Development of an HPLC-FLD method for rapid determination of Tyramine and Histamine in Tuna Fish

Introduction

Biogenic amines are basic nitrogenous compounds formed because of microbial decarboxylation of amino acid. Large amounts of biogenic amine can cause allergy like food poisoning, known as scromboid poisoning, which can lead to death in very sensitive subjects. Histamine content increases with the progress of fish decomposition at processing plants or even after catching. Tyramine is reported to cause food intoxication commonly associated with ripened cheeses, affecting health due to its capacity to potentiate sympathetic cardiovascular activity by releasing noradrenalin, called “cheese reaction”.

Decarboxylation of amino acid

Biogenic amines are decarboxylation products of amino acids and are formed during fermentation (e.g., cheese ripening and wine fermentation) and decomposition of protein, usually fish. These biogenic amines include Histamine, Tyramine, Cadaverine, Putrecine and related metabolites.

Decarboxylation of amino acid occurs by the removal of -carboxyl group to give corresponding amine.

Materials and methods

Standard histamine and Tyramine were purchased from sigma. Methanol of HPLC grade was supplied from Merck.

Apparatus and chromatographic condition

Quantitative analysis was performed by using Agilent HPLC system

by using Zorbax Eclipse amino acid column (4.6x150mm, 3.5µ) and methanol and phosphate buffer as mobile phase. Fluorescence Detector with excitation wavelength at 335nm emission wavelength at 440nm.

Fish samples preparation for biogenic amine extraction

10g of fish sample was extracted with 10ml 6% perchloric acid and was then centrifuged for 10 minutes at 3000rpm. The resulting supernatant was then filtered and finally injected into the system.

Results and Discussion

Linearity

Calibration curve for histamine

Calibration curve for Tyramine

Chromatogram of 5mg/kg mix standard

Recovery at 5mg/kg

Recovery at 20mg/kg

Precision and accuracy at 5mg/kg

Recovery 1 (mg/kg)

Recovery 2 (mg/kg)

Recovery 3

(mg/kg)

Average

Recovery %

RSD

Histamine

4.13

3.68

3.78

77.27

6.13

Tyramine

4.58

3.94

4.11

84.32

7.91

Precision and accuracy at 20 mg/kg

Recovery 1 (mg/kg)

Recovery 2 (mg/kg)

Recovery 3

(mg/kg)

Average

Recovery %

RSD

Histamine

17.47

17.85

17.87

88.68

1.27

Tyramine

20.21

19.02

18.12

95.59

5.49

Conclusion

The present study validated a rapid, sensitive and reproducible method using fluorescence detector to determine histamine and Tyramine concentration in tuna fish samples. 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 repeatability and could be useful in fish processing plants for routine analysis.

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Development of an HPLC-FLD method for rapid determination of Tyramine and Histamine in Tuna Fish

Testimonial

Mitra S.K. have added value to our business process by effective inspection, strict quality control and timely response. It is a pleasant experience to work with “Team Mitra S.K.”. We hope the same service level will continue in the future too....

Sirsendu Mukherjee

Mitra SK Inspection Gulf LLC has been appointed by us and the services provided by them has been up to the mark and as per our expectations....

Ajay Mathur

Amar Singh Gohil

The Inspection services provided by Mitra S. K. Inspection Gulf has been prompt and accurate and has been in the line with our expectations....

Naser Bustami

We would like to express our high appreciation of the business association our company had with Mitra S. K. as a testing and inspection house for their quality of service, good documentation and reporting during the job ....

R. Jeyakumar

Tulus Situmeang

Their job was excellent and showed Mitra S.K. differential from others....

Geraldo Pimenta

Konstantin Gertsen

We’ve been pleased with the improvement we have seen in CIQ results, and Mitra S.K’s reports have certainly helped us to resolve any disputes with our customers in a more timely manner….....

Brian Mcdonald

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