In view of the medical and social problem caused by the increasing number of drug addicts the determination of opium alka-loids is of special importance. Morphine is known as a highly addictive and potent narcotic but drug users prefer heroin because of its more intense immediate effect. As heroin is hydrolysed in the organism to morphine the knowledge of the morphine content of biological fluids and tissues is indispensable for forensic and therapeutic purposes. The methods recently used for the determination of morphine in urine are gas-liquid chromatography 1, high-pressure liquid chromatography with fluorimetric determination 2 and gas chromatography mass spectrometry 3. In this paper a simple and inexpensive kinetic method is presented , based on the decomposition of the coloured compound formed by the reaction of hydrogen peroxide with cobalt(II) and morphine, in the presence of carbonate buffer. There is a definite concentration range over which the decomposition rate of the compound mentioned is a linear function of the morphine concentration.

1. MikrochimicaActa [Wien] 1984III, 477--483
9 by Springer-Verlag1984
Chemical Institute, Faculty of Sciences, University of Belgrade
Kinetic Determination of Morphine in Urine""
By
G. A. Milovanovic and M. A. Sekheta
With 1Figure
(Received November 27, 1984)
In view of the medical and social problem caused by the in-
creasing number of drug addicts the determination of opium alka-
loids is of special importance.
Morphine is known as a highly addictive and potent narcotic
but drug users prefer heroin because of its more intense immediate
effect. As heroin is hydrolysed in the organism to morphine the
knowledge of the morphine content of biological fluids and tissues
is indispensable for forensic and therapeutic purposes.
The methods recently used for the determination of morphine
in urine are gas-liquid chromatography1, high-pressure liquid chro-
matography with fluorimetric determination2 and gas chromatog-
raphy-mass spectrometry3.
In this paper a simple and inexpensive kinetic method is pre-
sented, based on the decomposition of the coloured compound
formed by the reaction of hydrogen peroxide with cobalt(II) and
morphine, in the presence of carbonate buffer. There is a definite
concentration range over which the decomposition rate of the com-
pound mentioned is a linear function of the morphine concentration.
* Presented at the first International Symposium on Kinetics in Ana-
lytical Chemistry, C6rdoba, September 27--30, 1983.
31 Mikrochim. Acta 1984 1II/5-6

2. 478 G.A. Milovanovicand M. A.Sekheta:
Experimental
Apparatus
The reaction rate was followed photometrically with a Zeiss Specol by
measuring the solution absorbance at 350 nm in a 5-cm cell, every 15 sec
during the first 3 rain of the reaction. The temperature was kept constant
with an "Ultrathermostat nach HSppler", type NBE (VEB Prfifger~ite-
Werk, Medingen). The pH-measurements were performed by means of a
Radiometer 4C pH-meter.
Reagents
Analytical grade chemicals and redistilled water were used. Concen-
trations of the stock solutions were: hydrogen peroxide (Merck), 9.8 M;
cobalt(II) sulphate (Merck), 1• 10-~ M; morphine hydrochloride (Alkaloid),
1x 10-2 M. Carbonate buffer solution was made by mixing 1M sodium
bicarbonate and 1M sodium carbonate (Merck).
Procedure
The reaction was performed in a special vessel with three com-
partments. The solution of cobalt(II) was measured into one com-
partment, buffer and morphine into the second, and hydrogen per-
oxide and enough water to make a total volume of 25 ml into the
third compartment of the vessel. The vessel was brought to
25_+0.1o C in the thermostat and the reaction was started by mix-
ing all the solutions in the vessel.
Extraction of Free Morphine from Urine Samples
The urine sample (25 ml) was extracted with chloroform-iso-
propyl alcohol (95 : 5) mixture at pH 8.9 (carbonate buffer). After
extraction the mixture was centrifuged and the organic layer was
separated and evaporated. The residue was dissolved in 5 ml of
redistilled water, and analysed for morphine as just described.
Results and Discussion
To establish the optimum conditions for the determination of
morphine, the kinetics of the decomposition of the unstable product
formed between hydrogen peroxide, cobalt(II) and morphine has
been studied.
It was found that the rate of decomposition increases with in-
creasing concentration of morphine and of cobalt(II), and is maximal
when the two components are in 1 : 1 molar ratio; on this basis

3. Kinetic Determination of Morphine in Urine 479
it was concluded that the decomposition rate depends on the cata-
lytically active 1:1 complex which cobalt forms with morphine.
From the results obtained the kinetic equation of the reaction has
been postulated and the apparent rate constant calculated:
da
dt = ~ [Co-morphine] [H20211/4
/~= (0.51 • 0.03) mole-1/4.11/4. min-1.
This kinetic equation is valid for hydrogen peroxide concentra-
tions ranging from 1.2 x 10-~ M to 19.6 x 10-3 M, buffer concentra-
tions ranging from 1.6 x 10-2 M to 5.6 x 10-2 M, and pH=8.9;
T=25 +_0.10C.
From the rate of the reaction at various temperatures in the
range 20--35 o C the activation energy and thermodynamic param-
eters have been calculated and are given in Table L The activation
Table I. The Activation Energy and Thermodynamic Parameters for the Decom-
position of the Compound Formed Between Hydrogen Peroxide, Cobalt(II) and
Morphine
E A H ++ AS ++ A G ++ pK++
(kJ/mole) (kJ/mole) (J.K-l.mole-i) (kJ/mole)
37.8 35.3 - 131 74.5 13.1
energy is lower than that for the decomposition of hydrogen per-
oxide by inorganic ions, which is about 3 46 kJ/mole. It may be con-
cluded that a catalytically active complex, with catalyse-like action
in the decomposition of hydrogen peroxide, is probably formed
between cobalt(II) and morphine.
Table II. Kinetic Determination of Morphine (5 Determinations)
No. Taken Found Relative
standard
(~g/mI) (#g/ml) deviation (%)
5 1.50 1.66 + 0.13 7.8
5 7.50 7.16 __+0.17 2.4
5 12.3 12.07__+0.36 3.0
From the kinetic investigations the optimum conditions for the
determination of morphine have been established: hydrogen per=
oxide, 19.6 x 10 '3 M; carbonate buffer, 4.0 x 10-2 M (pH 8.9),
31"

4. 480 G.A. Milovanovic and M. A. Sekheta:
cobalt(II) sulphate, 4.0 x 10 .5 M. Determination of morphine by
the differential tangent method gives a calibration graph of the
type shown in Fig. 1. The results obtained are given in Table II.
I /t~. j
2
88 "'13" I'2 lg 20 Nor],pg/mt~
0 I 2 3 4 5 [Mor], M- 105
Fig. 1. Calibration graph for the determination of morphine
Initial concentrations: hydrogen peroxide, 19.6x10-aM; carbonate buffer
(pH 8.9), 4.0 x 10-~ M; cobalt(II) sulphate, 4.0 x 10-5 M
Morphine was determined in concentrations ranging from 1.5 to
12.3/~g/ml, with relative standard deviation up to 7.8%.
A study was made of possible interference by the related com-
pounds of the opium alkaloid group as well as by some medical
Table III. Tolerance Ratio for Foreign Substances in the Determination of
Morphine (2 x 10-~ M)
Narcotine interferes
Codeine 1 : I
Thebaine 5 : 1
Dionin 5 : 1
Papaverine 1 : 1
Methadone 10 : 1
Benzoctamine 10 : 1
Valium 10 : 1
Lasdol 2 : 1
drugs. Results presented in Table III show that narcotine interferes
with the determination, whereas papaverine and codeine (when
present at the same concentration as morphine) do not affect the

5. Kinetic Determination of Morphine in Urine 481
accuracy of the determination. Lasdol (lysine-acetylsalicylate +gly-
cine, 9 : 1) does not interfere when present at twice the concentra-
tion of morphine, and thebaine and dionin can be tolerated at
five times the morphine concentration. Other compounds examin-
ed do not interfere even when present at ten times the concentra-
tion of morphine. It may be concluded that other alkaloids of this
group under the same experimental conditions may also form cata-
lytically active complexes with cobalt. Table IV shows how dif-
Table IV. Relative Molar Coefficient of Catalytic Activity, Given as F Values*
of Complexesof Morphine and Its Derivativeswith Cobalt(II)
Compound R1 R2 F value
Morphine - OH - OH 1.000
Codeine - OCH~ - OH 0.065
Dionin - OC2Ha - OH 0.051
Thebaine - OCH~ - OCH3 0.032
* The F value is the quotient of the molar concentrations of a reference
catalyst (in this case the morphine complex) and of the complex considered,
which have the same catalytic activity under identical conditions5.
ferent substituents affect the catalytic activity of the complexes
formed. The catalytic activity decreases on the replacement of the
phenolic hydroxyl group of morphine by a methoxy group in codeine
or an ethoxy group in dionin, and thebaine, which contains two
methoxy groups, exerts the lowest catalytic activity.
Finally, the application of the method to the determination of
morphine in urine has been investigated. The calibration graph was
obtained by spiking blank urine samples with known amounts of
morphine to give concentrations from 0.0 to i5.0 ,ug/ml. It was
found to be linear (-tane=l.808 x 10-9' x Cmo~phin~+0.008) with a
linear regression correlation coefficient of 0.997.
The efficiency of the extraction was checked by extracting
known concentrations of morphine from quintreplicate urine sam-
ples. The mean recovery of morphine was 85%.
The precision and reproducibility were obtained by determining
morphine at three concentration levels (five replicates) (Table V).
The proposed method was also applied to the analysis of five
urine samples from persons suspected of having taken morphine or
heroin. As morphine is present in urine predominantly as the glu-
curonide, for determination of its total morphine content the urine
was hydrolysed before extraction.

6. 482 G.A. Milovanovicand M. A.Sekheta:
To 25 mI of urine 2 ml of concentrated hydrochloric acid were
added and the solution was digested in a boiling water-bath for
30 rain. After cooling, the solution was adjusted to pH 9 with
ammonia solution, then buffered and extracted as described for the
free morphine in urine.
Two of the samples investigated were found to be negative
and were eliminated from further testing.
Table V. ReproducibilityData for Morphine in Urine Samples
(5 Determinations)
Taken Found Relative standard
(,t~g/ml) (#g/ml) deviation (%)
1.50 1.70+0.13 7.6
7.51 8.20+__0.62 7.6
13.53 13.47__+0.57 4.2
In the other samples the free morphine determined ranged from
2.0 to 3.5 #g/ml and the total morphine from 5 to 9#g/ml. The
results obtained were compared with those of HPLC determina-
tions by the method of Jane and Taylorz. The correlation coefficient
of 0.94 for the two sets of data appeared to indicate satisfactory
overall agreement.
If codeine is present it could be expected partly to undergo
O-demethylation to form morphine. However, the main metabolic
pathway is conjugation with glucuronic acid, and free codeine inter-
feres with the determination only when present in concentrations
greater than that of morphine. Other alkaloids, which under the
experimental conditions react like morphine, do not interfere at
low concentrations.
Acknowledgement
The authors are grateful to the Serbian Republic Research Fund
for financial support.
Summary
Kinetic Determination o[ Morphine in Urine
A kinetic method for the determination of morphine in urine is
presented. It is based on the decomposition of the coloured com-
pound formed by the reaction between hydrogen peroxide, cobalt(II)

7. Kinetic Determination of Morphine in Urine 483
and morphine, in alkaline media. The decomposition rate of the
coloured reaction product was followed photometrically. Morphine
was determined in concentrations ranging from 1.5 to 13.5/~g/ml,
and the relative standard deviation was not higher than 8%. The
determination was performed by the differential tangent method.
The effect of related substances from the opium alkaloid group was
also examined. The method determines the free morphine present.
For total morphine the glucuronides have to be hydrolysed by acid
digestion first.
Zusammenfassung
Kinetische Bestimmung yon Morphin in Harn
Die beschriebene Methode beruht auf der Zersetzung des geffirbten
Produktes, das sich aus Morphin, Wasserstoffperoxid und Kobalt(II) in
alkalischer L6sung bildet. Die Geschwindigkeit dieser Zersetzung wurde
photometrisch verfolgt. Morphin wurde im Konzentrationsbereich 1,50--
13,53/~g/ml bestimmt. Die relative Standardabweichung war nicht h6her
als 8%. Die Bestimmung wurde mit der differentiellen Variante der Tan-
gentenmethode ausgefiihrt. Der Einflut~ verwandter Substanzen aus der
Gruppe der Opiumalkaloide wurde untersucht.
References
1 G. R. Nakamura and E. L. Way, Analyt. Chemistry 47, 775 (1975).
2 I. Jane and J. F. Taylor, J. Chromatography 109, 37 (1975).
8 E. J. Cone, C. W. Gorodetzky, S. Y. Yeh, W. D. Darwin, and W. F.
Buchwald, J. Chromatography 230, 57 (1982).
4 N. I. Kobozev, Chosen Works (Russian), Moscow (1978).
5 S. Pantel, Analyt. Chim. Acta 141, 353 (1982).
Correspondence and reprints: Dr. G.A. Milovanovid, Institute of
Chemistry, University of Belgrade, Studentski trg 16, P. O. Box 550, 11001
Belgrade, Yugoslavia.