Simultaneous stereoselective analysis of venlafaxine and O-desmethylvenlafaxine enantiomers in clinical samples by capillary electrophoresis using charged cyclodextrins

Abstract

Capillary electrophoresis (CE) was used for the simultaneous chiral determination of venlafaxine (Vx), a new antidepressant drug and its main active metabolite, O-desmethyl venlafaxine (ODV). Among the charged cyclodextrins (CD) tested, phosphated γ-CD was the most appropriate. Resolution of Vx and ODV was obtained with 50 mM phosphate buffer (pH 2.5) containing 20 mg/ml of phosphated γ-CD. After optimisation of the method (including robustness), validation was carried out. Vx and ODV concentrations, as well as the enantiomeric ratio, were investigated in clinical samples. Chiral determination of Vx and ODV was performed after a simple liquid–liquid extraction (LLE). In the tested concentration range (25–500 ng/ml), coefficients of correlation were superior to 0.996. Within-day and between-day accuracy and precision were determined at three different concentrations for each enantiomer. Analyses of clinical samples (n=16) exhibited non-racemic ratios for Vx and ODV, which suggests a stereoselective metabolism in humans.

Introduction

1-2-(dimethylamino)-1-(4-methoxyphenyl)ethyl cyclohexanol hydrochloride (venlafaxine) is a second generation antidepressant which proves to be particularly effective [1]. Venlafaxine (Vx) exerts a dual mechanism of action on the monoaminergic system. It principally blocks neuronal re-uptake of noradrenaline, serotonine and, to a lesser extent, dopamine [2], [3]. In humans Vx is metabolised into two minor metabolites, N-desmethyl and N,O-didesmethyl venlafaxine, and into a major active metabolite, O-desmethyl venlafaxine (ODV) which presents an activity profile similar to that of Vx [4], [5], [6].

As shown in Fig. 1, Vx has an asymmetric centre and both enantiomers exhibit different activities. In fact, the R (−) enantiomer inhibits both noradrenaline and serotonine re-uptake while the S (+) isomer inhibits primarily the serotonine re-uptake [7]. Furthermore, experimental data suggest that there is a difference potential in the O-desmethylation of the two Vx enantiomers [5]. It is important to determine the enantiomeric plasma concentrations for both Vx and its active O-desmethylated metabolite ODV in order to establish pharmacodynamic as well as pharmacokinetic relations and to understand the pharmacological contribution of each active enantiomer [4], [5].

The methods used for venlafaxine analysis include gas chromatography with a nitrogen-phosphorus detector (GC-NDP) [8], [9], [10], high-performance liquid chromatography (HPLC) with UV [4], [5], [10], fluorimetric [3] and coulometric detections [11]. Enantiomeric separations of venlafaxine and/or its impurities have been carried out by HPLC [12].

Capillary electrophoresis (CE) is known to be a powerful separation technique in a large number of analytical fields, and particularly for the determination of drugs in biological fluids [13], [14]. Compared to chromatographic techniques such as HPLC, its high-resolution power and efficiency present several advantages especially in the separation of complex drug metabolite mixtures [15], [16]. Moreover, CE exhibits excellent results in the field of optical isomer separation [17], [18], [19], [20]. Chiral discrimination by CE is generally achieved with the direct separation method where the chiral selector is simply added to the background electrolyte. Cyclodextrins and their derivatives have been predominantly used as chiral selector [21]. Due to the chemical reactivity of the hydroxy groups, native charged cyclodextrins (CD) have been modified to produce chiral selectors with properties different from those of the parent CD [22], [23]. Charged and ionisable CD have been widely and successfully employed [24], [25]. For positively charged compounds, negatively charged chiral selectors have already been used by several authors [26], [27], often leading to higher resolution than with neutral cyclodextrins [28], [29].

Recently, the enantiomeric separation of Vx by CE was achieved with neutral cyclodextrins [30], [31] but to our knowledge, no separation of Vx and its active metabolite enantiomers has been carried out with charged cyclodextrins as chiral selectors in biological samples. In this paper, a phosphated γ-CD was used for the simultaneous determination of Vx and ODV enantiomers.