekly. The working
standard solution prepared fresh daily on 0.5, 1.0, 1.5, 2.0 and 3.0 mg/L concentration.

(b) Ion-exchange resin (Amberlite CG-50): Amberlite CG-50 (100–200 mesh, H+ form, Fluka chemi-
cals, USA) resin is converted to the H-form as follows. Added 100 mL of 1 M HCl per 20 g of resin and
stir continuously for 10 min. Let it stand for another 10 min or more. Discarded the upper liquid layer.
Repeat this procedure three times. Thoroughly washed the resin with distilled water until free form
HCl.

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(c) 0.1% O-phthaladehyde (OPT) in methanol: Dissolved 100.0 mg of O-phataladehyde (Fluka

chemicals, Austria) in methanol in a 100-mL one-mark volumetric flask and made up to the mark
with methanol. Tore in an amber bottle in a refrigerator.

2.3. Sample preparation and extraction

Cut whole sample into small pieces and mashed mechanically. Mixed mass well and weighed a 10-g
sample into 100-mL beaker and added 20 mL of 10% tri-chloro acetic acid (TCA) and 20 mL of dis-
tilled water. Homogenized sample in 2 min using homogenizer (Heidolph-Q1) and transferred the
sample into 100-mL volumetric flask, made up using distilled water and stood 10 min. Then, the
sample was filtered through Whatman No. 1 filter paper and pipetted out 10 mL of filtrate to 50-mL
beaker and adjusted the pH to 4.6 (Hanna, pH 211, USA) and passed through the column filled by
Amberlite CG-50 resin. The 8 mL of eluted samples were taken into 25-mL beaker and adjusted the
pH to 7 and made up to 10 mL with distilled water.

2.4. Derivatization of sample extracts and standard

Pipetted out 5.0 mL of the column chromatography elute into a 10.0-mL volumetric flask and added
1.00 mL of 1.0 M NaOH and mixed. Then added 0.50 mL of 0.1% OPT and mixed. After that, added
1.50 mL of 1 M H2SO4 just after 4 min and mixed. Made up to the mark with distilled water and mixed
thoroughly.

2.5. Chromatographic separation

Set up the HPLC system and follow at least 30 min to stabilize. The HPLC conditions were maintained
as follows; column; ODS Hypersil (150 × 4.6 mm), mobile phase; NaCl:Methanol (20:80), adjust to pH
3.1, flow rate; 0.5 mL/min, detection; excitation 350 nm, emission 450 nm.

2.6. Calculation
Histamine content (mg/kg) is calculated using the area under the peaks of standard histamine solu-
tion chromatograms.

= [Measured concentration of histamine in extract (mg/L)/Sample weigh (g)] × 100

2.7. Method validation

Standard quality control materials (canned fish) T-2742 from FAPAS (Food Analysis Performance
Assessment Scheme, the Food and Environment Agency, Sand Hutton, York, UK) were used for qual-
ity control in the study. The method validation procedures were followed according to IUPAC techni-
cal report, harmonized guidelines for single laboratory validation of methods of analysis (IUPAC,
2002) and EURACHEM/CITAC guide CG 4, quantifying uncertainty in analytical measurement
(EURACHEM/CITAC, 2000). In the method validation following parameters were calculated, i.e. speci-
ficity, selectivity, precision, accuracy, linearity and range, LOD, LOQ, robustness/ruggedness and
uncertainty.

3. Results and discussion

The chromatographic separation of histamine standard, (upper) (1 μg/L) & fish sample (lower) is
shown in Figure 1.

The precision of the method was assayed by 6 replicate extraction of pure analytical standard,
which contained histamine in low, medium and high concentrations within the shortest possible
time period in the same instrument. Calculated amount, standard deviation and relative standard
deviation are listed in Table 1.

The accuracy and reproducibility were tested using histamine quality control material (FAPAS T
2742, assigned value is 30.3 mg/kg) at different time periods. The results are given in Table 2.

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Figure 1. Chromatographic
separation of histamine
standard (upper) and fish
sample (lower).

Table 1. Precision of method assayed by different histamine concentrations

Sample type Histamine concentration (mg/kg)
Concentration of histamine in the sample Low = 20.00 Medium = 100.00 High = 200.00
1 21.82 97.07 199.86
2 21.96 98.53 198.27
3 22.09 100.07 198.88
4 22.91 100.45 194.60
5 22.77 102.10 198.41
6 22.31 102.85 198.61
Mean 22.31 100.18 198.11
Standard deviation (s) 0.44 2.16 1.81
Relative standard deviation (RSD), % 2.0 2.2 0.9

The LOD was established with six independent sample blanks fortified at a lowest acceptable
concentration of histamine (1.00 mg/kg) and measured once each. LOD was calculated using ana-
lyte concentration corresponding to a mean blank value +3s. According to that, the LOD was 0.2 mg/
kg histamine and LOQ was calculated as LOD × 5 and it was 1 mg/kg of histamine.

The linearity and working range calculated by normal calibration curve was extended to higher
and lower ranges (Figure 2).

The dilution factor was 10, hence the working range of this method is calculated as 1.0–250.0 mg/
kg, with 0.99 or more correlation coefficient.

Identify variable/interferences which could have a significant effect on method performance. Set
up experiments (using reference materials or histamine standards) in order to monitor the effect of

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Table 2. Accuracy and reproducibility values in sample

Trial No. Histamine concentration (mg/kg)
1 26.2
2 24.1
3 25.5
4 26.4
5 28.6
6 26.8
Mean 26.3
Standard deviation (s) 1.5
Relative standard deviation (RSD), % 5.7

Figure 2. The graph of 160

x 100000

2
R = 0.9958
peak area vs. histamine 140
concentration. 120
100
80
Peak area

60
40
20
0
0 5 10 15 20 25 30
Histamine concentration (mg/kg)

each changed condition on the mean value (percentage of validation). Rank the variables in order of
significant effect on method performance. Maintain quality control data in order to control the effect
of critical variables. Interfering compounds in the analysis of histamine are cadeverine and putres-
cine (having a similar chemical structure of histamine) and the effect of those studied. The mixture
of histamine, cadeverine and putrscine was analysed by HPLC. Separate three peaks were observed
for histamine, cadeverine and putrescine. That means, this method is effective to separate hista-
mine from other similar amine compounds.

The pH of mobile phase and the ambient temperature were identified as critical variables.
According to the method, the pH of the mobile phase and ambient temperature are 3.1 and 25°C,
respectively. Six histamine standards (100.0 mg/kg) were analysed under normal and change condi-
tions (pH of mobile phase 3.4 and ambient temperature 30°C). The percentage of variation in be-
tween replicates was calculated and variation of mobile phase pH and ambient temperature were
2.7 and 4.1%, respectively.

From our work, the efficiency of OPT derivatives extraction calculated from the unfortified and
fortified fish samples, yellowfin tuna (fortified 50.0 mg/kg, histamine standard solution) at different
time periods were found and the results are given in Table 3. Average recoveries for fortified samples
were 86.3% and that is between the AOAC recommended ranges (75–120%).

The main component of uncertainty calculation was associated with the recovery (3.97%) and the
uncertainty associated with precision was 0.022%. The expanded uncertainty value of this method
was calculated as 11.0% (k = 2).

After validation the method, the analytical chemistry laboratory, national aquatic resources re-
search and development agency (NARA), Sri Lanka participated the two proficiency testing scheme
(low level and high level histamine in fish matrices) with this method and that results also confirmed
that the method is suitable for the determination histamine in fish and fishery products (z-score −1.4

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Table 3. Recovery percentage of histamine fortified samples

Trial No. Histamine concentration (mg/kg) Recovery (%)
1 43.95 87.9
2 42.76 85.5
3 40.42 80.8
4 53.39 106.8
5 38.99 77.98
6 39.37 78.74
Mean 86.3
Standard deviation (s) 10.8
Relative standard deviation (RSD), % 12.5

was obtained at an assigned value of 212 mg/kg, z-score 0.0 was obtained at an assigned value of
26.8 mg/kg in the Food Analysis Performance Assessment Scheme, Proficiency Testing Report 27137,
May–June 2014 and 27126, Oct. 2013, The Food and Environment Research Agency, Sand Hutton,
York YO41 1LZ, UK).

4. Conclusion
Considering the results of method validation criteria such as specificity, selectivity, precision, accu-
racy, linearity and range, LOD, LOQ and uncertainty, this method is suitable for the determination of
histamine in fish and fishery product samples. The method was also validated and results comply
with ISO 17025 laboratory accreditation criteria.

Acknowledgements References
Our thanks to Mrs. J.M. Chandrika (IPHT-NARA) and Mrs. Tan EU/EC. (2005). Commission Regulation (EC) No 2073/2005 on
Lu Hsia (SEAFDEC) for assistance. microbiological criteria for foodstuffs. Official Journal of the
European Union, L338, 1–26.
Funding EURACHEM/CITAC. (2000). EURACHEM/CITAC Guide CG 4. In
The authors received no direct funding for this research. S. L. R. Ellison & A. Williams (Eds.), Quantifying uncertainty
in analytical measurement. EURACHEM.
Author details FDA. (1998). Scombrotoxin (histamine) formation. In Fish and
B.K.K.K. Jinadasa1 fishery products hazards and control guide (pp. 73–90).
E-mail: jinadasa76@gmail.com Washington, DC: Department of Health and Human
G.D.T.M. Jayasinghe1 Services, Public Health Service, Nutrition, Office of Seafood.
IUPAC. (2002). Harmonized guideline for single laboratory
E-mail: thilini.madurangika123@gmail.com
validation method of analysis. Journal of pure applied
S.B.N. Ahmad1
chemistry, 74, 835–855.
E-mail: sbnaahmad@yahoo.com
Jana, L., Kathleen, T., & Christine, W. (2002). Comparison of a
1
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Citation information
S. R. (2005). The effect of storage temperature on
Cite this article as: Validation of high-performance
histamine production and the freshness of yellowfin tuna
liquid chromatography (HPLC) method for quantitative
(Thunnus albacares). Food Research International, 38,
analysis of histamine in fish and fishery products, B.K.K.K.
215–222.
Jinadasa, G.D.T.M. Jayasinghe & S.B.N. Ahmad, Cogent
Rocco, R., Lina, M., Nicola, U., & Paolo, R. (2006). Influence of
Chemistry (2016), 2: 1156806.
storage temperature and freezing time on histamine
level in the European anchovy Engraulis encrasicholus
Cover image
(L., 1758): A study by capillary electrophoresis. Journal of
Source: Kolita Jinadasa.
Chromatography B, 830, 161–164.

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