Food Control 16 (2005) 465–472

www.elsevier.com/locate/foodcont

A simple and rapid method for colorimetric determinationof histamine in fish flesh

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 significant chemical hazard in fish. It is derived from the bacterial decarboxylation of amino acid histidine, that is

present in large amounts in fish 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 fish. A simple and rapid chemical method for determination

of histamine in fish flesh is reported for use in seafood quality inspection laboratories. Good recoveries (>91%) were obtained for

histamine at spiking levels ranging 1–60 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 ¼ 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 deter-

mination 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 fish fulfilling 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 fish.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Histamine; Fish; Rapid method

1. Introduction

Histamine is a biogenic amine produced during

microbial decomposition of scombroid fish such as tuna

and mackerel (Halasz, Barath, Sarkadi, & Holzapfel,

1994; Pan & James, 1985). Histamine has been associ-

ated with scombroid poisoning, which resembles an

allergic reaction (Taylor, 1986). Several regulatoryagencies have, therefore, imposed a limit on histamine

content in fish 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 fish.

A variety of methods exist for analysis of histamine infish. Most involve chromatography of histamine deriv-

*Corresponding author.

E-mail address: sbpatange@rediffmail.com (S.B. Patange).

0956-7135/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.foodcont.2004.05.008

atives using expensive instrumentation such as HPLC or

GC (Hayman, Gray, & Evans, 1985; Henion, Nosan-

chuk, & Bilder, 1981; Jeyashakila, Vasundhara, &

Kumudavally, 2001; Ozogul, Taylor, Quantick, & Ozo-

gul, 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 exchangechromatography, and derivatisation by o-phthalaldehyde

and fluorometric quantitation. This method, while sen-

sitive and reproducible (Stratton, Hutkins, & Taylor,

1991), is complex and time consuming. The chemical

method of AOAC (Method 957.07, 2002) also involved

chromatographic purification of histamine and further

coupling it with a diazonium reagent, p-niroaniline.

The colorimetric assays reported involve extractionprocesses and the use of chromatographic purification of

histamine by carboxylic cation exchangers and further

coupling with a diazonium salt/2,4-dinitroflurobenzene

(Code&McIntire, 1956;Hardy&Smith, 1976;Kawabata,

466 S.B. Patange et al. / Food Control 16 (2005) 465–472

Uchida, & Akano, 1960). Bateman et al. (1994) reportedthe interaction between the purified 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 re-

ported (Lerke, Porcna, & Chin, 1983). This enzymic test

was further modified by Rodriguez-Jerez, Grassi, and Ci-

vera (1994) and recommended awavelength of 580 nmandincubation 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 fish

with UV detection at 210 nm. Other techniques used for

determination of histamine in fish include the use of

oxygen sensor-based assay using purified 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 aminecontents of foods (Draisci et al., 1998). Frebort, Skoupa,

and Pec (2000) developed an amine oxidase-based flow

biosensor for the assessment of fish freshness involving

spectrophotometric detection of enzymatically produced

hydrogen peroxide by a peroxidase/guaiacol system.

More recently, the use of flow injection determination of

histamine with a histamine dehydrogenase-based elec-

trode has been reported by Takagi and Shikata (inpress). Among these methods enzymatic assays are re-

ported to provide simplicity and rapidity, however,

these methods tend to overestimate histamine levels

(Ben-Gigirey, Craven, & An, 1998).

Histamine has been identified as a significant chemical

hazard in the execution of HACCP in fish processing

(FDA, 1998). A sensitive and rapid method for monitor-

ing its levels in scombroid fish is therefore needed to avoiddelay in the analysis in order to practice HACCP ensuring

safety of fish products. Our objective was to adopt a

simple extraction procedure coupling with an interaction

with the imidazole reacting and quantitatively color-pro-

ducing reagent, and to formulate a reference color scale

for rapid estimation of histamine, particularly in deter-

mination of the defect and hazard action levels in fish.

2. Material and methods

2.1. Reagents

Amine standards, p-bromoaniline, Amberlite Resin

(CG-50) and sulfanilic acid were from Sigma (St. Louis,

USA). Other chemicals and solvents used were of ana-lytical grade.

2.2. Fish samples

Fresh Little Tuna (Euthynnus affinis) (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 fishing 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 andan intelligent pump L-7100. LiChrospher 100, RP-18

column, 250 mm · 4.0 mm i.d., particle diameter 5 lmcoupled with guard cartridge 4 mm · 4 mm i.d., was

purchased from Merck, Germany. Chromatographic

conditions, sample preparation and derivatisation pro-

cedure were similar to as described by Ozogul et al.

(2002b). Histamine quantitation was carried out by

comparison of the analyte peak areas versus an exter-nally 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 pre-

pared according to Koessler and Hanke (1919) with

minor modifications. Chilled 1.5 ml 0.9% (w/v) sulfanilic

acid in 4% hydrochloric acid and 1.5 ml 5% (w/v) so-

dium nitrite were mixed in 50 ml standard flask and keptin 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 fish muscle, with little

modifications to the procedure given by McIntire, Roth,

and Shaw (1947) for the extraction and purification ofhistamine from blood plasma. Fish muscle (5 g) was

taken from the dorsal part of fillet 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

12000� 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

S.B. Patange et al. / Food Control 16 (2005) 465–472 467

trisodium phosphate monohydrate was added. Thetubes 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 briefly to break the protein gel.

The tubes were further shaken vigorously for few sec-

onds 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 astream of nitrogen. The residue was dissolved in 1 ml of

distilled water and then reacted with the reagent as de-

tailed 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 pro-duced was measured immediately after 5 min at 496

nm using distilled water as a reference. 1 ml aliquots of

standard histamine solutions containing 0–100 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) UV–visible spectrophotometer with glass

cuvettes was used for the purpose.The concentration of histamine in sample was ob-

tained 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 ðmg=100 gÞ ¼ A� 2� 25� 100

5� 1000

¼ A mg=100 g

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 fish given by

Hardy and Smith (1976) (HS method) comprises threesteps: (1) sample preparation using 10 g fish muscle with

2.5% trichloroacetic acid, (2) removal of interfering

compounds using an ion exchange column (weakly

acidic cation exchange resin, Amberlite––CG 50), and

(3) derivatisation of purified sample with diazo reagent

followed by measurement of absorbance at 495 nm. The

absorbance of sample and standards was measured

using Shimadzu (UV-1610) UV–visible spectrophotom-eter with glass cuvettes and histamine was estimated

from the standard curve of absorbance versus known

concentrations of histamine in the range 0–80 lg/ml by

regression analysis.

2.6. Recovery of added histamine

Recovery of histamine was performed by spiking the

known concentrations of histamine into the muscle ex-

tracts 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 fish muscle was

homogenized in a food processor; appropriate aliquots

of this sample were used for preparation of extracts. Toeach muscle extract appropriate quantity of 1 mg/ml free

base solution of histamine dihydrochloride was added to

get the spiked levels in the range 0–60 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 fish samples

To analyze histamine from fish samples, freshly lan-

ded 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 first analysis

was carried out immediately after the fish were brought

to the laboratory in fresh and iced condition and was

designated 0 h observation, and then the subsequentanalyses were carried out after 6, 12 and 24 h of storage

of fish. Each time an individual fish was drawn, required

quantity of muscle was homogenized and aliquots from

the homogenate were used for further analysis by the

different 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 dilutionswere then prepared to obtain aliquots of histamine

solutions containing 0–100 lg/ml in distilled water.

Similarly, tryptamine hydrochloride (12.28 mg), putre-

scine 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 (17.54mg) 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.

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 fit

between the two assays.

468 S.B. Patange et al. / Food Control 16 (2005) 465–472

3. Results and discussion

Using the procedure outlined in the new assay, a

linear relationship (correlation coefficient¼ 0.988) was

found between the color intensity at 496 nm and hista-

mine concentration in the range 0–100 lg/ml. A pink

color of increasing intensity with histamine concentra-

tion 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 30–40 s. In the 6th minute there was a fall (<1%) in

absorbance of the solution with the higher histamineconcentrations. The reagent, p-phenyldiazonium sulfo-

nate, 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 0–60 mg/100 g were

analyzed for histamine recovery by the new assay and

for comparison with the chemical assay of Hardy andSmith (1976). A high correlation (R2 ¼ 0:999) was ob-

served 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 sam-

ples overestimated the recovery that ranged from 2% to

4%. Although the values are non-significant, this may be

attributed to non-uniform distribution of histamine

originally present in the fish muscle. The recovery ofhistamine at all the levels tested was more than 91% and

there was no significant difference between the recoveries

obtained by the two assays for the respective levels of

spiked histamine. Precision of the assay was determined

Table 1

Recovery of added histamine in tuna fish samples in New assay and the ass

Parameter Spiked histamine levela

0 1 2 3

New assay

Histamine founda 1.85 2.76 3.73 4.90

Histamine recovereda – 0.91 1.88 3.05

S.D.a 0.05 0.07 0.18 0.19

Precision (R.S.D.%) 7.69 9.63 6.23

% Recovery 91.00 93.50 102.00

Trueness (%) )9.0 )6.5 +2.0

Assay of Hardy and Smith (1976)

Histamine founda 1.68 2.60 3.65 4.62

Histamine recovereda – 0.92 1.97 2.94

S.D.a 0.11 0.08 0.21 0.15

Precision (R.S.D.%) 8.70 10.70 5.10

% Recovery 92.00 98.50 98.0

Trueness (%) )8.0 )1.5 )2.0aValues in mg/100 g fish.

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 con-

centrations of analyzed samples (Cinquina et al., in

press). The overall precision (R.S.D., %) in the new as-say ranged from 2.61 to 9.63, and in the HS method it

was 3.86–10.7. 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 2–8 in the HS method. Cin-

quina 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 at5, 10 and 20 mg/100 g levels. The values of recovery

obtained in the new assay closely related to the HPLC

assay reported.

ay of Hardy and Smith (1976)

4 5 10 20 40 60

6.00 6.75 11.50 20.80 39.30 57.55

4.15 4.90 9.65 18.95 37.45 55.70

0.36 0.25 0.46 0.92 1.56 1.45

8.67 5.1 4.79 4.85 4.17 2.61

104.00 98.00 96.00 94.80 93.50 92.80

+4.0 )2.0 )4.0 )5.2 )6.5 )7.2

5.55 6.65 11.50 21.00 39.50 59.40

3.87 4.97 9.82 19.30 37.80 57.72

0.27 0.28 0.51 1.17 1.65 2.23

6.98 5.63 5.19 6.06 4.36 3.86

96.80 99.40 98.20 96.60 94.60 96.20

)3.2 )0.6 )1.8 )3.4 )5.4 )3.8

S.B. Patange et al. / Food Control 16 (2005) 465–472 469

The smallest concentration of histamine spiked insample 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 fish. The limit of quantitation, as cal-

culated from the procedure described in the new assay,

was also 1 mg/100 g. The color intensity recorded for

histamine at six different concentrations (0, 5, 10, 20, 30

and 50 lg/ml) is given in Fig. 2. The reference color scalecan also be developed in the laboratory with concen-

trations ranging 0–50 lg/ml with a difference 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 inten-

sity of concentrations exceeding 50 lg/ml did not facil-

itate 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 absor-

bance of histamine standard of similar concentration.

Extraction steps involving filtration of muscle extracts

and chromatographic purification where negligible

amounts of histamine might be lost (Hardy & Smith,

1976) have been avoided for obtaining better recovery ofadded histamine to satisfactory levels. Extraction of

histamine from muscles with 0.85% NaCl solution was

also observed to give the desired recovery. Higher con-

centrations of saline resulted in gelling of muscle pro-

teins 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 thatincludes sample preparation. On the contrary, the HS

method involving chromatographic purification re-

quired more than 2 h for a single assay. Kose and Hall

(2000) reported that the assay of Hardy and Smith

(1976) works well with fish samples and reported a

modification to the assay for determination of histamine

in fish meal. The AOAC chemical method (method 25,

1975 or method 957.07, 2002) is also reported to be te-dious 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 pro-

vides equal sensitivity.The new assay may prove to be comparatively eco-

nomical 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

affordable to low budget seafood quality inspectionlaboratories where histamine levels in fresh seafood are

required to be analyzed rapidly for examining fish

samples for the lower or higher levels than the defect/

hazard action levels of histamine. Comparatively un-

skilled technicians can perform the assay provided they

are trained under scientific supervision.

FDA (1998) guidelines for tuna, mahi–mahi and re-

lated fish specified 50 mg/100 g as the toxicity level and 5mg/100 g as the defect action level because histamine is

not uniformly distributed in a decomposed fish. Simi-

larly, 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 contenthigher 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 0–50 lg/ml of histamine works well with the color

scale and the spectrophotometric absorbance too.

The new assay was successfully applied to the deter-

mination of histamine in spoiling fish samples. The

histamine levels in the decomposing tuna and mackerelduring storage at 30 �C analyzed by the new procedure,

HS method and HPLC method are shown in Table 2.

The correlation coefficient 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 boththe fish 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

insignificant amounts of other biogenic amines in both

the fish in all samples analyzed. Histamine levels in

marine fish, especially the scombroid fish, are known to

rise continuously and can even reach toxic levels when

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.) Histamine in mackerel (mg/100 g) (Avg±S.D.)

Storage period (h) Storage period (h)

0 6 12 24 0 6 12 24

New assay 1.80± 0.12 23.50± 0.25 32.50± 0.64 51.25±0.90 2.50± 0.08 14.50± 0.35 19.50± 0.25 75.00± 1.22

Assay of Hardy

and Smith (1976)

1.60± 0.15 21.50± 0.38 29.70± 0.51 49.50±1.11 1.75± 0.05 13.75± 0.41 23.00± 0.31 77.50± 1.18

HPLC assay 2.17± 0.15 26.75± 0.66 35.45± 0.33 56.62±1.22 2.17± 0.15 16.50± 0.62 24.95± 0.60 78.76± 0.84

470 S.B. Patange et al. / Food Control 16 (2005) 465–472

fish are stored abusively. Histamine accumulation is

directly related to the kind and number of histidine

decarboxylating bacteria the fish might be contaminated

with (Eitenmiller, Orr, & Wallis, 1982). However, vari-

ous reports state that there are differences in the for-

mation of amines in fish that are mainly due to the type

and level of microflora present in fish (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 fish analyzed in the

present study was caught from offshore waters and

mackerel from coastal waters with purse seines. Al-though the kind and number of histamine-forming

bacteria in fish were not analyzed in the study, the

higher rise of histamine in mackerel than in tuna could

be presumably due to contamination with potent hista-

mine-forming bacterial flora that might be present in

coastal waters.

The new assay was also used in principle for the study

of properties of histidine decarboxylase purified fromRaoultella planticola 19-3 (Kanki, Yoda, Tsukamoto, &

Shibata, 2002). Our experience showed that the new

assay gave very satisfactory results in the enzyme stud-

ies.

The new assay utilized the reaction between the

imidazole ring and p-phenyldiazonium sulfonate as the

basis of a quantitative colorimetric method for estima-

tion of histamine. To examine possible interferencescaused 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 con-

centrations of 100 lg/ml each were reacted with the re-

agent. Except tyramine, the rest showed formation of alight lemony-yellow color. Tyramine formed a yellow to

light-pink color in the concentration range of 50–100 lg/ml whose intensity was far less than the color intensity

of histamine of similar concentration.

The interference of tyramine with histamine estima-

tion 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 difference 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 wasmeasured and histamine concentration determined from

the standard curve. Thus, the interference by tyramine

at 10 lg/ml concentration was insignificant as compared

with color intensity given by 50 lg/ml of histamine. 50

lg/ml of added tyramine did show the difference in the

color intensity and it added about 4.0 lg of additional

histamine to the standard 50 lg/ml histamine concen-

tration. The interference of tyramine in quantitativeestimation of histamine ranged between 1% and 8%.

Tyramine is reported to occur at higher concentra-

tions in cheese and cheese products (Stratton et al.,

1991). On the contrary, its occurrence in decomposing

fresh fish is comparatively very low (Shalaby, 1996).

Ozogul, Taylor, Quantick, and Ozogul (2002a) reported

about 400 ppm of histamine and 0–5 ppm of tyramine in

a decomposing herring (Clupea herengus). Similarobservations on tyramine levels have been reported in

respect of mackerel and Mediterranean hake (Baixas-

Nogueras, 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 fishes. Tyramine formationin fish is attributed mainly to decarboxylase activity

of Streptococcus faecalis and Pediococcus cerevisiae

(Eitenmiller, Koehler, & Reagan, 1978). The predomi-

nance of these organisms in a fresh fish may not be

considered as significant in relation to fish spoilage

organisms. It can be concluded that tyramine concen-

trations in fish like tuna, mackerel, herring etc. are not

likely to significantly influence the analysis of histamineby the new assay, although it may contribute to over-

estimation of histamine to levels less than 1% in a

decomposing fish. The new assay described here may

not be suitable for application to dried or salt-dried fish

S.B. Patange et al. / Food Control 16 (2005) 465–472 471

samples and may need standardization because in suchsamples tyramine could occur in relatively higher con-

centrations.

Acknowledgements

The authors are thankful to the Director, Central

Institute of Fisheries Technology, Cochin for the per-

mission to publish this paper.

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