ANALYSIS OF HISTAMINE AS INDICATOR OF SHELF-LIFE IN SEAFOOD

Abstract

In the present study, a reversed high performance liquid chromatographic method with diode array detection was developed to analyze histamine in sea food samples. The separation was performed on a Kromasil OD S (C18) (150x3.20 mm, 5 µm) column thermostated at 25°C and using a gradient of Milli-Q water and methanol as mobile phase. The biogenic amine was extracted from seafood samples by using 0.1 N HCl and derivatized with dansyl chloride. Histamine contents ranged from 1.0 to 2.5 mg/kg. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

In the present study, a reversed high performance liquid chromatographic method with diode array detection was developed to analyze histamine in sea food samples. The separation was performed on a Kromasil OD S (C18) (150x3.20 mm, 5 µm) column thermostated at 25°C and using a gradient of Milli-Q water and methanol as mobile phase. The biogenic amine was extracted from seafood samples by using 0.1 N HCl and derivatized with dansyl chloride. Histamine contents ranged from 1.0 to 2.5 mg/kg.

Key words: chromatographic analysis, derivatization, food deterioration, histamine, shelf-life.

INTRODUCTION

Histamine is a biogenic amine resulting of the enzymatic decarboxylation of the amino acid histidine. It is considered an indicator of deterioration in foods and frequently used as a biomarker for food quality control. The European regulation (CE) N. 1441/2007 establishes a caution level of 100-200 mg/kg in seafood.

Several undesirable effects on the human health such as, hypotension, nausea, headache, rash, cardiac palpitation, emesis and even intracerebral anaphylactic shock, haemorrhage and death in very severe cases caused by high levels of histamine have been reported in the literature (Chiacchierini et ed., 2006; Innocente et ed., 2007).

Numerous analytical procedures have been developed for the determination of histamine including, enzyme-linked-immunoassays (ELISA), colorimetrie methods and chromatographic methods. Among them, high performance liquid chromatography appears as the most suitable technique to analyze the biogenic amine. Due to these compounds do not have a suitable fluorophore or chromophore group a derivatization is essential in order to enhance the sensitivity. Several derivatizing agents have been employed; the most commonly used are o-phthaldialdehyde, dansyl chloride and fluorescamine (Dugo et ed., 2006; Saito et al., 1992).

The aim of this paper is to determine the histamine content in sea food samples by high-performance liquid chromatographic with diode array detection after derivatization with dansyl chloride.

MATERIALS AND METHODS

Reagents and standard solutions

All reagents were of analytical grade. Methanol, acetone and hydrochloric acid were from Merck (Darmstadt, Germany); sodium bicarbonate from Vorquimica (Vigo, Spain) and dansyl chloride (Dns-cl) from Fluka (Steinheim, Germany). Water used for all solutions was obtained from a Milli-Q water purification system (Millipore; Bedford, MA). Standard of histamine was purchased from Aldrich. Stock standard solution of histamine was prepared in 0.1 N HCl and stored at 4°C in the darkness. Working solutions were prepared by dilution.

Instrumentation

HPLC-analysis were performed on a HP1 100 system (Hewlett-Packard Waldbronn, Germany) equipped with quaternary pump, a degassing device, an autosampler, a column thermostatting system, a diode-array detector (DAD), a fluorescence detector and Agilent Chem-Station for LC and LC/MS systems software.

Samples and extraction procedure

Surimi was selected as representative seafood and was purchased in a local supermarket. Histamine was extracted as follow; 25 mL of 0. 1 ? HCl were added to 5 g of sample, the mixture was homogenized by magnetic stirring for 10 min. The supernatant was removed and the residue was re-extracted with 25 mL of 0.1 ? HCL. The supernatants were combined and made up to 50 mL. The solution was stored at 4°C overnight in order to precipitate lipids and proteins. Then, an aliquot of the solution was filtered and derivatized. The dansylated derivative of histamine was formed by adding to 1 mL of sample 1 mL of dansyl chloride solution (5 mg/mL) and 300 µL of saturated NaHC03 solution; then the mixture was incubated at 80°C 60 min. Detailed information regarding derivatization procedure is reported in a paper submitted for publication.

Chromatographic conditions

The chromatographic separation was performed on a Kromasil ODS (CI 8) (150x3.20 mm, 5 µp?) column thermostated at 25°C.The mobile phases consisted of A (Milli-Q water) and ? (Methanol). The gradient elution program was as follows: 0 min (50% A/50% B); 45 min (10% A/90% B) 50 min (50% A/50% B). The flow rate was 0.8 mL/min and the injection volume 20 µ?^. The diode array detector was set at 254 nm.

RESULTS AND CONCLUSIONS

Since histamine has neither suitable absorption in the UV-Vis region nor fluorescence characteristics a derivatization step is essential in order to improve the sensitivity. According to the literature dansyl chloride is one of the most suitable derivatizing agents for biogenic amines. A method that involves the derivatization of amine with dansyl chloride followed by a reversed phase high performance liquid chromatography with detection at 254 nm was used to analyze the histamine content in surimi samples.

Atypical chromatogram of the dansylated derivative of the amine is shown in fig. 1.

Identification of histamine was made by comparison of the retention time and UV spectra with that of pure standard. A spectrum of the dansylated derivative is presented in fig. 2.

Quantification was carried out with the external standard method. Calibration line was constructed based on four concentration levels of standard solutions within 1-23 mg/L range and it was fitted to a linear equation y = 132.67 ? - 45.102 (R2 0.9995).

The recovery of the method was estimated on the basis of determination after spiking the samples with known amounts of histamine (10 mg/L). Satisfactory recoveries were achieved (102.8±2.8%). Once the analytical conditions were established the method was applied to determine histamine in surimi samples. The concentrations found in the samples analyzed ranged from 1 .0 to 2.5 mg/kg.

ACKNOWLEDGEMENTS

The study was financially supported by the Ministerio de Ciencia e Innovación, réf. ?. AGL/2008-04146 "MIGRAMIN". Authors are grateful to "Ministerio de Ciencia e Innovación" for the Predoctoral fellowship FPI (réf. BES-2009-023016) awarded to Rafael Paseiro-Cerrato.

References

REFERENCES

Chiacchierini E., Restuccla D., Vinci G. 2006. Evaluation of two different extraction methods for chromatographic determination of bioactive aminés In tomato products. Talanta, 69:548.

Commission Regulation (EC) N. 1441 /2007 of 5 December 2007 amending Regulation (EC) N. 2073/2005 on microbiological criteria for foodstuffs.

Dugo G., Vilasi F., La Torre G.L., Pellicano T.M. 2006. Reverse phase HPLC/DAD determination of biogenic amines as dansyl derivatives in experimental red wines. Food Chem. 95:672.

Innocente N., Biasutti M., Padovese M., Moret S. 2007. Determination of biogenic amines in cheese using HPLC technique and direct derivatization of acid extract. Food Chem. 101:1285.

Saito K., Horie M., Nose N., Nakagomi K., Nakazawa H. 1992. Determination of polyamlnes in foods by liquid chromatography with on-column fluorescence derivatization. Anal. Sci. 8:675.