High Pressure Liquid Chromatography

Histamine in Fish
J. P. GOUYGOU,

of

C. SINQUIN, and P. DURAND

ABSTRACT
Histamine was determined by reversed-phase high-pressure liquid
chromatography in trichloroacetic acid extracts after derivatization with
o-phthaladehyde. Fluorescence was monitored at 350 nm excitation
and 450 nm emission wavelength after elution with water containing
40% acetonitrile. The advantage of the described method is a rapid
analysis in an automated system, where no selective extraction procedure is necessary and interfering substances are easily separated
from the histamine fluorophore.

INTRODUCTION
THE FLUORIMETRIC METHOD for determining histamine
is based on the reaction of o-phtalaldehyde, at highly alkaline
pH, which forms a fluorescent product after converting into a
more fluorescent and stable product by acidification (Shore et
al., 1959). During the last few years, the sensitivity and specificity of this method have been greatly improved. The sensitivity can now be measuredin nanogram amounts of histamine,
but the lack of specificity requires large and laborious purification of most tissue extracts prior to assay.
Many methods have been used to avoid interferences with
other naturally occurring substances,mainly histidine and spermidine (Green and Erickson, 1964; Kremzner and Pfeiffer,
1966; Michaelson, 1967) by specific purification techniques
such as ion-exchange chromatography (Ronnberg et al., 1984)
or selective extraction into alcohol (Anton and Sayre, 1969)
prior to the chemical derivatization with o-phtalaldialdehyde.
Fluorescencedetection coupled with high-pressureliquid chromatography offers possibilities to improve the specificity.
Many studies dealing with the determination of histamine in
fish by high-pressure liquid chromatography have been published (Mietz and Karmas, 1977; Volk and Gemmer, 1982;
Walters, 1984).
Histamine is generally present in fresh fish at levels less
than 50 mg/kg (Staruszkiewicz et al., 1977). Larger amounts
of histamine and other biogenic amines are formed during the
decomposition process by the action of decarboxylating bacteria on the corresponding free amino acids. A maximum allowable level of 100 mg histamine/kg fish seemsto be adequate
in order to prevent fish poisoning (Luten, 1983).
The purpose of this study was to develop a sensitive, simple,
and selective method to analyze histamine in fish after condensation with o-phthaldialdehyde, using HPLC under isocratic elution conditions.
MATERIALS

Determination

All other reagents were of analytical grade (E. Merck). All solvents
were degassed prior to use.
The HPLC system consisted of a LC Pump 414 and MS1 660 autosampler (KONTRON), and a Rheodyne injection valve with a loop
capacity of 20 pl. The detector used was a KONTRON SFM 25
fluorometer, equipped with a Xenon-High pressure Lamp. Fluorescence was monitored at 350 nm excitation and 450 nm emission wavelengths.
The KONTRON RP 18 column (4.6 x 220 mm) packed with a
particle size of 5 pm was used for reversed-phase liquid chromatography. The isocratic mobile phase consisted of 40% acetonitrile in
water with monosodium phosphate (50 mmoles/L) at a flow-rate of
0.7 mL/min. Chromatogram recordings and all calculations were performed on a ANACOMP 220 computer.
Derivatization was done by a modification of the method of Rice
et al., 1975. The OPA reagent was prepared by dissolving 10 mg
OPA in 1 ml methanol. Standard solutions of histamine ranging be-l

0 bos

061

0015

CONCENTRATION

Fig. l-Linearity
of fluorescence
intensity
centrations
of histamine
in TCA (100%).

for

k&rn~

increasing

con-

& METHODS

HISTAMINE dihydrochloride, extra pure, was obtained from E. Merck

(Darmstadt, G.F.R.); cadaverine dihydrochloride, putrescine dihydrochloride, spermine tetrahydrochloride, spermidine trihydrochloride, DL-Histidine were purchased from Sigma (St Louis, MO, USA);
o-phthalaldehyde (OPA) was obtained from United States Biochemical Corporation (Cleveland, OH).

The authors
are with the lnstitut
Francais
de Recherche
Pour
LExploitation
de la Mer, Rue de llle dYeu - BP 1049 44037
Nantes
Cedex 01, France.

Fig. 2-Stability
measured
over

Volume

52,

No.

of fluorescent
a 24-hr period

4, 1987-JOURNAL

OPA-histamine
derivative
at 4C and 23C (ambient).

OF

FOOD

SCIENCE-925

was

HPLC DETM OF HiSTAMiNE

IN FISH. . .

Histamine

.
.3

I
n

Retention

time

4
v)

"

(min)

Fig. 3-HPLC
separation
of OPA-TCA
fish extract
using
reversed-phase
column
with
monosodium
phosphate
mmolesll)
in the acetonitrile-water
(40.60) solvent.

RP 18
(50

tween 0.001 and 0.02 mg/mL in 10% TCA were filtered (0.45 urn)
and stored at 4C.
The histamine-OPA fluorophore was prepared by mixing 135 )LI
stock solution with 1.86 mL deionized water and 0.4 mL N sodium
hydroxide solution, and leaving it for 1 min in a tube protected from
light with aluminium foil. OPA reagent (0. 1mL) was added, the
solution mixed, and left 4 min before addition of 0.2 mL 3N HCI.
For a histamine solution containing 0.01 mg/mL in 10% TCA the
20 )LL injection volume corresponded to a quantity of 10 ng histamine.
The sample was prepared by blending 5Og fish with 100 mL 10%
TCA solution, centrifuging and filtering the clear extract through a
0.45 pm filter. The filtrate was stored at 4C.

RESULTS & DISCUSSION

THE HISTAMINE fluorophore is formed by the derivatization
of histamine with o-phthalaldehyde (OPA) at highly alkaline
pH (- 13):
, CHi-CHTNH2

CHO

HI

CH2
yCH2

Acidification makes this product more fluorescent and stable.

The chromatographic system was carefully studied to find a
suitable mobile phase with high resolution of the histamine
fluorophore without quenching the fluorescence.
A mobile phase consisting of 40% acetonitrile in water with
926-JOURNAL

OF FOOD SCIENCE-Volume

52, No. 4, 1987

Retention

I
0

time-(min)

Fig. AHPLC
separation
of histidine
standard
(5 mglmL)
using
RP 18 reversed-phase
column
with monosodium
phosphate
(50
mmoleslL)
in the acetonitrile-water
(40:6OJ solvent.

Table I-Range
and average
(parts per millionJ.
Samples of
canned tuna
N II 2
G II 1

values of histamine

Number of
analvsis
8
8

found in canned

Histamine
Ranae
17.5-18.2
5s
6.1

content

tuna

(ppm)
Averaae
17.9
5.9

monosodium phosphate (50 mmoles/L) was found to give a

satisfactory separation in less than 15 min.
OPA-derivatized histamine standard produced a major peak
at 9.7 min retention time. The peak area was proportional to
the concentration and a linear relationship was observed (Fig.
1). The detection limit, defined as a signal-to-noise ratio of 2,
was found to be 100 pg histamine per 20 PL loop volume
injected. The reproducibility was tested by nine repeated injections and was in the order of +_ 5%.
The stability of the histamine fluorophore was studied by
injecting the same sample into the column at various intervals
from 0 to 24 hr. The experiment was carried out at 4C and
at room temperature (23C). The complex was prepared with
a standard solution of histamine in 10% TCA (0.01 mg/mL).
The results are shown in Fig. 2.
During the first 4 hr, at 4C a fairly stable complex was
obtained; however, there was a decreasein fluorescence intensity at 23C. The histamine fluorophore complex in fish TCA
extract was stable for 24 hr at both 4C and room temperature.
After separation on the HPLC column, the OPA complex
obtained from fish TCA extract, produced five peaks: 2 major
peaks at 4 and 10 min, respectively, and 3 small peaks (Fig.

3). The 10 min peak corresponds to the retention time of histamine .

Experiments were done with histidine, the compound from
which histamine is formed. A typical chromatographic profile
of the OPA derivative of histidine standard shows a major peak
at 4.0 min. retention time and 3 smaller peaks. The similarities
of the HPLC profile of the histidine standard (Fig. 4) and the
one obtained with the OPA-TCA fish extract (Fig. 3) indicate
clearly that the 4.0 min. retention time peak can be attributed
to histidine.
Other biogenic amines, such as cadaverine, putrescine,
spermine and spermidine, were also investigated for possible
interference. These compounds also produce fluorophores with
OPA, however under the chromatographic conditions described no peaks appear before 20 min. retention time.
The present methodology was applied to commercial canned
tuna from different origins and the results showed good reproducibility and a relative standard deviation of 2% (Table 1).
Fortified tuna samples with 1 and 0.5 mg histamine/lOOg
fish showed a mean recovery of 94.2% with RSD of 2.18%.
CONCLUSION
THE HPLC METHOD described for the determination of histamine in fish is a sensitive, simple and selective method for
the analysis of histamine, where no specific extraction procedure is necessary. Analysis can be performed rapidly in an
automated system after a simple sample preparation and derivatization with o-phthalaldehyde.
From an analytical point of view, the HPLC allows the sep-

SALTED AND RIPENED ANCHOVIES.

. .From

REFERENCES
rocedure for
Anton, A.H. and Sayre, D.F. 1969. A modified fluorometric
tissue histamine and its distribution
in various animals. s: Pharmacol.
Exp. Ther. 166: 285.
Green, H. and Erickson, R.W. 1964. Effect of some drugs upon rat brain
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Kremsner, L.T. and Pfeiffer, C.C. 1966. Identification
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Pharmacol. 15: 197.
Luten, J.B. 1983. Analysis, toxicity and formation of histamine. Paper
presented at 13th Annual Meeting of Western European Fish Technologists Association, Iimuiden,, the Netherlands.
Michaelson, I.A. 1967. Spernudine: A contaminant
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Miets, J.L. and Karmas, E. 1977. Chemical quality index of canned tuna
as
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103.

Rice, S., Eitenmiller,

R.R., and Koehler, P.E. 1975. Histamine and tyramine content of meat products. J. Milk Food Technol. 38: 256.
RONNBERG, A.L., HANSSON, C., and HAKANSON,
R., 1984. Hi h erformance liquid chromatographic
determination
of histamine in %.10Pog
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Anal. Biochem. 139:
338.
Shore, P.A., Burkhalter,
A.? and Cohn, V.H., 1959. A method for the fluorometric assay of histamme in tissues. J. Pharmacol. Exp. Ther. 127:
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Staruszkiewics, W.F. Jr, Waldron, E.M., and Bond, J.F. 1977. Fluorometric
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of histamine in tuna: Development of method. J. Assoc.
Off. Anal. Chem. 60: 1125.
Volk, K. and Gemmer, H. 1982. Schneller Nachweis von histamin in &hen
und fischerseugnissen mittels HPLC. Fleischwirtsch.
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Walters, M.J. 1984. Determination
of histamine in fish by Liquid chromatography with post-column reaction and fluorometric detection. J. Assoc. Off. Anal. Chem. 57: 1040.
MS received 8/27/86; revised l/27/87; accepted l/31/87.

page 921

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COMPOSlTlON

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recovery values.

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MS reveived 4/l/85; revised l/16/87; accepted 1116187.

The author

thanks

La Campagnola

S.A., Mar del Plata

and Mr. Mario

Iribarren

for

providing the facilities for the initial stages of anchovy salting.

OF ATLANTIC COD. . .From page 924

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Composition of Foods. Her Majestys Stationery Office, London, 418~.
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and mollusks, I. Protein, fat, moisture, ash, carbohydrate, energy value

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MS received 11/8/86; revised 1130187; accepted 2/20/87.

The authors sincerely thank R. Benson, G. Bon&l, G. Browne, S. Cave, N. Cheeseman, A. Downey, J. Johnson, J. Lauder, W. Saint, H. Stagg, J. Summers and P. Wheeler
for their technical assistance.
The assistance of J. Rice and D. Stansbury
in analyzing
the data is greatly appreciated.
The authors are grateful to the commercial
fishermen
at Bonavista,
NF, for the
excellent co-operation that was received.

Volume 52, No. 4, 1987-JOURNAL

OF FOOD SCIENCE-927
1