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A simple and rapid method for colorimetric determination of histamine in sh esh

S.B. Patange *, M.K. Mukundan, K. Ashok Kumar
Quality Assurance and Management Division, Central Institute of Fisheries Technology, Willingdon Island, Cochin 682 029, India Received 20 October 2003; received in revised form 4 May 2004; accepted 6 May 2004

Abstract Histamine is a signicant chemical hazard in sh. It is derived from the bacterial decarboxylation of amino acid histidine, that is present in large amounts in sh of Scombridae family and its presence is considered as a good indicator of temperature abuse and the state of good manufacturing practices adopted in the handling of such sh. A simple and rapid chemical method for determination of histamine in sh esh is reported for use in seafood quality inspection laboratories. Good recoveries (>91%) were obtained for histamine at spiking levels ranging 160 mg/100 g. The overall precision (relative standard deviation, %) in the new assay ranged from 2.61 to 9.63. The interaction between the imidazole ring and p-phenyldiazonium sulfonate was made the basis of a quantitative colorimetric method for estimation of histamine. The results of the new assay showed a high correlation (R2 0:999) with the assay of Hardy and Smith [J. Sci. Food Agric. 27 (1976) 595] in the recovery of histamine. The limit of detection was 1 mg/100 g for the new assay and was comparable with the existing methods. A concentration-based reference color scale is provided for the determination of defect and hazard action levels set by the regulatory agencies. Visual comparison of color intensity of test samples with standard concentrations in reference color scale for determining these levels without the aid of a spectrophotometer was an important practical application for rapidly estimating histamine in fresh sh fullling one of the HACCP requirements. The assay was simple requiring no laborious treatments, and may be suitable for routine analysis in monitoring of histamine in sh. 2004 Elsevier Ltd. All rights reserved.
Keywords: Histamine; Fish; Rapid method

1. Introduction Histamine is a biogenic amine produced during microbial decomposition of scombroid sh such as tuna and mackerel (Halasz, Barath, Sarkadi, & Holzapfel, 1994; Pan & James, 1985). Histamine has been associated with scombroid poisoning, which resembles an allergic reaction (Taylor, 1986). Several regulatory agencies have, therefore, imposed a limit on histamine content in sh used for human consumption (EU Directive No. 91/493; FDA, 1998). Since histamine is neither volatile nor destroyed by cooking, a simple and convenient method of detecting it in seafood samples is needed, particularly where decomposition is suspected due to temperature abuse of sh. A variety of methods exist for analysis of histamine in sh. Most involve chromatography of histamine deriv*

Corresponding author. E-mail address: sbpatange@redimail.com (S.B. Patange).

atives using expensive instrumentation such as HPLC or GC (Hayman, Gray, & Evans, 1985; Henion, Nosanchuk, & Bilder, 1981; Jeyashakila, Vasundhara, & Kumudavally, 2001; Ozogul, Taylor, Quantick, & Ozogul, 2002b; Redmond & Tseng, 1979; Suzuki, Kobayashi, Noda, Suzuki, & Takama, 1990; Yen & Hsieh, 1991). The method of AOAC (Method 977.13, 2002) involves extraction of histamine with hot methanol, ion exchange chromatography, and derivatisation by o-phthalaldehyde and uorometric quantitation. This method, while sensitive and reproducible (Stratton, Hutkins, & Taylor, 1991), is complex and time consuming. The chemical method of AOAC (Method 957.07, 2002) also involved chromatographic purication of histamine and further coupling it with a diazonium reagent, p-niroaniline. The colorimetric assays reported involve extraction processes and the use of chromatographic purication of histamine by carboxylic cation exchangers and further coupling with a diazonium salt/2,4-dinitrourobenzene (Code & McIntire, 1956; Hardy & Smith, 1976; Kawabata,

0956-7135/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2004.05.008

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Uchida, & Akano, 1960). Bateman et al. (1994) reported the interaction between the puried histamine and copper and a dye to form an easily visualized red complex. An enzymic test using diamine oxidase (DAO), horse-radish peroxidase and leuco-crystal violet to visualize a purple compound upon oxidation of histamine has also been reported (Lerke, Porcna, & Chin, 1983). This enzymic test was further modied by Rodriguez-Jerez, Grassi, and Civera (1994) and recommended a wavelength of 580 nm and incubation time of 15 min for the reaction. Several newer methods have been developed in the past decade for the analysis of histamine. Mopper and Sciacchitano (1994) reported on the use of capillary zone electrophoresis for determination of histamine in sh with UV detection at 210 nm. Other techniques used for determination of histamine in sh include the use of oxygen sensor-based assay using puried amine oxidase (Ohashi et al., 1994), a solid phase assay based on the coupling of DAO to a peroxidase/dye system (Hall, Eldridge, Saunders, Fairclough, & Bateman, 1995), monoclonal antibody-based ELISA (Serrar, Brebant, Bruneau, & Denoyel, 1995), DAO-based amperometric biosensor for total histamine, putrescine and cadaverine (Male, Bouverette, Loung, & Gibbs, 1996), and the use of an electrochemical biosensor for biogenic amine contents of foods (Draisci et al., 1998). Frebort, Skoupa, and Pec (2000) developed an amine oxidase-based ow biosensor for the assessment of sh freshness involving spectrophotometric detection of enzymatically produced hydrogen peroxide by a peroxidase/guaiacol system. More recently, the use of ow injection determination of histamine with a histamine dehydrogenase-based electrode has been reported by Takagi and Shikata (in press). Among these methods enzymatic assays are reported to provide simplicity and rapidity, however, these methods tend to overestimate histamine levels (Ben-Gigirey, Craven, & An, 1998). Histamine has been identied as a signicant chemical hazard in the execution of HACCP in sh processing (FDA, 1998). A sensitive and rapid method for monitoring its levels in scombroid sh is therefore needed to avoid delay in the analysis in order to practice HACCP ensuring safety of sh products. Our objective was to adopt a simple extraction procedure coupling with an interaction with the imidazole reacting and quantitatively color-producing reagent, and to formulate a reference color scale for rapid estimation of histamine, particularly in determination of the defect and hazard action levels in sh.

USA). Other chemicals and solvents used were of analytical grade. 2.2. Fish samples Fresh Little Tuna (Euthynnus anis) (average weight 1.8 kg) and Indian mackerel (Rastrelliger kanagurta) (average weight 0.18 kg) landed in iced condition were purchased from Cochin Fishing Harbor immediately after landing by the shing vessels. 2.3. HPLC method High-performance liquid chromatography (HPLC) analyses used Merck-Hitachi Model D-7000 apparatus equipped with UV detector Merck-Hitachi L-7400 and an intelligent pump L-7100. LiChrospher 100, RP-18 column, 250 mm 4.0 mm i.d., particle diameter 5 lm coupled with guard cartridge 4 mm 4 mm i.d., was purchased from Merck, Germany. Chromatographic conditions, sample preparation and derivatisation procedure were similar to as described by Ozogul et al. (2002b). Histamine quantitation was carried out by comparison of the analyte peak areas versus an externally generated calibration curve. The concentration of amine standards for calibration ranged from 0.5 to 1.0 mg/ml. 2.4. New assay The reagent, p-phenyldiazonium sulfonate was prepared according to Koessler and Hanke (1919) with minor modications. Chilled 1.5 ml 0.9% (w/v) sulfanilic acid in 4% hydrochloric acid and 1.5 ml 5% (w/v) sodium nitrite were mixed in 50 ml standard ask and kept in ice bath for 5 min. 6 ml more of 5% sodium nitrite solution was added and after 5 min volume was made up with chilled distilled water. The reagent stored in ice bath was used 15 min after dilution with water and was stable for 12 h. Histamine was extracted from sh muscle, with little modications to the procedure given by McIntire, Roth, and Shaw (1947) for the extraction and purication of histamine from blood plasma. Fish muscle (5 g) was taken from the dorsal part of llet without skin and transferred to 75 ml centrifuge tube. The sample was homogenized with 20 ml of 0.85% NaCl solution (saline) for 2 min using a high-speed blender and centrifuged at 12 000 g for 10 min at 4 C. The supernatant was made up to 25 ml with saline. The muscle extract was used immediately for further analysis. In a glass-stoppered test tube, 1 ml of the extract was diluted to 2 ml with saline and 0.5 g of salt mixture containing 6.25 g of anhydrous sodium sulfate to 1 g

2. Material and methods 2.1. Reagents Amine standards, p-bromoaniline, Amberlite Resin (CG-50) and sulfanilic acid were from Sigma (St. Louis,

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trisodium phosphate monohydrate was added. The tubes were stoppered and shaken thoroughly. 2 ml of n-butanol was then added and the tubes shaken vigorously for 1 min and allowed to stand for 2 min and then shaken briey to break the protein gel. The tubes were further shaken vigorously for few seconds and then centrifuged at 3100 g for 10 min. The upper butanol layer (only 1 ml) was transferred into a clean and dry test tube and evaporated to dryness in a stream of nitrogen. The residue was dissolved in 1 ml of distilled water and then reacted with the reagent as detailed below. In a clean tube 5 ml of 1.1% sodium carbonate solution was taken and 2 ml of the chilled reagent was added slowly and mixed. It was then added to the tube containing 1 ml solution of the residue collected in the extraction process. The absorbance of the color produced was measured immediately after 5 min at 496 nm using distilled water as a reference. 1 ml aliquots of standard histamine solutions containing 0100 lg/ml in distilled water were reacted in a similar manner to obtain the reference color scale and standard curve of absorbance against histamine concentration. Shimadzu (UV-1610) UVvisible spectrophotometer with glass cuvettes was used for the purpose. The concentration of histamine in sample was obtained from the standard curve for the corresponding absorbance measured at 496 nm by regression analysis. The histamine concentration in sample was estimated using the following formula. Histamine mg=100 g A 2 25 100 5 1000 A mg=100 g

2.6. Recovery of added histamine Recovery of histamine was performed by spiking the known concentrations of histamine into the muscle extracts of tuna and analyzing the samples for histamine content by the new assay and by the assay of Hardy and Smith (1976). The required quantity of sh muscle was homogenized in a food processor; appropriate aliquots of this sample were used for preparation of extracts. To each muscle extract appropriate quantity of 1 mg/ml free base solution of histamine dihydrochloride was added to get the spiked levels in the range 060 mg/100 g. The extracts were vortex stirred for 1 min and the volume was made up to 25 ml with saline for the new assay and 100 ml with 2.5% trichloroacetic acid for the method of Hardy and Smith (1976). Assay for both the methods were carried out in triplicate. 2.7. Analysis of histamine from spoiling sh samples To analyze histamine from sh samples, freshly landed whole tuna and mackerel were abusively stored at 30 C for 24 h. Histamine content in the test samples were analyzed using the new assay, HS method and the HPLC method (Ozogul et al., 2002b). The rst analysis was carried out immediately after the sh were brought to the laboratory in fresh and iced condition and was designated 0 h observation, and then the subsequent analyses were carried out after 6, 12 and 24 h of storage of sh. Each time an individual sh was drawn, required quantity of muscle was homogenized and aliquots from the homogenate were used for further analysis by the dierent methods. Assay for the three methods were carried out in triplicate. 2.8. Preparation of standard amine solutions Histamine dihydrochloride (16.55 mg) was dissolved in 10 ml of distilled water to obtain a concentration of 1000 lg/ml of histamine free base. Appropriate dilutions were then prepared to obtain aliquots of histamine solutions containing 0100 lg/ml in distilled water. Similarly, tryptamine hydrochloride (12.28 mg), putrescine dihydrochloride (18.29 mg), 2-phenylethylamine hydrochloride (13.01 mg), cadaverine dihydrochloride (17.14 mg), spermidine trihydrochloride (17.53 mg), spermine tetrahydrochloride (17.20 mg), tyramine hydrochloride (12.67 mg) and agmatine sulphate (17.54 mg) were dissolved separately in 10 ml distilled water to obtain a concentration of free base for each amine at 1000 lg/ml. Appropriate dilutions were then prepared to obtain aliquots of each amine solution containing 100 lg/ml in distilled water and 1 ml each was used for examining the cross reactions with the reagent prepared in the new assay.

where A is the value of histamine obtained in lg/ml from the standard curve.

2.5. Assay of Hardy and Smith (1976) The method for histamine analysis in sh given by Hardy and Smith (1976) (HS method) comprises three steps: (1) sample preparation using 10 g sh muscle with 2.5% trichloroacetic acid, (2) removal of interfering compounds using an ion exchange column (weakly acidic cation exchange resin, AmberliteCG 50), and (3) derivatisation of puried sample with diazo reagent followed by measurement of absorbance at 495 nm. The absorbance of sample and standards was measured using Shimadzu (UV-1610) UVvisible spectrophotometer with glass cuvettes and histamine was estimated from the standard curve of absorbance versus known concentrations of histamine in the range 080 lg/ml by regression analysis.

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3. Results and discussion Using the procedure outlined in the new assay, a linear relationship (correlation coecient 0.988) was found between the color intensity at 496 nm and histamine concentration in the range 0100 lg/ml. A pink color of increasing intensity with histamine concentration was observed. The color of the reaction between histamine and the reagent initially becoming yellow, lasting for about 30 s was followed by pink color development. Although most of the color developed within 1 min, the maximum intensity reached only after 5 min. With dilute solutions, 20 lg/ml or less, the color of maximum intensity persisted for 2 min. With more concentrated solutions, the stability of color intensity was 3040 s. In the 6th minute there was a fall (<1%) in absorbance of the solution with the higher histamine concentrations. The reagent, p-phenyldiazonium sulfonate, was found to have the sensitivity to form visible color with 1 lg/ml of histamine. In order to examine the sensitivity and accuracy of the new assay, muscle extracts of fresh tuna spiked with concentrations of histamine ranging 060 mg/100 g were analyzed for histamine recovery by the new assay and for comparison with the chemical assay of Hardy and Smith (1976). A high correlation (R2 0:999) was observed for the recovery of added histamine between the two methods (Fig. 1). Recoveries are shown in Table 1. Among the nine samples tested for recovery, two samples overestimated the recovery that ranged from 2% to 4%. Although the values are non-signicant, this may be attributed to non-uniform distribution of histamine originally present in the sh muscle. The recovery of histamine at all the levels tested was more than 91% and there was no signicant dierence between the recoveries obtained by the two assays for the respective levels of spiked histamine. Precision of the assay was determined

Fig. 1. Scatter plot of histamine recovery estimated by the New assay and the assay of Hardy and Smith (1976). The line shows the linear t between the two assays.

by calculating the relative standard deviation (R.S.D., %) for the repeated measurements, and the accuracy of the method (trueness, %) was determined by assessing the agreement between the measured and nominal concentrations of analyzed samples (Cinquina et al., in press). The overall precision (R.S.D., %) in the new assay ranged from 2.61 to 9.63, and in the HS method it was 3.8610.7. The values for the new assay can be considered very satisfactory. The trueness denoting the percent loss of histamine during the assay ranged from 2 to 9 in the new assay and 28 in the HS method. Cinquina et al. (in press) observed an average recovery of more than 92% with R.S.D. less than 4% for HPLC assay determined for the recovery of added histamine at 5, 10 and 20 mg/100 g levels. The values of recovery obtained in the new assay closely related to the HPLC assay reported.

Table 1 Recovery of added histamine in tuna sh samples in New assay and the assay of Hardy and Smith (1976) Parameter New assay Histamine founda Histamine recovereda S.D.a Precision (R.S.D.%) % Recovery Trueness (%) Spiked histamine levela 0 1.85 0.05 1 2.76 0.91 0.07 7.69 91.00 )9.0 2.60 0.92 0.08 8.70 92.00 )8.0 2 3.73 1.88 0.18 9.63 93.50 )6.5 3.65 1.97 0.21 10.70 98.50 )1.5 3 4.90 3.05 0.19 6.23 102.00 +2.0 4.62 2.94 0.15 5.10 98.0 )2.0 4 6.00 4.15 0.36 8.67 104.00 +4.0 5.55 3.87 0.27 6.98 96.80 )3.2 5 6.75 4.90 0.25 5.1 98.00 )2.0 6.65 4.97 0.28 5.63 99.40 )0.6 10 11.50 9.65 0.46 4.79 96.00 )4.0 11.50 9.82 0.51 5.19 98.20 )1.8 20 20.80 18.95 0.92 4.85 94.80 )5.2 21.00 19.30 1.17 6.06 96.60 )3.4 40 39.30 37.45 1.56 4.17 93.50 )6.5 39.50 37.80 1.65 4.36 94.60 )5.4 60 57.55 55.70 1.45 2.61 92.80 )7.2 59.40 57.72 2.23 3.86 96.20 )3.8

Assay of Hardy and Smith (1976) Histamine founda 1.68 Histamine recovereda S.D.a 0.11 Precision (R.S.D.%) % Recovery Trueness (%)
a

Values in mg/100 g sh.

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The smallest concentration of histamine spiked in sample with 91% of average recovery was 1 mg/100 g. With higher concentrations the recovery ranged between 94% and 98%. The assay had the detection limit of 1 mg histamine/100 g sh. The limit of quantitation, as calculated from the procedure described in the new assay, was also 1 mg/100 g. The color intensity recorded for histamine at six dierent concentrations (0, 5, 10, 20, 30 and 50 lg/ml) is given in Fig. 2. The reference color scale can also be developed in the laboratory with concentrations ranging 050 lg/ml with a dierence of 5 lg/ml in the successive concentrations and can be used in the visual examination of test samples. This will make the assay more amenable and rapid, and will not be requiring the use of a spectrophotometer. Color intensity of concentrations exceeding 50 lg/ml did not facilitate visual comparison of histamine concentrations. The color intensity produced in each concentration of histamine in spiked samples was comparable with the reference color scale and spectrophotometric absorbance of histamine standard of similar concentration. Extraction steps involving ltration of muscle extracts and chromatographic purication where negligible amounts of histamine might be lost (Hardy & Smith, 1976) have been avoided for obtaining better recovery of added histamine to satisfactory levels. Extraction of histamine from muscles with 0.85% NaCl solution was also observed to give the desired recovery. Higher concentrations of saline resulted in gelling of muscle proteins during homogenization that hindered obtaining a clear aqueous extract. The new assay is rapid because the test-to-result duration is approximately 45 min for a single assay that includes sample preparation. On the contrary, the HS method involving chromatographic purication required more than 2 h for a single assay. Kose and Hall (2000) reported that the assay of Hardy and Smith (1976) works well with sh samples and reported a modication to the assay for determination of histamine in sh meal. The AOAC chemical method (method 25, 1975 or method 957.07, 2002) is also reported to be tedious and time consuming and could not be considered practical for routine analysis of large number of samples as the time required for a single determination, not

Fig. 2. Reference color scale for histamine (concentrations in lg/ml).

including sample extraction, may range up to 2 h (Arnold & Brown, 1978). Among the colorimetric methods reported, DAO-based assays are comparatively rapid. However, enzymatic assays in general have a tendency to overestimate histamine levels compared with the AOAC method (Ben-Gigirey et al., 1998). In comparison with the AOAC chemical method, the new procedure is comparatively simple and rapid, and provides equal sensitivity. The new assay may prove to be comparatively economical as the operational cost involved would be less than US$ 1.0 for analysis of one sample. Apart from the chemicals and reagents required as mentioned earlier, other material required involve a refrigerated centrifuge, ice production facility and a source of pure nitrogen. The new assay, therefore, appears to be simple and aordable to low budget seafood quality inspection laboratories where histamine levels in fresh seafood are required to be analyzed rapidly for examining sh samples for the lower or higher levels than the defect/ hazard action levels of histamine. Comparatively unskilled technicians can perform the assay provided they are trained under scientic supervision. FDA (1998) guidelines for tuna, mahimahi and related sh specied 50 mg/100 g as the toxicity level and 5 mg/100 g as the defect action level because histamine is not uniformly distributed in a decomposed sh. Similarly, European Union Directive No. 91/493 stipulated that nine independent samples from each batch should correspond to: (1) an average histamine concentration lower than 10 mg/100 g, (2) no more than two samples out of nine with a concentration of between 10 and 20 mg/100 g and (3) no sample with a histamine content higher than 20 mg/100 g. The limits imposed by these regulatory agencies have been taken into consideration for developing the color scale. The color intensity range of 050 lg/ml of histamine works well with the color scale and the spectrophotometric absorbance too. The new assay was successfully applied to the determination of histamine in spoiling sh samples. The histamine levels in the decomposing tuna and mackerel during storage at 30 C analyzed by the new procedure, HS method and HPLC method are shown in Table 2. The correlation coecient for values obtained by the three procedures ranged between 0.995 and 0.999. These values again were considered very satisfactory. Values obtained by the new procedure and HS method were closely related but HPLC analyses showed slightly higher values. The histamine levels accumulated in both the sh from 0 h of storage increased steadily with time and at the end of 24 h storage, mackerel had higher histamine level than tuna. The HPLC analysis showed insignicant amounts of other biogenic amines in both the sh in all samples analyzed. Histamine levels in marine sh, especially the scombroid sh, are known to rise continuously and can even reach toxic levels when

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Table 2 Histamine levels in tuna and mackerel abusively stored at 30 C as analyzed by the New assay, the assay of Hardy and Smith (1976) and HPLC method (Ozogul et al., 2002b) Name of assay Histamine in tuna (mg/100 g) (Avg S.D.) Storage period (h) 0 New assay Assay of Hardy and Smith (1976) HPLC assay 1.80 0.12 1.60 0.15 2.17 0.15 6 23.50 0.25 21.50 0.38 26.75 0.66 12 32.50 0.64 29.70 0.51 35.45 0.33 24 51.25 0.90 49.50 1.11 56.62 1.22 Histamine in mackerel (mg/100 g) (Avg S.D.) Storage period (h) 0 2.50 0.08 1.75 0.05 2.17 0.15 6 14.50 0.35 13.75 0.41 16.50 0.62 12 19.50 0.25 23.00 0.31 24.95 0.60 24 75.00 1.22 77.50 1.18 78.76 0.84

sh are stored abusively. Histamine accumulation is directly related to the kind and number of histidine decarboxylating bacteria the sh might be contaminated with (Eitenmiller, Orr, & Wallis, 1982). However, various reports state that there are dierences in the formation of amines in sh that are mainly due to the type and level of microora present in sh (Lopez-Sabater, Rodriguez-Jerez, Roig-Sagues, & Mora-Ventura, 1996; Middlebrooks, Toom, Douglas, Harrison, & McDowell, 1988). Higher than toxic levels of histamine in skipjack tuna (Katsuwonus pelamis) and mackerel (Scomber scombrus), when subjected to temperature abuse, have been reported by Frank, Yoshinaga, and Nip (1981) and Ritchie and Mackie (1980). The tuna sh analyzed in the present study was caught from oshore waters and mackerel from coastal waters with purse seines. Although the kind and number of histamine-forming bacteria in sh were not analyzed in the study, the higher rise of histamine in mackerel than in tuna could be presumably due to contamination with potent histamine-forming bacterial ora that might be present in coastal waters. The new assay was also used in principle for the study of properties of histidine decarboxylase puried from Raoultella planticola 19-3 (Kanki, Yoda, Tsukamoto, & Shibata, 2002). Our experience showed that the new assay gave very satisfactory results in the enzyme studies. The new assay utilized the reaction between the imidazole ring and p-phenyldiazonium sulfonate as the basis of a quantitative colorimetric method for estimation of histamine. To examine possible interferences caused by the reaction between the reagent and other amines that can be extracted in the assay along with histamine, amines other than histamine were reacted with the reagent in the similar way. Amines such as tryptamine, putrescine, 2-phenylethylamine, cadaverine, spermidine, spermine, tyramine and agmatine at concentrations of 100 lg/ml each were reacted with the reagent. Except tyramine, the rest showed formation of a light lemony-yellow color. Tyramine formed a yellow to light-pink color in the concentration range of 50100 lg/ ml whose intensity was far less than the color intensity of histamine of similar concentration.

The interference of tyramine with histamine estimation was studied by mixing each 0, 10 and 50 lg/ml of tyramine separately with 50 lg/ml solutions of histamine and conducting the test as described above. 10 lg/ml of additional tyramine showed no color dierence when examined visually with 50 lg/ml of histamine, while it added to about 0.4 lg of additional histamine in a total concentration of 50.4 lg/ml when absorbance was measured and histamine concentration determined from the standard curve. Thus, the interference by tyramine at 10 lg/ml concentration was insignicant as compared with color intensity given by 50 lg/ml of histamine. 50 lg/ml of added tyramine did show the dierence in the color intensity and it added about 4.0 lg of additional histamine to the standard 50 lg/ml histamine concentration. The interference of tyramine in quantitative estimation of histamine ranged between 1% and 8%. Tyramine is reported to occur at higher concentrations in cheese and cheese products (Stratton et al., 1991). On the contrary, its occurrence in decomposing fresh sh is comparatively very low (Shalaby, 1996). Ozogul, Taylor, Quantick, and Ozogul (2002a) reported about 400 ppm of histamine and 05 ppm of tyramine in a decomposing herring (Clupea herengus). Similar observations on tyramine levels have been reported in respect of mackerel and Mediterranean hake (BaixasNogueras, Bover-Cid, Vidal-Carau, & Veciana-Nogues, 2001; Wendakoon, Murata, & Sakaguchi, 1990). Jeyashakila and Vasundhara (2001) reported negligible levels of tyramine in market samples of tuna, mackerel and sardine, however considerable higher levels were reported in only salt-dried shes. Tyramine formation in sh is attributed mainly to decarboxylase activity of Streptococcus faecalis and Pediococcus cerevisiae (Eitenmiller, Koehler, & Reagan, 1978). The predominance of these organisms in a fresh sh may not be considered as signicant in relation to sh spoilage organisms. It can be concluded that tyramine concentrations in sh like tuna, mackerel, herring etc. are not likely to signicantly inuence the analysis of histamine by the new assay, although it may contribute to overestimation of histamine to levels less than 1% in a decomposing sh. The new assay described here may not be suitable for application to dried or salt-dried sh

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samples and may need standardization because in such samples tyramine could occur in relatively higher concentrations.

Acknowledgements The authors are thankful to the Director, Central Institute of Fisheries Technology, Cochin for the permission to publish this paper. References
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