Z Lebensm Unters Forsch A (1998) 206: 246 ± 250
O R I G I N A L PA P E R
Meta Mahendradatta ? Georg Schwedt
Sample preparation for photometric determination of histamine
in foodstuffs compared with capillary electrophoresis
Received: 3 September 1997 / Revised version: 23 October 1997
AbstractmTo determine levels of histamine, two methods
were used, photometry in conjunction with two sample
clean-up procedures, and capillary zone electrophoresis
(CZE). The two sample clean-up procedures used were
liquid liquid extraction (LLE) with n-butanol and solid
phase extraction (SPE). Using CZE, the separation of
histamine from the matrix was good. The other method,
photometry, represents a classic and simple method, that
can be employed for in situ measurement of histamine. We
found that it was necessary to clean up the samples prior to
photometry; if this was not done, the recorded levels of
histamine were higher than those determined by CZE. In
order to determine levels of histamine, both of these rapid
tests were applied to ten different foodstuffs. The levels of
histamine measured using photometry following either LLE
or SPE were compared. The results indicated that photo-
metry is a suitable method for the measurement of hista-
mine, although the sample solutions have to be purified by
either LLE or SPE. Samples do not need to be cleaned up
before CZE because there is no interference between
histamine and attendant material. Both sample clean-up
procedures were applied to the following foodstuffs: toma-
toes, sauerkraut, tuna, leaf spinach, cream spinach, white
wine and mackerel. The differences of the measured values
vary between 3% and 18% for LLE and 6% and 27% for
SPE. For the other foodstuffs, such as beef, beer and non-
alcoholic beer, only one sample clean-up procedure is
suitable. LLE used for beef and beer leads to differences
in measured levels of histamine between 18% and 50%,
respectively, whereas SPE used for non-alcoholic beer leads
to differences of 20%.
M. Mahendradatta
Department of Agriculture Product Processing, Faculty of Agriculture,
Hasanuddin University, Ujung Pandang 90245, Indonesia
G. Schwedt ( )
Institut fuÈr Anorganische und Analytische Chemie der Technischen
UniversitaÈt Clausthal, Paul-Ernst-Strasse 4,
D-38678 Clausthal-Zellerfeld, Germany
Ó Springer-Verlag 1998
Key wordsmHistamine ? Capillary electrophoresis
Photometric determination ? Liquid liquid extraction
Solid phase extraction
Introduction
Histamine (1H-imidazol-4-ethanamine) is a heterocyclic
amine and is obtained through decarboxylation of free
amino acid histidine. Because of its various effects, hista-
mine is of physiological as well as pharmacological inter-
est. Histamine is also used as an indicator of food quality
and hygiene [1]. The importance of histamine in food
poisoning of food is well-known, in particular where
poisoning occurs after consuming fish. The well-known
type of food poisoning, ªscombroid fish poisoningº, is
caused by fish from the family Scomberesocidae and
Scombridae (mackerel and tuna) [1, 2]. Today, the descrip-
tion ªscombroid poisoningº is no longer used, because of
the high content of histamine in other fish and even other
foodstuffs, which can lead to the same type of food
poisonings.
Numerous techniques for the determination of histamine
contents in foodstuffs have been described, namely photo-
metry, fluorimetry, chromatography, flow injections analy-
sis, enzyme immunoassays, amino acid analysis and elec-
trophoresis [3±10]. Photometric procedures for the deter-
mination of histamine in foodstuffs have not been further
developed because of their low sensitivity. Moreover,
interference due to the matrix can lead to incorrect results.
In this study, two kinds of sample clean-up procedures
were applied before making measurements ± liquid liquid
extraction (LLE) and solid phase extraction (SPE). The
results of histamine contents in foodstuffs obtained by these
methods were subsequently compared with those derived
from capillary zone electrophoresis (CZE).

Materials and methods
Photometry
Apparatus
± Spectral photometer CADAS 50 (Dr. Bruno Lange)
± SPE system (Baker)
± Three-millilitre LiChrolut-SCX cartridges (Merck)
Materials
All foodstuffs were obtained from different supermarkets.
Canned sauerkraut. 100-g sample was mixed with 100 ml water by
means of a household mixer. Twenty-five millilitres of this suspension
was then mixed with 25 ml of 5% trichloroacetic acid, centrifuged at
4000 rpm for 10 min and filtered through a 0.2 mm membrane.
Tomato stored for 1 week. A 5-g sample was homogenized with 40 ml
doubly distilled water by means of a stirring machine, decanted into a
50-ml volumetric flask, filled up to the mark with doubly distilled
water and filtered through a 0.2 mm membrane.
Beef (deep frozen). A 10-g sample was homogenized with 5% trichloro-
acetic acid by means of a stirring machine, and centrifuged at 4000 rpm
for 10 min. The supernatant was decanted into a 50-ml volumetric
flask. The residue was homogenized again with 25 ml of 5% trichlor-
oacetic acid and centrifuged. The combined extracts were filled up to
the mark with 5% trichloroacetic acid and filtered through a 0.2 mm
membrane.
Tuna, leaf spinach, cream spinach (all deep frozen) and canned
mackerel. Prepared in the same manner as the sample of beef.
White wine. After filtration through a 0.2 mm membrane the white wine
was directly analysed.
Non-alcoholic malt beer and beer. Prepared in the same manner as the
sample of white wine. All of the samples were adjusted to pH 3.0.
Reagents
n-Butanol and KCl were obtained from Riedel-de HaeÈn; NaOH, NaCl,
methanol, KOH, citric acid, isopropanol and Na2CO3?H2O from Merck
(Darmstadt); n-heptane and trichloroacetic acid from Fluka; 4-nitro-
benzene-diazonium-tetrafluoro-borate from Aldrich.
Preparation of buffer solutions
Potassium citrate buffer at a concentration of 0.1 mol/l. The buffer was
prepared by dissolving 1.4 g citric acid, 1.12 g KOH and 24.6 g KCl in
doubly distilled water and made up to 100 ml. A volume of 2.857 ml
from the resulting 3.5 mol/l potassium citrate buffer was pipetted into a
100 ml volumetric flask and filled up to the mark with doubly distilled
water.
Potassium citrate/isopropanol buffer. A volume of 92 ml of 3.5 mol/l
potassium citrate buffer was mixed with 8 ml isopropanol (92%) and
shaken.
Methods
Liquid liquid extraction
The LLE procedure was carried out in the same manner as that
described by Rice [5]. A 1-ml sample was transferred to a centrifuge
247
tube containing 5 ml n-butanol, 0.25 ml of 5 mol/l NaOH and 0.75 g
NaCl. The tube was shaken for 3 min and then centrifuged at 4000 rpm
for 10 min. To eliminate any free histidine, the butanolic layer was
transferred to a second centrifuge tube containing 2.5 ml of NaCl-
saturated 0.1 mol/l NaOH, shaken and centrifuged in the same way as
before. A 4-ml aliquot of the washed butanol extract was transferred to
a third centrifuge tube containing 2.5 ml of 0.1 mol/l HCl and 7.5 ml n-
heptane. The tube was shaken for 1 min and centrifuged. The organic
phase was removed. The acid phase containing histamine was mea-
sured by photometry.
Solid phase extraction
For the SPE, the LiChrolut-SCX column was conditioned with 3 ml
methanol followed by 3 column volumes of doubly distilled water and
3 ml of 1 mol/l HCl. Three millilitres of the treated sample was then
passed through the column. The column was washed with 0.1 mol/l
potassium citrate buffer (the volume required depends on the kind of
foodstuffs), and air in the column was evacuated until the column was
dry. Elution was performed with 3 ml of the potassium citrate buffer/
isopropanol buffer. The eluate was measured photometrically.
Photometric analysis
To 1 ml of the acid phase containing histamine, 1 ml of 0,2% (w/v)
4-nitrobenzene-diazonium-tetrafluorborate was added, followed by
1 ml of a 10% (w/v) Na2CO3 solution. The solution was maintained
at room temperature for 5 min. The absorbance was then measured at
470 nm and 480 nm for the solutions treated with LLE and SPE,
respectively.
Capillary zone electrophoresis
Apparatus
BioFocus 3000 capillary electrophoresis system (BioRad).
Column. The column was an uncoated fused silica capillary tube,
length 50 cm, with an internal diameter of 50 mm (BioRad).
Electrolyte
The electrolyte comprised 20 mmol/l sodium citrate buffer at pH 2.5
and 25 mmol/l sodium phosphate buffer at pH 6.5.
Experimental conditions
These were the same as those described by Mahendradatta and
Schwedt [10].
Samples
These were the same as those analysed by photometry. The pH was not
adjusted.
Reagents
Tri-sodium citrate-2-hydrate (Merck) and di-sodium hydrogen phos-
phate-2-hydrate (Riedel-de HaeÈn).