Tyramine in Cocoa and Derivatives

Abstract

ABSTRACTA method is described for the analysis of tyramine in cocoa and derivatives by sand-column extraction with alkaline ethyl acetate as eluant and later transference to a 0.2N HCl acid phase. Tyramine is determined quantitatively by fluorometry after reaction with -nitrous-β-naphthol. Qualitative confirmation is carried out by spectrofluorometry and thin layer chromatography. Average recovery of the method was 88.7% and relative standard deviation was 4.61%. The method was applied to samples of derivatives of cocoa sold in Spain and to intermediate substances in the industrial processes used in the production of sweetened cocoa powder. The amounts of tyramine found ranged between 0.1 and 2.8 μg/g.

Full text

Tyramine in Cocoa and Derivatives
M. JALON, C. SANTOS-BUELGA, J. C. RIVAS-GONZALO, and A. MARINE-FONT
ABSTRACT
A method is described for the analysis of tyramine in cocoa and
derivatives by sand+olumn extraction with alkaline ethyl acetate
as eluant and later transference to a 0.2N HCl acid phase. Tyramine
is determined quantitatively by fluorometry after reaction with
ar-nitrous&naphthol. Qualitative confirmation is carried out by
spectrofluorometry and thin layer chromatography. Average
recovery of the method was 88.7% and relative standard deviation
was 4.61%. The method was applied to samples of derivatives of
cocoa sold in Spain and to intermediate substances in the indus-
trial processes used in the production of sweetened cocoa powder.
The amounts of tyramine found ranged between 0.1 and 2.8 pg/g.
INTRODUCTION
OF THE MANY INTERACTIONS between ingested foods
and the health of individuals, the one which occurs between
the intake of certain foods and migraine is well known.
Among such foods, the derivatives of cocoa, especially
chocolate, are outstanding and the related migraine has
been attributed to the presence of tyramine in them (Smith
et al., 1970; Rivas-Gonzalo et al., 1978). This has not been
entirely confirmed, however, and other vasoactive amines
have also been implicated, principally serotonin (Ghose
et al., 1978; Hardebo et al., 1978) and phenylethylamine
(Sandler et al., 1974; Gonsalves and Stewart, 1977).
Tyramine in foods is also of importance because it is
able to interact with mono-amineoxidase inhibitor drugs
(IMAO), giving rise to hypertensive crises of variable con-
sequence (Marine-Font, 1978).
Other authors have described the relationship between
the fermentation process undergone by certain foods and
their tyramine content (Rice et al., 1976; Rice and Koehler,
1976). In the particular case of cocoa, fermentation takes
place during the manipulations which the seeds are sub-
jected to in exporter countries (Minifie, 1980). Kenyhercz
and Kissinger (1977) reported greater amounts of tyramine
in fermented seeds (11.5 f 0.1 Erg/g), than in unfermented
seeds (3.9 + 0.1 gg/g), which seems to support this re-
lationship between tyramine and fermentation in cocoa,
too.
The few studies appearing in the literature (Kenyhercz
and Kissinger, 1977; Ingles et al., 1978; Hurst and Toomey,
1981) report amounts of tyramine in cocoa derivatives
ranging from undetectable to 14.6 pg/g; relatively low
when compared with the amounts detected in cheese (un-
detectable to 2170 pg/g) (Rivas-Gonzalo et al., 1978)
or meat derivatives (undetectable to 1500 pg/g) (Santos-
Buelga et al., 1981). These latter have been related to the
appearance of migraine to a much lesser extent, though
they have frequently been implicated in cases of interac-
tion with IMAO drugs (Blackweel and Mabbit, 1965; Rice
et al., 1976).
Authors Jalon. Santos-Buelga, Rivas-Gonzalo. and Marine-Font are
affiliated with the Dept. of Bromatology, Toxicology & Chemical
Analysis, Faculty of Pharmacy, Univ. of Salamanca, Salamanca.
Spain.
This work includes data on the tyramine content in
cocoa derivatives commercialized in Spain as well as data
on intermediate products of the industrial production of
cocoa powder.
Even though moisture is very low in this kind of pro-
duct, the water content was analyzed in all samples,
MATERIALS & METHODS
THE METHOD used was an adaptation of that proposed
by Santos-Buelga et al. (1981) for the analysis of tyramine
in meat products.
Preparation of sample
Two grams of cocoa or derivative were weighed into a
250 ml beaker (when the products are compact it is neces-
sary to freeze and then grate until a granular texture is
achieved). Ten ml borate buffer pH = 10.4 (50 ml of O.lN
solution in boric acid and potassium chloride + 46.5 ml of
O.lN sodium hydroxide) and l-1.2g of sodium carbonate
were added to obtain a pH value of 10.4 + 0.1. The alka-
linized substance was then mixed with 70-8Og fine sand
and 50-60g anhydrous sodium sulphate to obtain a dry
mass.
Extraction and separation
One hundred milliliters of ethyl acetate were added to
the dry mass and left to stand for 30 min, stirring occa-
sionally to avoid aggregation of the sand. The mass was
placed in a 3 cm diameter column provided with a stop-
cock. The beaker was washed with 100 ml ethyl acetate,
which was added to the column. The 200 ml ethyl acetate
were eluted over a period of approximately 2 hr. The eluate
was extracted three times with 8 ml 0.2N HCl in a sep-
aratory funnel. The acid extracts were combined and
adjusted to 25 ml.
Quantitative determination
Two milliliters of the final extract were transferred
into a test tube. One milliliter of the solution of a-nitrous-
fl-naphthol (0.1% [w/v] in 95% ethyl alcohol) and 1 ml of
the nitric solution (1 M nitric acid containing 2% [v/v] so-
dium nitrite at 2.5% [w/v], prepared fresh) were added.
This was mixed well and heated at 60°C for 1 hr, cooled
to room temperature and 10 ml 1,2-dichloroethane added.
The mixture was shaken vigorously for 1 min to extract
the excess cu-nitrous-/3-naphthol. The top layer was re-
covered and used for fluorometric determination of tyra-
mine at 545 nm resulting from activation at 450 nm.
Tyramine concentrations were calculated from a com-
parison of the fluorescence intensities with respect to a
calibration curve, based on varying concentrations of free
tyramine subject to the ar-nitrous-fl-naphthol reaction. A
blank was used to correct native fluorescence of the re-
agents.
Qualitative identification
Spectrofluorometry.
Activation and emission spectra
Volume 48 (1983kJOURNAL OF FOOD SCIENCE-545

TYRAMINE IN COCOA.. . .
were determined to ensure that they coincided with those
of the fluorophore formed from standard tyramine.
Thin-layer chromatography.
The method used was that
of Rivas-Gonzalo et al. (1979). Specifications were: The
final acid phase was brought to dryness in a rotovapor at
4O’C. The residue was redissolved in 1 ml 0.2N HCl, and
20 ~1 were spotted onto TLC plates.
- Support: Cellulose MN-300.
- Eluent: Butanol/Acetic acid/Water (12:5 :3).
- Color development: 0.1% ol-nitrous&naphthol in 95%
ethyl alcohol t 3M nitric acid containing 0.05% so-
dium nitrite.
Determination of moisture
The extract was dried at 100-l 05OC (Casares, 1967).
RESULTS & DISCUSSION
THE METHOD EMPLOYED was subjected to recovery and
accuracy assays before being applied to the sample. Re-
covery assays were carried out by adding different amounts
of standard tyramine (1, 2, 5, 10, and 15 pg/g) to a sample
of sweetened cocoa powder. The method was applied five
times for each of the amounts shown, simultaneously carry-
ing out an assay with a sample of the same sweetened cocoa
powder to which no tyramine had been added in order to
Table 1-Tyramine content (referring to overall product) and mois-
ture of Spanish products derived from cocoa
Sample Tyramine (@g/g1 Moisture (%I
Dark chocolate
a
b
c
d
i
Drinking chocolatea
a
b
C
d
e
f
9
h
Milk chocolate
a
b
c
d
e
f
Substitute chocolate
a
b
C
d
Sweetened cocoa powder
a
b
C
d
i
Chocolate aranules
0.5 2.4
0.5 1.2
0.7 1.4
0.7 1.6
0.9 4.1
1.1 2.3
0.2
0.4
0.5
0.5
0.6
0.7
0.8
0.8
0.1 1.7
0.2 1.6
0.3 2.0
0.3 1.6
0.4 1.2
0.5 1.5
0.1 2.6
0.1 2.4
0.5 2.0
0.6 2.0
0.3
0.4
0.4
0.7
0.8
0.8
2.7
2.5
2.8
1.0
2.8
2.2
1.3
1.3
3.6
2.3
7.8
1.4
4.4
1.8
a 0.4 1.6
a “Drinking chocolate” IS Plain or milk chocolate, in bar form, to
which cereal flour has been added to a maximum of 15%, for grat-
ing and heating before CoITIsumPtiorI.
correct, in each case, the amount of.the amine present in
the sample. Average recovery values ranged between 83.7%
and 93.8% overall recovery being 88.7%.
To test accuracy, nine succesive analyses for tyramine
content were carried out on a sample of sweetened cocoa
powder. Relative standard deviation was 4.61%.
The method employed was then used to analyze two
main groups of samples:
(a) Samples of different cocoa derivatives purchased
from food stores, including dark chocolate, drinking choc-
olate, milk chocolate, chocolate substitute, sweetened
cocoa powder and chocolate granules (see Table 1). The
highest tyramine contents were found in dark chocolate
(average content 0.7 pg/g) and the lowest in milk chocolate
and chocolate substitute (average content 0.3 pg/g), which
is logical since these latter have a lower proportion of co-
coa. Sweetened cocoa powder and drinking chocolate have
intermediate values (average content 0.6 pg/g). Though
references to the tyramine content in this kind of product
is scarce, our findings are in general lower than those re-
ported by other workers. Hurst and Toomey (1981) found
that for milk chocolate levels ranged between 3.76 and
12.02 pg/g and Kenyhercz and Kissinger (1977) found
8.3 pg/g for sweetened cocoa powder. It should be noted
that we found tyramine in all the samples studied, in dis-
agreement with the findings of Ingles et al. (1978), who
found none in dark chocolate, milk chocolate and white
milk chocolate. They did, however, report levels of 2 pg/g
in drinking chocolate.
(b) Samples obtained during the industrial manufactur-
ing processes of sweetened cocoa powder. Figure 1 out-
lines the principal technological processes and intermediate
products together with the tyramine content at each stage.
Tyramine content does not seem to be markedly affec-
ted by the techniques involved and the small alterations it
does undergo could be explained by the separation of frac-
tions and the addition of other products. It may be seen
that tyramine is already present in the original product
Fig. 1 -Tyramine con tent (referring to overall product) and moisture
of intermediate products in the process of making cocoa pow&r.
,546-JOURNAL OF FOOD SCIENCE- Volume 48 (7983)

(the fermented and roasted cocoa bean) and it must there-
fore have first appeared earlier, possibly during fermenta-
tion. This is plausible since the formation of tyramine is
always associated with a fermentative or maturation proc-
ess; furthermore, this is in agreement with the findings of
Kenyhercz and Kissinger (1977) who reported that fer-
mented beans had a greater tyramine content (11.05 +
0.1 pg/g) than unfermented beans (3.9 f 0.1 pg/g). Hurst
and Toomey (1981), reported great variations in the tyra-
mine content of cocoa liquor of different origins (0.73-
14.6 pg/g). Our own results (1.9 pg/g) are of the same order
as the lowest values reported by other workers.
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MS received 4/19/82’; revised 10/12/82; accepted 10/15/82.
The authors express their gratitude to S.A. Nutrexpa, Barcelona
(Spain) for the samples supplied.
This work was made possible by grant from the “Comisioh
Asesora de Investigacioh Cientifica y Te’cnica de1 Ministerlo de
Education y Ciencia”.
Translation by Nick Skinner, B.A.
BROWNING KINETICS OF ASPARTAME. . . From page 544
by Warmbier et al. (1976). In addition, the QIo’s are listed
with glucose/aspartame having the higher Qro of 2.38
as compared to the glucose/glycine system which gave a
Qre of 1.89. Thus the aspartame system is more sensitive to
temperature change at higher temperature levels.
From data at accelerated temperature, one can predict
what the shelf life of a particular product will be at a lower
temperature (Labuza, 1982). The predicted shelf life at
45’C for the aspartame/glucose system gives a value of 62
days. Fig. 3 shows that the actual time to an O.D. of 0.1
for the aspartame/glucose system held at 45’C was 60 days,
indicating that the Arrhenius relationship holds very well
in this model system. Whether this would be true in a real
food is not known. The accelerated storage data presented
here can also be applied to processed food products such as
reduced calorie syrups and soft drinks which are sterilized
in boiling water, in predicting the browning that would
occur with aspartame as part of the formulation. We are
currently investigating the kinetics of the loss of sweeten-
ing power during the Maillard reaction. This work will be
published at a later date.
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MS received g/8/82: revised l/10/82: accepted l/10/83.
This paper is scientific journal series No. 13062 from the Univer-
sity of Minnesota Agric. EXP. Station. This project was supported
in Part by the University of Minnesota Agricultural Experiment
Station Grant No. 18-78.
Mr. Stamp won first prize in undergraduate research paper compe-
tition for this paper at the 32nd Annual Meeting of the Institute of
Food Technologists, Las Vegas, NV, June 22-25, 1983.
Volume 48 (1983hJOlJRNAL OF FOOD SCIENCE-547