Can Food Quality be Enhanced by Chromatography?

H. Steinhart* / G. B i e r n o t h
University of Hamburg, Institute of Biochemistry and Food Chemistry, Grindelallee 117, 20146 Hamburg, Germany

For the producer and the consumer, food quality has

several aspects: - chemical, physical, micro-biological,
and even psychological ones. Firstly, foods must be
guaranteed with respect to health. This is preferably
realised by chemical and microbiological quality parameters. Foods must also be tasty; this is realised by raw
material and processing parameters which result in an
optimal concentration of specific minor components acting as taste and flavour compounds. Off-flavour and
off-taste compounds must also be absent.

Key Words
Gas chromatography
Column liquid chromatography
Foodstuffs quality
Trace analysis

Summary
Quality of food is the utmost goal of all food producers.
It is determined by chemical, physical, microbiological,
and even psychological aspects. The chemical and
microbiological quality aspects are especially responsible for health and welfare of the consumer. Quality of
food, therefore, at all times has to be ensured and be enhanced as far as possible. From the chemical point of
view, foods contain a great number of major and minor
components. That determine the nutritional value of a
food product. Furthermore, taste and flavour are the
result of many food minor components. Foods also may
contain unwanted compounds like residues and contaminants. Which represent undesirable aspects of food
quality.
With chromatography as a powerful analytical tool in
food chemistry, the quality of food products cannot only
be characterized but also be enhanced. Examples of
enhancing quality of food by applying chromatography
to cheese, margarine, meat, and other products is reported.

Introduction
Food Quality
For all food producers the quality of their products is an
important goal. Especially with foods, quality is what the
consumer asks for and by selling quality the producers
can keep and gain market shares.

Presented at the 21st ISC held in Stuttgart, Germany,

15th-20th September, 1996

Apart from taste and flavour compounds, other minor

components play an important role for food quality.
Food must not only provide energy, but also components vital for the organism like vitamins and essential fatty and amino acids. On the other hand, food must
not contain detrimental compounds which may injure
the consumer's health. During food processing illegally
added compounds or residues of added compounds
higher than the permitted value or detrimental contaminants from the environment are examples of unwanted materials in foods. Assuring and enhancing
quality of food means, to a large extent, controlling
minor compounds with both positive and negative
qualities in a great variety of products.

Chromatography as VersatileMethodforFood
Analysis
By chromatographic methods, nearly all volatile and
soluble compounds in foods can be identified and quantified. This makes chromatography a powerful tool in
food analysis. In some areas like flavour research and
control, chromatographic methods are essential, because chromatographic analysis is the method of choice
for volatile compounds. But due to its versatility,
specificity, sensitivity, and simplicity, chromatography is
applied to all areas of food analysis. Nowadays its high
separation potential combined with sensitive detection
techniques made chromatography the most widely used
analytical method in food analysis. It can be used especially for trace analysis with very low detection limits.

Chromatographia Vol. 45,1997

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0009-5893/97

Role of Minor Components in Foods

11-07

$ 03.00/0

9 1997 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH

11

Therefore, by applying chromatographic methods within food analysis, not only control of food quality became
possible but also its enhancement. For these reasons
chromatographic methods are widely applied in food research and development.

Complex and Individual Nature of Food Analysis

Food analysis comprises specific features which distinguish it from the more general type of chemical analyses.
In foods, minor components are bound in a matrix of
main components and the type of matrix varies from
product to product within wide range of foods. Therefore, before analysis, the compounds to be analysed
have to be separated from the matrix of main components. This has to be achieved as completely as possible and without less or decomposition of the compounds to be analysed. Furthermore, the compounds to
be analysed often have to be separated from other
minor components which are of no interest in a specific
case and which may interfere with the analysis to be carried out. This separation of the minor compounds from
the main components and other interfering compounds
often involves a sometimes labour and time consuming
work-up procedure. Work-up techniques also depend on
the analytical method to be applied. So, in food analysis
there is always a relationship that has to be considered
between the components to be analysed, matrix, and the
analytical method to be employed. Because of these
considerations, nearly all analytical tasks within food
analysis have to be tackled individually. Fortunately, the
great versatility of chromatographic methods can accommodate the analytical needs of most food products
and types.

Enhancement of Food Quality by

Chromatography
Not only for reasons of competition but also because
more and more foods are manufactured industrially, enhancement of the quality of food is essential. New
products are developed which need to be optimized and,
new knowledge of the physiological effects of specific
food components may require alteration and change in
food component composition. So the question whether
there is a necessity for enhancement of food quality is
clearly in the affirmative. As mentioned, chemical food
analysis and especially chromatographic methods play a
dominant role in this field.
There are many examples of enhancement of food quality by chromatography (Table I). They underline the
possibilities and the potential of all the different chromatographic methods.
Some of these examples will be treated in more detail.
For hormones, trans fatty acids, and flavours, capillary
gas chromatography is the preferred chromatographic
method; for biogenic amines and especially tryptophan,
HPLC was used.
12

Table I. Recent examples of e n h a n c e m e n t of food quality by

chromatography (*:discussed)
-

reduction of pesticide residues in vegetable and fruit

control of illegal additives, e.g. c h l o r o a m p h e n i c o l in m e a t
control of permitted value of food additives
identification of natural and naturally identical flavour
influencing flavour of carp by feed
allergenes: identification of allergenes
reduction of biogenic amines in f e r m e n t e d foods
h o r m o n e analysis in m e a t
reduction of trans fatty acids in fat containing foods
control of traces of b e n z e n e in baby food (I0 p p m )
tryptophan p r o d u c e d by gen technology
flavoar of boar m e a t
identification of n o t allowed processes like irradiation

.o

pregnenolone

progesterone

OH

testosterone

Figure 1
Chemical structures of some steroidal hormones.

Steroidal Hormones in Meat

Quality of beef is influenced by sex. For instance, meat
from young cows is preferred to that of bulls. Meat contains very low concentrations of sex hormones from
ng/kg up to ~tg/kg. From the chemical point of view, sex
hormones are steroidal hormones (Figure 1).
Since highly sensitive methods of analysis for steroidal
hormones are available, sex and thereby quality of meat
can be determined. To this end a suitable method of
analysis was developed [1] comprising capillary GC
coupled with MS detection in the selected ion monitoring (SIM) mode. Before analysis by GC-MS, the samples
must be prepared by isolating the hormones from the
bulk substances and other interfering compounds. This
work-up procedure comprises
1. enzymatic hydrolysis of the homogenized meat
sample,
2. extraction of the crude hormones by methanol, defatting the methanol solution with hexane, and extraction of the hormones with dichloromethane,
3. separation of phenolic compounds with aqueous
alkali,
4. solid phase extraction applying both silica and
alumina columns.
Before the steroidal hormones are separated by capillary gas chromatography, they are derivatized to
trimethylsilyl ethers.

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By this method, 7 steroidal hormones and 14 of their

metabolites can be detected in meat with a detection
limit in the order of 0.2 to 0.3 gg/kg. Progesterone and
pregnenolone were quantitatively dominant (43.7 and
6.5 gg/kg, respectively). The highest hormone concentrations were determined in female cattle. The
progesterone/pregnenolone-ratio in female cattle is
about 3, but is below 1 in male cattle.
By application of GC-MS in the form of capillary gas
chromatography and MS in the SIM mode it is now possible to identify meat from male and female cattle and
as a consequence, statements on the quality of meat
samples are now possible. Furthermore, the occurrence
of illegally added hormones in meat is readily detected.

Trans Fatty Acids in Foods

In natural unsaturated fatty acids the cisisomers dominate. Trans fatty acids occur in hardened fats as
manufactured industrially/chemically or produced naturally by bacteria in the first stomach of ruminants.
Trans fatty acids are found in margarine and frying fats
and in products containing milk fat, e.g. milk, butter, and
cheese. Recently, trans fatty acids have been found to increase low density lipoproteins (LDL) and to decrease
high density lipoproteins (HDL) in serum, so possibly
causing arteriosclerosis [2]. Therefore a study has been
carried out to measure the content of trans fatty acids in
fat-containing food products. The sum of trans fatty
acids can be measured by infrared spectrometry, but in
order to determine individual trans fatty acids in foods
a new method had to be developed capable of giving
reliable results at low concentrations. Capillary gas
chromatography was chosen for the separation of the
fatty acids [3].

CGC of trans fatty acids

Fat is extracted from food by hexane and transesterified with methanol. The methyl esters obtained are
separated on 30 to 100 m capillary columns with cyanopropyl silicone stationary phases. Depending on fat type,
different temperature regimes must be run. The compounds are detected by FID.
Using this method, many trans fatty acids can be
detected and quantified. Not only can trans fatty acids
be separated from cis acids, but trans fatty acids differing in the position of the double bond e.g. elaidinic acid
C18:1t9 from trans vaccenic acid C18:ltll can be determined. Furthermore, trans linoleic acids and trans
linolenic acids can be identified, as can mixed cis trans
fatty acids.
More than 200 fat-containing foods have been analysed
for trans fatty acids by GC with contents ranging between 0.5 and 34.9 %. High contents were found in
some margarines and frying fats with partly hardened
vegetable oil as raw material. Due to these results, the
content of trans fatty acids in e. g. margarine has been
drastically reduced for health reasons [4]. In 1994 it was
Original

found to average 8.5 %, by 1996 it was only 1.5 %. This

can be seen as a convincing example of the enhancement of quality of this type of food by chromatographic
analysis.

Flavour Compounds
Flavour compounds are especially suited for chromatographic analysis due to their volatility. Numerous food
have been identified by gas chromatography.
In the meantime, more tricky flavour problems can be
solved by chromatography, e.g. the question whether a
flavour component is natural or synthetic. Special forms
of chromatography may be necessary to solve such a
problem, making use of differences in the molecule like
chirality or the isotope ratio of specific atoms in the
compound [5].

Enantiospecific Gas Chromatography

Natural flavour compounds are normally synthesized
enzymatically e.g. in plants. In this case, one enantiomer
is produced when the compound contains an asymmetrical C-atom. In chemical synthesis both enantiomers are formed in a 1:1 ratio. In flavours and aromas
the enantiomers contribute to the flavour in different
ways. For instance, (R)-(+)-limonene gives a pleasant
fresh orange flavour, whereas (S)-(-)-limonene has a
weak terpentine note. (R)-(+)-p-menthen-8-thiol gives
the flesh grapefruit note, whereas its (S)-(-)-enantiomer
is neutral.
With the aid of enantiospecific capillary gas chromatography the (R)/(S)-ratio can be determined by which the
question "natural or naturally identical ?" can be
answered.
The prerequisite of enantiospecific GC is a chiral stationary phase like special cyclodextrins. Enantiomers
are separated by such chiral phases by means of inclusion chromatography. To separate from interfering
compounds, GC is carried out after a first chromatographic separation step. This step can be thin-layer
chromatography or capillary gas chromatography. The
techniques are called "off-line DC-GC" and "MDGC"
(Multidimensional Capillary Gas Chromatography).
With MDGC, the first separation is carried out with a
normal capillary column and the second with an alkylcyclodextrin column. With the recent introduction of 6O-tert.-butyldimethylsilyl-cyclodextrins as chiral stationary phases, enantioselective GC can be carried out simultaneously (enantioselective simultaneous analysis).
With the help of enantiospecific capillary GC many
flavours like that of butter with its content of enantiomeric y(8)-lactones [6] or new flavours like the odour
of orchids [7] or of passionfruits like maracuja [8] can
now be characterized. This plays an important role in
quality control in the aroma industry. Today the GC of
enantiomers is even used as a routine method of
analysis for the characterization of essential oils.

Chromatographia Vol.45,1997

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Table II. examplesof biogenicamines

Biogenic amines

Chemical formula

Original amino acids

methylamine
ethylamine
propylamine
butylamine
ethanolamine
2-phenylethylamine
4-arninobutyrica c i d
putrescine
cadaverine
dopamine
histamine
tryptamine
serotonine
tyramine

CH3-NH2
CH3-CH2-NH2
CH3-CH2-CH2-NH2
CH3-CH2-CH2-CH2-NH2
HO-CH2-CH2-NH2
C6H5-CH2-CH2-NH2
NHE-CH2-CH2-CH2-COOH
NH2-(CH2)4-NH2
NH2-(CHE)5-NH2
3,4-(OH)2-C6H5-CH2-CH2-NH2
4-imidazolyl-CH2-CH2-NH2
3-indolyl-CH2-CHE-NH2
3-(5-OH-indolyl)-CH2-CH2-NH2
3-HO-C6H5-CH2-CH2-NH2

l-glycine
l-alanine
t~-aminobutyricacid
norvatine
1-serine
1-phenylalanine
1-glutamic acid
l-omithine
l-lysine
l-dopa
l-histidine
l-tryptophan
5-hydroxy-l-tryptophan
l-tyrosine

GC-Isotope Ratio Mass Spectrometry (GC-IRMS)

A second m e t h o d to distinguish between natural and
non natural flavour compounds is "isotope ratio mass
spectrometry" (IRMS). The ratio of stable isotopes of
atoms like 13C/12C, 2H/1H, or 15N/14N in a given compound can be determined and the products of plants like
lemon oil are often characterized by a typical isotope
ratio [9]. By G C - I R M S with a combustion interface, the
question, natural or synthesized ?, can be answered by
determining e. g. the 13C/12C ratio.

The flavour of the wheat-fed carp contained more octanal, methional, and (E,Z)-2,6-nonadienal, whereas the
carp living on plankton had more (Z)-4-heptenal, 1octen-3-on, and 2-acetyl-2-thiazolin. This is a result of
differences in the composition of fatty and amino acids
in the two types of carp. Fatty and amino acids act as
flavour precursors because they are partially metabolized to flavour active c o m p o u n d s . - It was thus established by chromatographic analysis, that the quality of
carp flavour can be influenced and improved by the nature of the feed.

Recently, enantioselective G C has been coupled with

IRMS directly. In this way the distribution of enantiomers (R)/(S) and the isotope ratio of the enantiomers
can be determined simultaneously.

Reduction of Biogenic Amines in Fermented

Foods

Influence of Feed on Aroma of Steamed Carp

Biogenic amines are aliphatic or aromatic mono- or

diamines (Table II), the chemical structure of which
depends on the natural amino acid from which they are
metabolized by enzymatic decarboxylation.

(Cyprinus carpio L.)

Carp farmed in pools have b e c o m e an important food
product. Normally, carp live on natural material like
plankton. In some places they obtain additional feed
like wheat. It was noticed that steamed carp fed with
wheat had a somewhat nutty, potato-like aroma not possessed by carp not fed on wheat, but the latter developed a somewhat fishy and mouldy aroma. Because
aroma is seen as an important quality aspect this influence of feed on carp aroma was evaluated [10].

GC-Olphactometry of Carp Aroma

1-2 ~tl aroma extract samples were injected on polar and
apolar 30 m x 0.32 mm, temperature programmed capillary columns. A t the column exit the gas stream was
split, one part passing to the FID, the other to a sniffing
port. 10 trained testers gave a signal when they detected
the presence of an aroma substance.
By sniffing, 42 flavour compounds were found, with
hexanal as the main compound. The pattern of other important flavour compounds were found to be quantitatively different between the carp on different diets.
14

Decarboxylation takes place in fermentation processes

which several types of food undergo during their
manufacture. Typical examples are cheese, the last
processing step of which is cheese ripening, but there are
other foods involving fermentation processes like
sausages and pickled cabbage, and of course beer and
wine. Bacterial cultures which grow within the product
and metabolize components of the substrate are responsible for these fermentation and ripening processes.
Many of the compounds so produced are wanted, others
like the biogenic amines are not wanted. Biogenic
amines occur in these foods in amounts of some
hundred mg kg -1 in total, in extreme cases up to several
grams per kg.
Biogenic amines in foods [11] are unwanted because
some people, especially those who lack the enzyme
monoaminooxidase, react sensitively towards these
compounds. In the absence of the oxidase, the consequences are abnormally high concentrations of amines
in the body. In higher concentrations, biogenic amines
are physiologically active substances and people lacking
the enzyme suffer from headache, dizziness and other

Chromatographia Vol. 45,1997

Original

symptoms after eating foods high in amine concentration. To avoid this, foods with low concentrations of
biogenic amines are aimed at.
A study was carried out on the biogenic amine concentration in Emmental cheeses, together with an
examination of the influence of cultures and bacteria
strains on the production of amines, and finally the effect of processing parameters.
These investigations required a lot of reliable analyses
of biogenic amines in cheese. A work-up procedure was
developed comprising an extraction step, which
separated the amines from the protein and fat, followed
by a solid phase extraction step, to separate interfering
substances. Interfering amino acids were separated by
elution on cationic exchange columns. Prior to the final
HPLC analysis amines were derivatized with phthalic
acid dialdehyde and removed by gradient elution
[12].The separated amines were detected fluorometrically. The detection limit of amines was found to be 525 pmol/ml.
As result of the measurements on Emmental cheese
samples, a variation in the content of the main biogenic
amines histamine, tyramine, putrescine, and cadaverine
from 30 up to 1800 mg/kg were found. Samples from the
inner part of cheese generally showed a lower amine
content than samples from outer part. The bacteriological status of the raw milk used to make the cheese was
identified as the main factor in the difference in amine
content. A positive correlation was found between
enterobacteria content of the milk and biogenic amines
content of the cheese. Furthermore an effect of different
types of enterobacteria on amine formation was found.
Although Emmental cheese produced in summer contained larger amounts of amines on average compared
with cheeses produced in winter time, these differences
were not marked [13].
The analytical data showed that both the ability of the
bacteria strains to form and to metabolize the biogenic
amines are responsible for the total content of amines.
This means that the amine content of the cheeses is the
sum of amine formation and decomposition. Bacteria
strains with high formation potential for specific
biogenic amines were identified and others were identified which had a high decomposition potential. Furthermore, in mixtures of bacteria strains, as usually applied in Emmental production, synergism with respect
to increased or decreased amine formation was measured. The selection of the cultures used was found to
have a strong influence and could be chosen to reduce
biogenic amine content in the cheese.
Process parameters were also investigated and it was
found that traditional methods like working in copper
rather than steel vessels and rind ripening in place of foil
ripping proved favourable to low amine content. Ripening time and temperature were also found to have a distinct influence on amine formation [14].

Original

Similar work to that on cheese was carried out with

sausages. Here, too, parameters could be identified by
which the content of biogenic amines formed during fermentation could be reduced.
The work described enables the quality of food products
to be improved with respect to those compounds in food
which might have an adverse influence on health.

EMS and Tryptophan

Tryptophan is produced as sleeping aid and antidepressant, but also for supplementation of food to bilanced
diets.
In 1989 the outbreak of a new disease, eosinophiliamyalgia-syndrome (EMS), was traced back to the intake of L-tryptophan biotechnologically produced by a
company in Japan [15]. A widely accepted hypothesis is
that contaminants of this tryptophan are responsible for
EMS [16]. World-wide more than 1600 people have been
affected by EMS and 38 have died [17].
About 60 contaminants were detected in the Japanese
tryptophan by RP-HPLC. Six of them were associated
with EMS [18]. Three contaminants are characterized as
1,1-ethylidenebistryptophan (EBT), 3-phenyl-aminoalanine (PAA) and 2-(3-indolylmethyl)-tryptophan
(IMT) (Figure 2).
EBT and PAA are most intensively discussed to contribute to the pathogenesis of EMS, although there is so
far no valid animal or in-vitro model that could establish
EMS-like effects [19]. RP-HPLC showed that the
responsible Japanese tryptophan contained a total of
about 2300 mg/kg contaminants.
There are many indications that EBT and PAA and
other compounds are artefacts which are formed during
purification of the synthesized batch material [20]. PAA
can be synthesized from dehydroalanine and aniline
under acidic conditions. EBT is formed from acetaldehyde and tryptophan at low temperatures.
An RP-HPLC method was developed to determine
PAA and EBT in tryptophan with fluorescence and UV
detection [21]. 16 contaminants could be identified in a
single HPLC run. For PAA the limit of detection was
found to be 1 mg PAA/kg tryptophan, for EBT 6 mg
kg-1. By this method, 105 mg kg -1 EBT and 220 mg kg -1
PAA were found in the suspect material whereas in recently produced tryptophan batches from two different
manufacturers no EBT or P A A were detected.
Since 1994 according to the German Pharmacopoeia
DAB 10, the permitted value for EBT in tryptophan is
8 mg kg-1, and the total for all other compounds detectable by RP-HPLC/UV (calculated as acetyltryptophan) is 400 mg kg-1. Since the beginning of 1996, it is
not allowed for EBT and PAA to be formed during
production of the final product if it is to be used in drugs.
Although this investigation mainly deal with tryptophan
in medicine, the work is also important for food analysis
because tryptophan is used for food supplementation.

Chromatographia Vol.45,1997

15

NH2

N,,~ -~COOH
NH2

tryptophan

1,1"-ethylidenebistryptophan(EBT)

3-phenylaminoalanine
(PAA)

Figure 2
Chemical structures of tryptophan, 1,1-ethylidenebistryptophan (EBT) and 3-phenylaminoalanine (PAA).

[~~NH2
Oxlndolylalanlne

oxidationof

pyrro~

COOH
(OIA)
OH

COOH

cOOH

hydroxylation
Tryptophan
oxidative

ofindole

ofindole

(TRP)

Hydroxytryptophan
(OH-TRP)

cleavage~
~_~ L ~ 2

O NH2

N:c o coo" deformylation

(NFKI

N..Fonnylkynurenlne

~COOH
Kynurenlne

(Kyn)

Figure 3
Irradiation products of tryptophan [22]

Furthermore, in the context of biotechnological production, the analytical investigations were of great importance in indicating that the EMS-contributing contaminants are formed during purification and not during
biosynthesis with gene-modified microorganisms.

Investigation of ~,-lrradiated Protein Rich Food

y-Irradiation is used for conservation of food. Irradiated
foods are not allowed to be distributed and/or sold in
Germany. For control, there is a lack of methods to
determine irradiation via protein degraded compounds.
Tyrosin and its degradation products have been used as
irradiation markers and tryptophan has also been investigated for this purpose [22].
As model substances free tryptophan, tryptophan containing tripeptides and the enzyme lysozyme were irradiated in solution.

16

The following compounds were identified by RP-HPLC

with UV and fluorescence detection: N-formylkynurenine (NFK), oxindolylalanine (OIA), and 4-, 5-, 6-, and
7-hydroxytryptophan (OH-TRP) (Figure 3).
Because N-formylkynurenine and 5-hydroxytryptophan
are natural tryptophan metabolites and oxindolylalanine is formed during the normal oxidation of tryptophan, the non-physiological hydroxytryptophan
isomers 4-, 6-, and 7-hydroxytryptophan can be seen as
possible markers for irradiated protein-rich food. This
must be confirmed with y-irradiated food. If successful,
HPLC then can be used as a control method for irradiated foods of high protein content.

Chromatographia Vol.45,1997

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Conclusions
Examples have been given where chromatographic
methods have been successfully applied to the control
and enhancement of food quality. There is no doubt that
advanced chromatographic methods will also be used
for this end in the future.
In modern quality control, analysis is carried out more
and more during production at the different production
steps. The Green light for continuation of production is
only given when various analytical parameters of the intermediates are shown to be within specification. For
this purpose chromatographic methods have to be
adopted to the demands of on-line control. Because
there is not much time for analytical work during production, rapid methods are needed. Further work on
method development seems to be necessary to achieve
this goal.
Time can especially be saved by shortening or even
omitting the work-up procedure before chromatographic analysis. This can, for instance, be achieved by
intelligent coupling of chromatographic separation and
detection methods. There are detection methods available which detect substances to be analysed very selectively so that otherwise interfering substances do not
disturb or at least to a lesser extent. In this case these
substances need not to be separated before chromatographic analysis, i. e. work-up can be shortened or
omitted.

HPLC-IMER (ImmobilizedEnzyme Reactor)

One example of such an intelligent coupling of chromatography and detection is coupling the chromatograph
with a biosensor or, more specifically, with an enzyme
reactor [23]. H P L C - I M E R stands for coupling of H P L C
with an Immobilized Enzyme Reactor. The applied enzymes are in most cases oxidases. They are compound or
group specific, e.g. alcohol oxidase. Hydrogen peroxide
is formed by the oxidase. This is electrochemically
detected with a platinum cell according to H202 -4 02 +
2H + 2e.
With H P L C - I M E R , the high separation power of H P L C
is combined with the specificity of the enzymes and the
sensitivity of electrochemical detection. Due to the specificity of the enzyme, work-up is simplified very much.
In case of the analysis of beer, work-up is reduced to
merely dilution of the sample. Known examples of the

Original

application of H P L C - I M E R are the analysis of natural

acids like oxalic acid in fruit, amino acids in juices, and
sugar and oligosaccharides in beer.
It is important to point out that modern stepwise
production control also results in an enhancement of
food quality.

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Chromatographia Vol. 45,1997

Received: Sep 16, 1996

Accepted: Oct 9, 1996

17