studying histamine in seafood products by high performance liquid chromatography

Comparison of dansylation procedure and benzoylation procedure in histamine analysis in fish products.. Comparison of dansylation procedure and benzoylation procedure in histamine analys

MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY

BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS

THE ADVANCED PROGRAM IN BIOTECHNOLOGY

STUDYING HISTAMINE IN SEAFOOD PRODUCTS

BY HIGH PERFORMANCE LIQUID

Can Tho, 2013

APPROVAL

SUPERVISORS STUDENT

Msc DO TAN KHANG LE ANH VU

Can Tho, May 6, 2013

PRESIDENT OF EXAMINATION COMMITTEE

2.2.2 Comparison of dansylation procedure and

benzoylation procedure in histamine analysis

in fish products

6

2.2.3 Changes in histamine and microbial analyses in

tuna muscle by time and temperature

6 2.2.4 Study the histamine content in some samples of

seafood products

7

3.2 Comparison of dansylation procedure and benzoylation

procedure in histamine analysis in fish products

11 3.3 Changes in histamine and microbial analyses in tuna

muscle by time and temperature

15 3.4 Study the histamine content in some samples of

ABSTRACT

The study was undertaken to asses the effect of different solvents in histamine analysis in seafoods The results showed that histamine in seafood samples was efficiently extracted with perchloric acid 0.6M and trichloroacetic acid 6% Moreover, the commonly applied HPLC method to determine biogenic amines in seafoods after dansylation was compared with an alternative benzoylation procedure Results showed a significant difference

in analysis efficiency among these two procedures for histamine quantitation in tuna samples The effects of storage time and temperature at 0, 4 o C on the development of histamine and microbial in tuna were also studied Tuna stored at 4 o C results in higher histamine level than tuna stored at 0 o C Sample stored at

0 o C contained potentially toxic histamine concentrations (49.1 mg/kg after 11 days of storage), whereas when stored at 4 o C, the sample contained highly toxic histamine concentrations (161.29 mg/kg after 7 days of storage) An increase overtime of microbial population was observed in all samples tested Bacteria counts increased from 4.54 log10 cfu/g to 5.88 log10 cfu/g in the samples stored at 0°C, while for the samples stored at 4°C, an increase was shown dramatically in counts from 4.54 log10 cfu/g to 8.88 log10 cfu/g The histamine levels in some common seafoods were also reported

Keywords: HPLC, histamine, seafoods, extracted solvents,

temperature storage

1 INTRODUCTION

Currently histamine poisoning is one of the most common threats to the health of consumers Histamine poisoning occurs after consuming seafoods containing high level of either histamine or other biogenic amines This foodborne intoxication was originally called “scombroid poisoning” because it was primarily associated with the consumption of fishes belonging to Scombridae and Scomberesocidae families such as tuna, mackerel, bonito, bluefish, and the like These species contain high levels of free histidine in their muscle that is decarboxylased

to histamine When the conditions for the development of bacteria are favorable (for example when the fish are kept in improper temperatures), decarboxylase enzyme produced by the bacteria will metabolize histidine to histamine Other biogenic amines produced during the development of bacteria may increase the toxicity of histamine The toxicity of histamine depends on the ability of individuals to metabolize normal dietary intakes of histamine In some places of the world, histamine poisoning accounted for the largest proportion of cases of poisoning associated with fish and fish products (FAO/WHO, 2012) In Vietnam, histamine poisoning often occurs and the fish usually associated with these cases are tuna and mackerel (Ministry of Fisheries, 2003)

Normally, after catching, fish has very low levels of histamine, but this amount increases with the decomposition of fish Thus, histamine has also been used as an indicator to assess the quality of the fish US Food and Drug Administration (USFDA, 2012) has set this histamine level at 50 mg/kg, above

which it is considered a potential health hazard Considering the importance of histamine in fish and fish products for legal, toxicological, and quality purposes, it is essential to have accurate analytical methods Histamine in different foods, including fish and fish products, have traditionally been determined by means of standard chromatographic techniques such as thin layer chromatography, gas chromatography, capillary electrophoresis,

chromatography (HPLC)

Histamine has a very important role in the food industry In recent years, many studies appear to study on histamine due to large potential applications of this amine in the assessment of quality seafood and seafood products In addition, high levels of histamine in seafood is also a health hazard for consumers.For these reason, the thesis "Studying histamine in seafood products

by high performance liquid chromatography" has been carried out

Thesis objectives:

- Selecting the appropriate solvent for extraction process and analysis procedure to determine histamine in seafoods by

high performance liquid chromatography

- Studying the development of histamine in seafood over

time and storage temperature

- Quantification of histamine content in some common seafoods

2 MATERIALS AND METHODS

2.1 Materials

- Seafood products were purchased in supermarkets in Can Tho City

- Reagents: Methanol, ethanol, perchloric acid (PCA),

2.2 Methods

2.2.1 Optimization of histamine extracting process

- Preparation of samples: Fresh tuna (Thunnus albacares) was

was large amount of histamine in tuna samples

- Extraction of Histamine:

• Extraction using 0.6M perchloric acid (PCA): Extraction

was carried out following the method of Cseinati and Forgacs (1999) A 10 g sample was homogenized with 20 ml 0.6M PCA for 10 min The homogenate was centrifuged at 7000 rpm for 10 min at 4°C, after which the supernatant was made up to 50 ml with 0.6M PCA, filtered and stored at 0°C until use

• Extraction using trichloroacetic acid 6%: Samples

extraction were done according to the method of Yung-Hsiang et

al (2001) A 5 g sample was homogenized with 20 ml trichloroacetic acid (TCA) 6% for 10 min The homogenates were

then placed in volumetric flasks, and TCA was added to a final volume of 20 ml and stored at 0°C until use

• Extraction using methanol (99%): A modified method of

Lin et al (1976) was used for the process of methanol extraction

A 10g sample was homogenized with 50 ml methanol then transferred to a volumetric flask, after which it was immersed in a water bath at 60ºC for 15 min The sample was cooled and then

decanted, filtered and stored at 0°C until use

- Determination of Histamine:

 Derivation of extracted solutions using benzoyl chloride: The benzoyl derivatives of all samples were prepared

according to a method of Hwang et al (1997) One milliliter of 2M sodium hydroxide and 10 µl of benzoyl chloride were added sequentially to 2 ml of samples extracted solution.The resulting

min Benzoylation was stopped by cooling the test tubes in an ice bath for 30 min, and the mixed solution was extracted with 3ml of diethylether After centrifugation at 3000 rpm, the supernatant was filtered through a 0.45 µm filter

 Separation of Histamine with HPLC: Histamine in test

samples were determined with a Hitachi liquid chromatograph (Hitachi, Tokyo, Japan), consisting of a Model L-6200 pump, a Rheodyne Model 7125 syringe loading sample injector, a Model L-4000 UV-Vis detector (set at 254 nm), and a Model D-2500 Chromato-integrator A Lichrospher 100RP-18 reversed-phase column (125x4.6 mm, E Merck, Damstadt, Germany) was used for separation The gradient elution program began with 50:50 (v/v) methanol:water at a flow rate of 0.8 ml/min for the first 0.5

min, followed by a linear increase to 85:15 methanol:water (0.8ml/min) during the next 6.5 min The methanol:water mixture was held constant at 85:15 (0.8ml/min) for 5 min and then

decreased to 50:50 (0.8ml/min) during the next 2 min

 Derivation of extracted solutions using dansyl chloride:

Derivation was carried out following the method of Earola et al (1993) A 1ml sample extract was made alkaline by adding 200 µl

of 2N NaOH A 300 µl saturated sodium bicarbonate and 2 ml Dns-Cl was added to the alkaline extract The reaction mixture was transferred to a 40°C incubator for 45 min Residual Dns-Cl was removed by adding 100 µl ammonia After centrifugation for

30 min at 2500 rpm, the supernatant was filtered through a 0.45

µm filter

 Separation of Histamine with HPLC: Histamine in test

samples were determined with a Shimadzu Prominence HPLC apparatus (Shimadzu, Kyoto, Japan) equipped with a SPD-M20A diode array detector (set at 254 nm) and two binary gradient pumps (Shimadzu LC-10AT), auto sampler (SIL 20AC), column oven (CTO-20AC), and a communication bus module (CBM-20A) with valve unit FCV-11AL was used The column was a reverse-phase, Spherisorb 5 Si C18 pH-St, 250x4.6 mm (Phenomenex, Macclesfield, Cheshire, UK) The mobile phase was 95:5 (v/v) acetonitrile : water, delivered at a flow rate of 1.0 ml/min

- Indicator for Assessment: Histamine concentration (mg/kg)

2.2.2 Comparison of dansylation procedure and benzoylation procedure in histamine analysis in fish products

- Preparation of samples: Fresh tuna (Thunnus albacares) was

4 and 7 days during storage procedure) to make sure there was a wide range of amount of histamine in tuna samples

- Extraction of Histamine: Histamine in fish samples was extracted by using optimum solvent derived from previous experiment (2.2.1)

- Determination of Histamine: Histamine was determined by using benzoylation and dansylation procedure – as described in 2.2.1

- Indicator for Assessment: Histamine concentration (mg/kg)

2.2.3 Changes in histamine and microbial analyses in tuna muscle by time and temperature

- Preparation of samples: Fresh tuna (Thunnus albacares) was

times (Table 2) and stored at -20°C to minimise bacterial activity that could influence the results prior to analysis For all samples, the histamine and microbiological analysis were assayed collectively to optimise efficiency and to limit possible day-to-day variability of analyses

Table 2: Sample taken times of tuna stored at 4 o C and 0 o C

st

day

2ndday

3rdday

4thday

7thday

nd

day

4thday

7thday

9thday

day

- Determination of Histamine: Histamine was determined by using optimum histamine analysis procedure, which derived in experiment 2.2.2

- Indicator for Assessment: Histamine concentration (mg/kg)

- Microbial Analysis: The total viable count (TVC) test was done according to the method of Guinn et al., (1999)

2.2.4 Study the histamine content in some samples of seafood products

- Preparation of Samples: Seven seafood samples were purchased from local supermarkets in Can Tho City, descriptions

of each sample are given in Table 3

Table 3: Description of seafood samples

- Determination of Histamine: Histamine was determined by using optimum histamine analysis procedure, which derived in experiment 2.2.2

- Indicator for Assessment: Histamine concentration (mg/kg)

2.2.5 Statistical analysis methods

The experiment was conducted in triplicates The results were reported as mean values ± standard deviation The ANOVA test was used for data analysis The differences between the mean values were considered significant when p<0.05 All data were analysed using Statgraphic Centurion XV statistical package for windows (Statgraphics Centurion XV, Manugistics, Inc., Rockville, USA, 2009)

3 RESULTS AND DISCUSSIONS

3.1 Optimization of histamine extracting process

Complete and selective extraction of target analytes from complex food samples is of great importance in food analysis Solid samples are most frequently extracted with acidic solvents which also act as deproteinisation agent during the liquid solid extraction Because of the high content of fat in fish samples, extraction solvents that can remove fat or protein are frequently selected for achieving high extraction efficiency In this study, perchloric acid (0.6M), trichloroacetic acid (6%, m/v), methanol (99%) was tested as extraction solvents for the samples Results revealed that histamine concentrations of different levels were obtained after the extraction of samples with different solvents (Figure 4)

Figure 4: Mean of histamine concentrations in tuna samples extracted by using Met - methanol; PCA - perchloric acid (0.6M) and TCA - trichloroacetic acid (6%)

As shown, the highest histamine concentration was from 0.6M perchloric acid for samples analysed by dansylation procedure The samples extracted by trichloroacetic acid 6% and analysed by benzoylation procedure resulted in higher yield of histamine concentrations as compared to the samples extracted in all the extract medium These results coincide with those of Ben-Griggrey et al (2001), who suggested perchloric acid and trichloroacetic acid are more effective than methanol, acetonitrile and acetone in extracting histamine in fish products Ruiz-Capillas and Moral (2001) also reported that PCA and TCA are

highly efective biogenic amine extractors for fish and fish products because of their efect on protein precipitation Since most of the biogenic amines present in fish are in bound form, the histamine concentration was low when extracted by methanol (Shalaby et al., 1996) In this study, histamine concentrations obtained in the tuna fish was quite high in the acid fraction, suggesting that histamine in fish are acid solube Because perchloric acid and trichloroacetic acid provided good peak shape and effective separation, it was selected as the optimized extraction solutions in the following experiments The suitable acid for extraction of histamine from fish samples and analysed

by dansylation procedure was 0.6M perchloric acid, while trichloroacetic acid 6% was suitable acid for extraction histamine from fish samples analysed by benzoylation

3.2 Comparison of dansylation procedure and benzoylation procedure in histamine analysis in fish products

Amongst the available analytical techniques, HPLC is by far the most frequently used to separate and quantify histamine Since histamine present in the food samples neither show an adequate absorption, nor exhibit significant fluorescence, derivatisation has

to be performed in order to increase the sensitivity needed for a subsequent UV, VIS or fluorescence detection (Onal, 2007) In this work the amines were determined by HPLC using two procedures – dansylation procedure and benzoylation procedure – based on pre-column derivatisation In both methods, the reversed-phase column was used Typical chromatograms of dansyl chloride and benzoyl chloride derivatives of the standard and of the extract of sample are shown in Figure 5 As shown,

histamine are well separated in the nearly 3 min runtime for benzoyl procedure, where as this seperation time was 5 min for dansyl procedure The peaks of histamine were satisfactorily resolved with good peak resolution, sharpness, and symmetry, and are consistent with previous report (Hwang et al., 1997) However, the histamine levels determined on six test samples revealed the significant difference in analysis efficency between two procedures (Figure 6) In general the histamine values analysed by benzoylation procedure were higher than the histamine levels determined by dansylation procedure (5 of 6 test samples) The histamine values determined by benzoylation procedure that had lower histamine values from dansylation procedure was 52.51 (mg/kg) on the third test The dansylation procedure result was 52.96 (mg/kg) for this test However, this difference was not statistically significant

Figure 5: Typical chromatograms of the histamine standard and samples (A), (C) Histamine standard (1 µg/ml); (B), (D) tuna sample at day 7 of storage time (A) and (B): chromatography derived by benzoylation procedure; (C) and (D): chromatography derived by dansylation procedure

Figure 6: Mean of histamine concentrations in tuna samples determined by dansylation procedure and benzoylation

procedure