1. Mikrochimica Acta [Wien] 1981I, 241--248
9 by Springer-Verlag 1981
Chemical Institute, Faculty of Sciences, University of Belgrade
A Kinetic Determination of Ultramicro Quantities of
Serotonin, 5-Hydroxyindolacetic Acid, L-Dopa, Methyl-
dopa and Carbidopa*
By
G. A. Milovanovid, M. A. Sekheta and T. J. Janjid
With 1 Figure
(Received October 1, 1980)
In a previous paper 1 we noted that in the presence of molyb-
denum(VI) and hydrogen peroxide, gallic acid and rutin in carbonate
buffer solution (pH=8.2) form coloured unstable compounds. Fur-
ther investigations have shown that certain other biologically active
substances, i. e., serotonin, 5-hydroxyindolacetic acid, L-dopa, meth-
yldopa and carbidopa, behave in a similar way under the same
conditions. In view of the fact that a linear dependence between
the concentration of the organic substance investigated and the rate
of formation of the coloured product has been found, a kinetic
method was sought for the determination of these physiologically
important compounds.
Since an increased amount of serotonin, which is formed in the
body as a tryptophan metabolite, may indicate the presence of car-
cinoid tumors, an understanding of its levels as well as a knowledge
of the content of its metabolite, 5-hydroxyindolacetic acid, in urine,
would be of great importance for diagnosis. Further, L-dopa, in
combination with carbidopa, which plays the role of decarboxylase
inhibitor, is applied in the treatment of Parkinson's disease, and
methyldopa is used as an antihypertensive agent. The content of
* Presented at the 8th International Microchemical Symposium, Graz,
August 25--30, 1980.
16"
0026-3672/81/8101/0241/$ 01.60

2. 242 G.A. Milovanovld et al.:
these substanses in biological fluids is therefore also of special im-
portance. Analysis of the related drugs is indispensible for control
of their purity.
Of the above-mentioned substances, L-dopa has been determined
kinetically in admixture with adrenaline 2 in concentrations of up
to 5 x 10 -6 M and in admixture with adrenaline and noradrenaline
in the concentration range from 1 • 10 -~ to 1 x 10 -4 M by the appli-
cation of a simultaneous kinetic analysis 3. In those investigations,
measurements were made with a stopped-flow spectrophotometer.
Experimental
The reaction rate was followed photometrically with Zeiss Specol, at
a wavelength of 350 nm, in 5-cm cells. The absorbance was measured
every 30 sec in the first 5 min of the reaction.
All solutions were prepared from reagent-grade chemicals and redistil-
led water. The standard ammonium molybdate solution was 0.001 M rela-
tive to molybdenum(VI), and was diluted to the required concentration
with 0.001M potassium nitrate. A 9.8% solution of hydrogen peroxide
was used and the solutions of serotonin-creatinine sulphate (Fluka), 5-hy-
droxyindolacetic acid (Fluka), L-dopa (MSD), methyldopa (Pharmaco-
chemical Works, Budapest) and carbidopa (MSD) were all 0.001M. All
the organic substances investigated were dissolved in 0.001 M hydrochloric
acid. Carbonate buffer solution was made by mixing 1 M sodium bicar-
bonate and 1 M sodium carbonate. A Radiometer PHM 4 C was used for
pH measurements.
The reaction was carried out in a special vessel with three compart-
ments; a known volume of hydrogen peroxide was measured into one,
known volumes of molybdate solution and analyte solution were placed
in the second, and enough buffer and water to make a total of 25 ml in
the third compartment of the vessel. The cell was brought to 25_+0.1o C in
a thermostat and the reaction was started by mixing all the solutions in
the vessel.
All glassware was washed with 6 M hydrochloric acid at 60o C and
then rinsed successively with tap, distilled and redistilled water.
Results and Discussion
Kinetic investigations have shown that in all the reactions
studied, except in the reaction with serotonin, the reaction rate
increases linearly with increasing concentration of organic substance
and molybdenum, respectively, up to 1 : 1 mole ratio. On this basis
it might be concluded that the rate of the formation of the yellow
reaction product is dependent on formation of a 1 : 1 complex be-
tween molybdenum and the substance investigated. In the reaction

3. A Kinetic Determination of Ultramicro Quantities of Serotonin 243
with serotonin this dependence is linear for mole ratios of serotonin
to molybdenum up to 1 : 6. It has been established that the rate of
all the reactions studied increases linearly with increase of hydrogen
peroxide concentration up to a certain value above which it remains
constant.
On the basis of all the kinetic data obtained the following ex-
pressions are proposed for the formation of the yellow product be-
tween hydrogen peroxide, molybdenum(VI) and the organic sub-
stances:
dx
dt =hi [H202] [Mo~-Serot] [buffer] [HsO+] ~ (a)
(Serot = serotonin creatinine sulphate)
ht = 27.9 + 0.8 mole 2.1.1~.1. min-1
dx
dt -ha [H202] [Mo-HIAA] [buffer] [H30+] ~ (b)
(HIAA = 5-hydroxyindolacetic acid)
h2 -- 31.9 + 2.9 mole -~-1 912 9min -1
dx
dt =ha [H202] [Mo-dopa] [buffer]
ha = (1.5 + 0.2) x 102 mole -2 912-1- min -1
(c)
dx
dt --h4 [H202] [Mo-methyldopa] [buffer] (d)
h4 = (2.9 _+0.3) x 102 mole -2 9129min-1
dx
dt =h5 [H202] [Mo-carbidopa] [buffer]
ha = (3.7 + 0.3) x 109' mole -2 912. min -1
(e)
These kinetic expressions are valid for hydrogen peroxide con-
centrations up to 2.7 x 10 -2 M [for reaction (a)], 3.9 x 10 -2 M [for
reaction (b)] and i x 10-2M [for reactions (c), (d) and (e)]; carbonate
buffer concentrations ranging from 4 x 10 -2 to 4 x 10 -1 M, pH range
8.5--10.5 [for reactions (a), (b) and (c)] and pH 9.8 [for reactions
(d) and (e)].
Since it was found that the reactions can be applied as indicator
reactions, these biologically active substances were determined under
the optimum experimental conditions obtained from the kinetic data.
For all determinations the differential form of the tangent method
was used.

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Fig.1.Calibrationgraphsforthedeterminationofserotonin(a),5-hydroxyindolaceticacid(b),L-dopa(c),methyldopa(d),and
carbidopa(e).Initialconcentrations:carbonatebuffer4x10-1M;hydrogenperoxide4x10.2M(a,b),1x10-eM(c,d,e);molyb-
denum24x10-5M(a),8x10.5M(b),2x10-5M(c,e),1x10.5M(d);pH8.8(a,b),9.8(c,d,e)
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5. A Kinetic Determination of Ultramicro Quantities of Serotonin 245
Kinetic Determination of Serotonin and 5-Hydroxyindolacetic Acid
Serotonin and 5-hydroxyindolacetic acid were determined under
the following optimum experimental conditions: hydrogen peroxide
4 x 10 -2 M, carbonate buffer 4 x 10 -1 M (pH 8.8), molybdenum
24 x 10 -5 M (for serotonin determination) and 8 x 10-5 M (for 5-hy-
droxyindolacetic acid determination). Calibration graphs for these
determinations are given in Fig. 1 (a and b), and the results obtained
are shown in Table I. From Table I it may be seen that serotonin
Table I. A Kinetic Determination of Some Biologically Active Substances
(5 determinations)
Compound Taken Found Relative standard
(/~g/ml) (/~g/ml) deviation (%)
Serotonin 0.70 0.81 + 0.04 5.2
2.80 2.87+0.09 3.2
5.60 5.75+ 0.14 2.5
5-Hydroxyindol- 1.15 1.12+ 0.04 3.6
acetic acid 11.6 11.3_+0.15 1.3
17.4 17.7_+0.11 0.6
L-Dopa 0.39 0.38• 0.01 2.6
1.57 1.62• 0.06 3.7
3.15 3.08_+0.09 2.9
Methyldopa 0.40 0.40+_0.02 4.2
1.00 0.97• 0.02 2.0
1.60 1.51_+0.02 1.4
Carbidopa 0.90 0.98_ 0.02 2.2
2.26 2.37• 0.14 6.1
3.62 3.55_+0.13 3.7
concentrations were determined in the range 0.70--5.60 #g/ml with
a relative standard deviation of up to 5.2%, while 5-hydroxyindol-
acetic acid concentrations ranging from 1.15 to 17.4#g/ml were
determined, with a relative standard deviation of up to 3.6%.
Finally, a study was made of possible interference by some of
the substances most often found in biological fluids. Results presented
in Table II show that most of those investigated have no effect
even when present at concentrations considerably greater than those
of the compounds to be determined. Adrenaline interferes when
present in a 1 : 1 ratio to the analyte. Most metal ions studied did
not exhibit any effects at 10 : 1 ratio to analyte, except cadmium,
cobalt(II), iron(III), copper(II) and lead in serotonin determination.

6. 246 G.A. Milovanovid et al.:
Kinetic Determination o[ L-Dopa, Methyldopa and Carbidopa
L-dopa and its derivatives were determined under the follow-
ing optimum experimental conditions: hydrogen peroxide i x 10-2M,
carbonate buffer 4 x 10 -1 M (pH 9.8), molybdenum 2 x 10 -5 M for
Table II. Tolerance Ratio for Foreign Substances in Determination of 4 x 10-5 M
Serotonin and 5-Hydroxyindolacetic Acid and 1 x 10-5 M L-Dopa, Methyldopa
and Carbidopa
Foreign substance Sero- 5-Hydroxyindol- L-Dopa Methyl- Carbi-
tonin acetic acid dopa dopa
Urea ............. 50 5 10 50 50
Creatine .......... 500 500 500 500 500
Creatinine ........ 50 100 50 50 50
Glucose .......... 500 500 1000 500 500
Glycine .......... 500 500 1000 500 500
Ascorbic acid ..... 100 5 5 5 5
Salycilate ......... 500 100 1000 500 100
Acetate ........... 1000 100 500 500 500
Phosphate ........ 1000 1000 1000 1000 1000
Chloride .......... 1000 1000 1000 1000 1000
Nitrate ........... 1000 1000 1000 1000 1000
Formic acid ....... 1000 100 100 50 100
Tartaric acid ...... 10 10 100 100 50
Lactic acid ....... 5 10 500 50 100
Malic acid ........ 50 10 500 100 500
Succinic acid ...... 1000 1000 100 1000 1000
Citric acid ........ 50 10 1000 100 100
Adrenaline ........ -- -- 1 1 1
Serotonin ......... -- -- 5 5 5
5-Hydroxyindol-
acetic acid ...... -- -- 5 5 5
Calcium .......... 50 50 5 5 500
Magnesium ....... 100 100 500 500 500
Copper(II) ........ 5 10 5 5 5
Cadmium ......... 5 10 100 50 10
Cobalt(II) ......... 5 10 10 5 5
Mercury(II) ....... 10 10 5 5 10
Iron(III) .......... 5 10 5 10 5
Zinc ............. 10 10 100 50 50
Lead(II) .......... 5 10 5 10 10
L-dopa and carbidopa determinations and i x 10 -5 M for methyl-
dopa determination. Calibration graphs for these determinations are
given in Fig. 1 (c, d and e). Results of these determinations are

7. A KineticDeterminationof UltramicroQuantitiesof Serotonin 247
presented in Table I. It may be seen that L-dopa concentrations
were determined in the range 0.39--3.15 ffg/ml, relative standard
deviation 3.7%; methyldopa concentrations were determined in the
range 0.40--1.60 #g/ml, relative standard deviation 4.2%, and carbi-
dopa in the range 0.90--3.62 #g/ml, relative deviation 6.1%.
The investigation of interferences showed that most of the sub-
stances investigated are without effect even at concentrations con-
siderably higher than that of the compound to be determined
(Table II). Adrenaline does not interfere when present in 1 : 1 ratio
to analyte, while serotonin and 5-hydroxyindolacetic acid exhibit
no effect at 5 : 1 ratio to analyte. Table II also shows that some
of the metal ions investigated interfere when present in more than
5 : 1 ratio to analyte.
On the basis of the results obtained it may be concluded that
the proposed very sensitive and simple kinetic method can be suc-
cessfully applied to the determination of ultramicro quantities of
the substances investigated.
The kinetic investigations show that the indicator reactions in-
volved are extremely complex in view of the fact that in addition
to hydrogen peroxide, molybdenum, buffer and the organic com-
pounds, pH also has an effect on the reaction.
Acknowledgements
The authors are grateful to the Serbian Republic Research Fund
for financial support.
Summary
A Kinetic Determination of Ultramicro Quantities of Serotonin,
5-Hydroxyindolacetic Acid, L-Dopa, Methyldopa and Carbidopa
A kinetic method is presented for the determination of the bio-
logically important substances serotonin, 5-hydroxyindolacetic acid,
L-dopa, methyldopa and carbidopa. The method is based on the
reaction between hydrogen peroxide, molybdenum(VI) and the sub-
stance to be determined, in carbonate buffer solution, the reaction
rate being followed photometrically. In order to find optimum
experimental conditions for these determinations the kinetics of the
reactions were examined. The substances investigated were deter-
mined at concentrations ranging from 0.40 to 17.4 #g/ml, with
relative standard deviations ranging from 0.6 to 6.1%. The effect
of some foreign species usually present in biological material has
also been examined.

8. 248 G.A. Milovanovid et al.: Determination of Ultramicro Quantities
Zusammenfassung
Kinetische Bestimmung von Uhramikromengen Serotonin, 5-Hydroxyindol-
essigsh'ure, L-Dopa, Methyldopa und Carbidopa
Eine kinetische Methode zur Bestimmung der biologisch wichtigen
Substanzen Serotonin, 5-Hydroxyindolessigs~iure, L-Dopa, Methyldopa
und Carbidopa wurde vorgeschlagen. Sie beruht auf der Reaktion zwischen
Wasserstoffperoxid, Molybd~in(VI) und der zu bestimmenden Substanz in
carbonat-gepufferter LSsung. Das Reaktionsergebnis wird photometrisch
gemessen. Um optimale Reaktionsbedingungen zu ermitteln, wurden die
kinetischen Daten gepriift. Die genannten Substanzen wurden in Konzen-
trationen zwischen 0,40 und 17,4#g/ml bestimmt, wobei relative Standard-
abweichungen zwischen 0,6 und 6,1% auftraten. Die Wirkung mancher,
in biologischem Material iiblicherwiese anwesender Fremdsubstanzen
wurde geprfift.
References
1 M. A. F. Sekheta and G.A. Milovanovid, Glasnik Hem. Drugtva
Beograd 45, 41 (1980).
2 E. M. Pelizzetti, E. Pramauro, and G. Giraudi, Analyt. Chim. Acta
85, 161 (1976).
3 G. M. Rider and D. W. Margerum, Analyt. Chemistry 49, 2090 (1977).
Correspondence and reprints: Docent Dr. Gordana Milovanovid, In-
stitute of Chemistry, Faculty of Sciences, University of Belgrade, Stu-
dentski trg 16, P. O. B. 550, YU-11001 Belgrade, Yugoslavia.