Voltammetric Oxidation of Drugs of Abuse I. Morphine and Metabolites

CORE Metadata, citation and similar papers at core.ac.uk Provided by Estudo Geral 1419 Full Paper Voltammetric Oxidation of Drugs of Abuse I. Morphine and Metabolites J. M. P. J. Garrido,a C. Delerue-Matos,a F. Borges,b T. R. A. Macedo,c A. M. Oliveira-Brett*d a CEQUP/Dep. Engenharia QuÌmica, Instituto Superior de Engenharia do Porto, Rua S. Tome¬, 4200-485 Porto, Portugal b CEQOFFUP/Dep. QuÌmica Orga√nica, Faculdade de Farma¬cia, Universidade do Porto, 4050-047 Porto, Portugal c Faculdade de Medicina, Universidade de Coimbra, 3004-535 Coimbra, Portugal d Dep. QuÌmica, Faculdade de Cie√ncias e Tecnologia, Universidade de Coimbra, 3004-535 Coimbra, Portugal *e-mail: brett@ ci.uc.pt Received: July 21, 2003 Final version: November 13, 2003 Abstract A detailed study of the electrochemical oxidative behavior of morphine in aqueous solution is reported. Through the synthesis of several metabolites and derivatives, pseudomorphine, morphine N-oxide, normorphine, dihydromorphine and 2-(N,N-dimethylaminomethyl)morphine, and their voltammetric study it was possible to identify the oxidation peaks for morphine. The anodic waves are related with the oxidation of phenolic and tertiary amine groups. It is also possible to verify that a poorly defined peak observable during morphine oxidation is not a consequence of further oxidation of pseudomorphine but due to formation of a dimer during phenolic group oxidation. The results obtained and especially those regarding the formation of a new polymer based on a CÀO coupling could be useful for clarifying the discoloration phenomenon occurring during storage of morphine solutions as well as leading to a better understanding of its oxidative metabolic pathways. Keywords: Morphine, Pseudomorphine, Morphine N-oxide, Normorphine, Dihydromorphine, 2-(N,N- Dimethylaminomethyl)morphine, Oxidation, Voltammetry, Drugs of abuse 1. Introduction Among the commonly encountered types of drugs of abuse, some of the most important are opium alkaloid drugs The use of medicinals by humans is as old as the human race and their derivatives. Commonly used as therapeutic agents, itself, since the need to find measures to combat sickness was a number of these compounds are also frequently abused as as important as the need for food and shelter. However, illicit drugs. The most abundant and economically important some of these ancient drugs are also among those that pose opium alkaloid is morphine (Fig. 1). The stability of serious problems in our contemporary culture through their morphine in aqueous solution has been extensively inves- extensive use for nonmedical purposes. Since these abused tigated [2]. It is generally accepted that several factors substances could turn into a substantial risk to the user and including oxygen from the air, sunlight and some organic to those with whom he or she may interact, extensive impurities can catalyze the degradation of morphine. It is controls have been placed upon the distribution, possession already known that morphine degrades by oxidation and and use of these substances. In addition, detailed chemical that the most important products are pseudomorphine and and metabolic studies, of a natural or synthetic narcotic, morphine N-oxide [2]. were found to be crucial for both preventive and repressive The oxidative electroactivity of morphine has been purposes. studied by voltammetry using platinum, graphite and glassy A common aspect of drug metabolism is the existence of carbon electrodes [3 ± 5]. It has been determined that the one or more oxidative pathways that convert the native phenolic group present is responsible for its electroactivity. molecular form of the compound into reactive species The existence of some gaps in knowledge regarding the capable of chemically modifying vital cellular constituents oxidative behavior of morphine prompted the detailed [1]. The knowledge of these intermediate metabolites and study described in this paper. In order to identify the the investigation of their reactivity is therefore very oxidation processes and the products formed, several important. morphine derivatives were synthesized and their electro- In this context, electrochemical techniques could play a chemical behavior studied, pseudomorphine, morphine N- very important part since besides allowing the detection of oxide, normorphine and 2-(N,N-dimethylaminomethyl)- low levels of drugs and metabolites in biological fluids morphine (Fig. 1). such as plasma and urine they could also be used for in vitro studies of the metabolic pathways of these compounds. Electroanalysis 2004, 16, No. 17 ¹ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/elan.200302966 1420 J. M. P. J. Garrido et al. 2.2. Other Conditions Thin-layer chromatography (TLC) was performed on pre- coated silica gel 60 F254 and cellulose plates (E. Merck). The layer thickness was 0.2 and 0.1 mm, respectively. The following chromatographic systems were used: n-butanol/ water/acetic acid (4:1:1), water/methanol/acetic acid (8:2:0.1), acetone/water/ammonia (8:1.5:0.5), chloroform/ methanol/diethylamine (8:1.5:0.5), carbon tetrachloride/ butanol/methanol/ammonia (4:3:3:0.3), chloroform/methanol (9:1), dichloromethane/diethylamine (9:1). The spots were visualized under UV detection (254 and 366 nm) and iodine vapor. Purification of the compounds was performed by column chromatography using Merck silica gel 60 (0.2 ± 0.5 and 0.040 ± 0.063 mm, E. Merck) or aluminium oxide, activated basic, Brockmann I (ca. 150 mesh, 58 ä, Aldrich). Solvents were evaporated in a B¸chi Rotavapor. 2.3. Reagents and Solutions Morphine hydrochloride was obtained from Uquipa (Lis- bon, Portugal) and was used without further purification. Dihydromorphine hydrochloride was kindly donated by the United Nations Drug Control Program (Vienna, Austria). Reagents used in the synthetic procedures were obtained from Sigma-Aldrich Quimica (Sintra, Portugal). All other reagents and solvents were pro analysis grade and purchased Fig. 1. Structural formulae of a) morphine, b) pseudomorphine, from Merck (Lisbon, Portugal). m-Chloroperbenzoic acid c) morphine N-oxide, d) normorphine, e) dihydromorphine and f) (free of m-chlorobenzoic acid) was purified according the 2-(N,N-dimethylaminomethyl)morphine. procedure of Bortolini et al. [6]. Deionized water with conductivity less than 0.1 mScmÀ1 was used throughout. Buffer solutions employed were 0.2 M in the pH range 1.2 ± 2. Experimental 12.2 [7]. 2.1. Apparatus 2.3.1. Synthesis of Pseudomorphine (2,2'-Bimorphine; Oxydimorphine) Voltammetric measurements were performed using an Autolab PGSTAT 10 potentiostat/galvanostat (EcoChimie, The synthetic procedure was adapted from the literature Netherlands) and a one-compartment glass 663 VA Met- [8 ± 10]. rohm cell with a three-electrode configuration (Metrohm). Morphine hydrochloride (1.0 g) was dissolved, with heat- The electrodes used were a glassy carbon working electrode ing, in 200 mL of 20% potassium hydroxide. After cooling, with a diameter of 2 mm (Metrohm), a glassy carbon rod 40 mL of a 3% potassium ferricyanide solution was slowly counter electrode (Metrohm) and an Ag/AgCl (3 M KCl) added dropwise. The reaction was stirred during 30 min. and reference electrode (Metrohm). The working electrode was refrigerated overnight. The solid obtained was filtered and polished with alumina (BDH) on a microcloth pad and washed with warm methanol. In TLC analysis pseudomor- rinsed with water before use. phine appears as a fluorescent spot [11 ± 14]. A Metrohm E-520 pH-meter and a Metrohm glass Due to the low solubility of the compound in water, the electrode were used for pH measurements. Melting points sulfate salt was prepared by dissolving the free base in warm were measured using a Kˆfler microscope (Reichert Ther- glacial acetic acid and adding sulfuric acid until the movar). Infrared spectra were recorded on an ATI Mattson formation of a precipitate. The product was filtered and Genesis Series FTIR spectrophotometer using potassium recrystallized from water. The compound was filtered, bromide disks (Uvasol, Merck). 1H and 13C NMR (1H thoroughly washed with water, then with ethyl ether and decoupled) data were acquired, at room temperature, on a dried under vacuum. Br¸ker AMX 300 spectrometer operating at 300.13 and The analytical data obtained for pseudomorphine was 75.47 MHz, respectively. Electron impact mass spectra (EI- similar to that found in the literature [10, 11, 13, 15]. MS) were obtained on a VG AutoSpec instrument. Electroanalysis 2004, 16, No. 17 ¹ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Voltammetric Oxidation of Drugs of Abuse I. 1421 2.3.2. Synthesis of Morphine N-oxide (Morphine Oxide) and redissolved in water. After alkalinization with concentrated aqueous ammonia a precipitate was formed, filtered Morphine free base was first prepared in order to be used in and washed with water. The compound has the same the subsequent preparation of morphine N-oxide. Morphine chromatographic behavior as normorphine standard. hydrochloride (1.0 g) was dissolved in 25 mL of water containing a trace of sodium metabisulfite at 108C. One 2.3.4. Synthesis of 2-(N,N-Dimethylaminomethyl)mor- equivalent of concentrated aqueous ammonia was added phine (2-(N,N-dimethylaminomethyl)-4,5-a-epoxy- dropwise, with stirring. The cooling was continued for an 17-metyl-morphin-7-en-3,6-a-di ol) additional 15 min. and during this time a white solid appeared in the solution. The crystals were filtered and This synthesis was adapted from a reported procedure with washed with ice-cold water. slight modifications [22]. Morphine (0.58 g) was dissolved in The synthesis of morphine N-oxide was then carried out 30 mL of anhydrous acetonitrile and N,N-dimethylmethy- using two different procedures adapted from the literature: leneiminium chloride (0.4 g) was added to the solution. The mixture was refluxed for 12 hours. After evaporation of the 1) Morphine base (0.6 g) was added to 1.5 mL of an ice- solvent, the remaining residue was purified by flash cooled 30% hydrogen peroxide solution.

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