Enantioselective analysis of (R)- and (S)-atenolol in urine samples by a high-performance liquid chromatography column-switching setup

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Abstract

An HPLC column-switching method for the enantioselective determination of (R,S)-atenolol in human urine was developed and validated. Diluted urine samples were injected onto a LiChrospher ADS restricted access column and atenolol was separated from most of the matrix components using 0.01 M Tris buffer. The atenolol peak was sharpened by a step gradient of 30% acetonitrile and the atenolol-containing fraction was switched onto an enantioselective column. Separation of the atenolol enantiomers was carried out on a Chirobiotic T (Teicoplanin) column using acetonitrile–methanol–acetic acid–triethylamine (55:45:0.3:0.2, v/v/v/v) as eluent. Detection of the effluent was performed by fluorescence measurement. Several experiments were carried out to suppress the high blank reading, which was efficiently achieved using Tris buffer in the first dimension. For the enantioselective analysis of (R)- and (S)-atenolol in plasma under the same conditions the sample capacity of the ADS column is considerably lower.

Introduction

(R,S)-Atenolol (4-(2-hydroxy-3-isopropylaminopropoxy)phenylacetamide) is a selective β1-adrenoceptor antagonist [1] applied in the treatment of numerous cardiovascular disorders such as hypertension, angina pectoris, etc. As known from previous studies, the main pharmacological activities reside in the (S)-enantiomer [2], [3] and therefore it is mandatory to analyse the individual enantiomers in body fluids to arrive at convincing pharmacokinetic and pharmacodynamic results.

Several methods have been reported in the literature for the enantioselective assay of (R)- and (S)-atenolol in body fluids [4], [5], [6], [7]. A comprehensive overview of this subject was given by Egginger et al. [8]. The direct injection of biological samples, in particular of serum or plasma samples, onto the chromatographic column, often a reversed-phase (RP) column, without any sample preparation, is in most cases problematic and may lead to irreversible contamination of the separation column, mainly by proteins. This effects not only the selectivity but also the column performance and efficiency, leading to unacceptable conditions. As a consequence, most of the bioanalytical methods, including the enantioselective method, need adequate sample pre-treatment steps, which are: an extraction and analytical enrichment step — usually performed by liquid–liquid- or liquid–solid-phase extraction (SPE) — followed by the direct enantioseparation of the pretreated sample on a chiral stationary phase (CSP) or by an indirect approach involving derivatisation with a chiral reagent and the analysis of the pair of diastereomers on, for example, a RP system.

The objective of the present work was to develop an on-line sample preparation and analysis method to be used for the analysis of (R)- and (S)-atenolol in urine and, possibly, also in plasma. A powerful tool to accomplish this relies on the implementation of restricted access material (RAM) for sample preparation [9], [10], [11]. The RAMs and particles, respectively, are characterised by a hydrophilic outer surface, a small pore size and a lipophilic inner surface. Proteins of high molecular weight cannot penetrate into the pores, thus being excluded on the hydrophilic outer surface. Hence they can easily be separated from sample matrices consisting of small molecules, including drug compounds and metabolites [12].

The present paper describes the elaboration of an enantioselective HPLC column-switching method for (R)- and (S)-atenolol RAMs for on-line sample preparation and a Teicoplanin-derived chiral column to be applied for final analysis.

Section snippets

Chemicals

For chromatography, Lichrosolv acetonitrile and methanol were purchased from Merck (Darmstadt, Germany) and racemic (R,S)-atenolol from Schweizerhall (Basel, Switzerland). All other chemicals were of analytical grade. Enantiomerically pure (S)-atenolol (ee >98%) and (R)-atenolol (ee >99%) were prepared according to a previously published method [13].

Biological samples

The urine samples represent aliquots of morning urine collected the following morning after the intake of two times 25 mg, or two times 50 mg

Chromatography in the first dimension (ADS column)

(R,S)-Atenolol is a highly hydrophilic drug and elutes on an ADS column under isocratic conditions as a broad peak. On a new ADS column the peak volume of atenolol with an aqueous 0.01 M Tris buffer is 10 mL (peak maximum at 15 mL elution volume), which is too high for an effective on-line column-switching protocol. Therefore, peak compression is necessary in order to facilitate the quantitative transfer of the compound in a small effluent volume. A sufficient peak sharpening effect can be

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