Simultaneous determination of amphetamine and one of its metabolites by HPLC with electrochemical detection

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Abstract

A high-performance liquid chromatographic method coupled with electrochemical detector was developed for the separation and quantitation of amphetamine and one of its metabolites, the 4-hydroxynorephedrine. The pre-column derivatisation of these compounds was carried out with 2,5-dihydroxybenzaldehyde as electroactive labelling reagent, in presence of Borohydride Exchange Resin. The new synthetic method developed was fast, clean and high yielding. The analysis was performed in isocratic mode on a reversed phase column 5 μm Hypersil ODS RP-18, 15 cm, using as a mobile phase methanol-NaH2PO4 buffer (50 mM, pH 5.5)(30:70 v/v) containing trietylamine (0.5% v/v) and the products were detected by a porous graphite electrode set at an oxidation potential of +0.6 V. The linearity of response was examined for each derivatised compound and was analysed using solutions in the range 10–40 nmol/ml. The correlation coefficients of the linear regression of the standard curves were greater than 0.99. The method developed in this study was sensitive and very selective. Because of the specificity for primary phenylethylamines, it could be applicable for the assay of other related substances in toxicology and drugs abuse.

Introduction

Amphetamine (AMP) and other related phenylethylamines are drugs of abuse as well as doping agents in many sports. In the last years, these substances have been extensively used among teenagers because of their psychoactive properties [1]. The ability of stimulants with an essential phenylethylamine structure cause neurotoxicity, particularly during chronic administration, was well established in experimental models [2]. Phenylethylamine-induced neurotoxicity was primarily characterized by degeneration of dopaminergic nerve terminal fields within the nucleus accumbens, olfactory area and frontal cortex. In addition, substantial destruction of serotonergic terminals in the hippocampus, cerebral cortex, amygdala and striatum was associated with administration of high dose of phenylethylamine stimulants [3]. Recent evidence indicates that production of phenylethylamine stimulant-induced dopaminergic neurotoxicity requires interaction with the glutamatergic neurotrasmission [4].

The main metabolic pathways of AMP [5] is shown in Scheme 1. The compounds formed in the first phase of metabolism of AMP are produced from (i) oxidative deamination; (ii) one and twofold ring hydroxylation, followed by methylation of one of the hydroxy group; (iii) N-demethylation. In the subsequent phase they are mainly excreted as glucoronide and/or sulphate conjugates. The hydroxy-derivatives metabolites of AMP are the principal compounds excreted in urine that can be detected for 7/8 days after ingestion, whereas the parent compounds are only detectable for about 2/3 days. Some of these metabolites may have activity that contribute to the effects seen after AMP administration, in particular 4-hydroxyamphetamine (4-HNE) acts as a false neurotransmitter.

Many analytical methodologies used in toxicological studies and forensic science have been described and the great number of publications on AMP analysis published in the last 5 years indicates that it was necessary to improve the methods of analysis [5], [6], [7]. However, only few chromatography methods were described for the simultaneous analysis of AMP and 4-HNE [8], [9].

Gas chromatographic (GC) methods are the most widely used for AMP and related substances analysis in biological samples [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21] and are traditionally recommended, especially if coupled to mass spectrometric (GC/MS) detection. Recently, capillary electrophoresis (CE) methods were also developed [22], [23], [24].

In high performance liquid chromatography (HPLC) several methods have been described for the determination of AMP and derivatives but HPLC procedures without derivatisation reactions have not been widely applied to the analysis of phenylethylamines because they show low UV absorbances and very little natural fluorescence [25], [26], [27]. In addition, primary and secondary amines often show poor chromatographic performance which could be improved by derivatisation. To improve both chromatographic behaviour or detectability of the AMP and derivatives, a great number of procedures involving precolumn or postcolumn derivatization using different reagents were developed [28], [29], [30], [31], [32], [33], [34], [35], [36].

HPLC with electrochemical detection represents a very sensitive method, providing enhanced selectivity as a result of the limited number of substances which could undergo redox reactions under certain conditions [37], [38], [39].

The aim of this study was the development of a sensitive and selective method for the simultaneous determination of AMP and one of its metabolites (4-HNE) by HPLC with electrochemical coulometric detection (HPLC-ECD).

A new derivatization method using 2,5-dihydroxybenzaldehyde (2,5-DBA) as an electrochemical probe, selected for the very low oxidation potential was developed. The 2,5-DBA was rapidly aminated with the primary amines AMP, 4-HNE and phenylethylamine (PHE), using borohydride exchange resin (BER) as a chemoselective reducing agent to give the electroactive secondary amines AMP-DBA, 4-HNE-DBA and PHE-DBA, the latter working as an internal standard (IS), respectively. The procedure is shown in Scheme 2.

In order to optimise the detection of the electroactive derivatives several parameters such as oxidation potential, pH and ionic strength of mobile phase were examined.

Section snippets

Apparatus

The HPLC apparatus comprised two Model 510 pumps, a Model 712 WISP auto-injector and a Model 490E absorbance detector (Waters Assoc., Milford, MA, USA) set at 276 nm and 0.05 absorbance units full scale. The UV detector was connected in series with the electrochemical detector (Model 5100A Coulochem; ESA, Bedford, MA, USA) which consists of a control module and an analytical cell (Model 5010) containing two on-line porous graphite coulometric electrodes.

The analysis was performed in the

Derivatization procedure

Scheme 2 shows the procedure of derivatization using 2,5-DBA as a derivatizing agent. This aromatic aldehyde was selected for its easy reactivity and its lower oxidation potential compared with other isomers such as 3,4-dihydroxybenzaldeide [40]. The electrochemical probe was reductively aminated by reaction with the primary aromatic amines AMP, 4-HNE and PHE to give the corresponding secondary amines using BER as a chemoselective reducing agent [41]. The most commonly used hydride reducing

Conclusions

The HPLC-ECD method described in this study was applied for the determination of AMP and its metabolite 4-HNE. The selective and easy derivatization of AMP, 4-HNE and PHE by the electroactive labelling 2,5-DBA yields stable and highly sensitive electroactive secondary amines that can undergo specific redox reaction under the condition reported. The HPLC-ECD method developed is a versatile technique because other primary phenylethylamines of forensic interest can be determinated with this system.

Acknowledgements

This work was supported by ‘Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST)’, Rome, Italy.

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