Published on October 20, 2016
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.