High-performance liquid chromatography with electrochemical detection applied to the analysis of 3,4-dihydroxymethamphetamine in human plasma and urine

Abstract

Metabolic activation in the disposition of 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”) has been implicated in some of its pharmacological and toxicological effects, with the major metabolite 3,4-dihydroxymethamphetamine (HHMA) as a putative toxicant through the formation of thioether adducts. We describe the first validated method for HHMA determination based on acid hydrolysis of plasma and urine samples, further extraction by a solid-phase strong cation-exchange resin (SCX, benzenesulfonic acid), and analysis of extracts by high-performance liquid chromatography with electrochemical detection. The chromatographic separation was performed in an n-butyl-silane (C₄) column and the mobile phase was a mixture of 0.1 M sodium acetate containing 0.1 M 1-octanesulphonic acid and 4 mM EDTA (pH 3.1) and acetonitrile (82:18, v/v). Compounds were monitored with an electrochemical cell (working potentials 1 and 2, +0.05 and +0.35 V, respectively, gain 60 μA). A mobile phase conditioning cell with a potential set at +0.40 V was connected between the pumping system and the injector. Calibration curves were linear within the working concentration ranges of 50–1000 μg/L for urine and plasma. Limits of detection and quantification were 10.5 and 31.8 μg/L for urine and 9.2 and 28.2 μg/L for plasma. Recoveries for HHMA and DHBA (3,4-dihydroxybenzylamine, internal standard) were close to 50% for both biological matrices. Intermediate precision and inter-day accuracy were within 3.9–6.5% and 7.4–15.3% for urine and 5.0–10.8% and 9.2–13.4% for plasma.

Introduction

There is widespread misuse of 3,4-methylenedioxymethamphetamine (MDMA, commonly called “ecstasy”) among young people, particularly in Europe and the United States. Neurodegeneration of serotonergic pathways of the central nervous system has been postulated as the major neurotoxicological effect after its continued consumption [1]. It seems that some of the pharmacological and toxicological effects of MDMA in humans are related to metabolic activation in the disposition process of the drug [2], [3], [4]. A metabolite of MDMA, 3,4-dihydroxymethamphetamine (HHMA), has been hypothesized as a putative toxicant through the formation of thioether adducts [5], [6]. The presence of HHMA has been demonstrated in in vitro studies and appears as a major metabolite of MDMA in human microsomal preparations [7]. HHMA would result from the O-demethylenation of MDMA, whereas O-methylation of HHMA would result in 4-hydroxy-3-methoxymethamphetamine (HMMA), which appears to be a major metabolite of MDMA in vivo. On the other hand, 3,4-methylenedioxyamphetamine (MDA), an N-demethylated metabolite of MDMA, and 4-hydroxy-3-methoxyamphetamine (HMA), an O-methylated metabolite of 3,4-dihydroxyamphetamine (HHA), have been identified as minor metabolites [8], [9], [10].

Fig. 1 shows a summary of the main metabolic pathways described for MDMA in humans. The analysis of O-methylenedioxy derivatives of amphetamine-related substances in different biological samples has been the subject of a number of publications [9], [11], [12] and there are several excellent reviews on this analytical topic [13], [14]. HHMA, however, has catecholamine-like physicochemical properties and, probably for this reason, the analysis of this compound has consistently been omitted from the analytical methods described for the detection of less-polar MDMA metabolites (MDA, HMMA, and HMA) [9], [15]. HHMA is an unstable substance, light- and temperature-sensitive, that auto-oxidises easily to its corresponding quinone. When, instead of following more ordinary analytical approaches in the analysis of amphetamine-like substances, one concentrates on the catecholamine structure of HHMA, high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) (HPLC–ECD) seems a suitable technique. In fact, HPLC–ECD is considered one of the techniques of choice for the analysis of catecholamines in biological fluids [16], [17], [18], [19], [20]. Several methods for the extraction of catecholamines from plasma and urine have been developed. These include alumina (Al₂O₃), the formation of cyclic boronates, with further extraction by liquid–liquid extraction procedures and solid-phase extraction methods using phenylboronic acid (PBA) or strong cation-exchange resins (SCX, benzenesulfonic acid) [16], [17], [18], [19], [20].

We describe here the first validated method for HHMA determination in human plasma and urine samples by strong cation-exchange solid-phase extraction and HPLC–ECD analysis.