Available online at www.sciencedirect.com Journal of Chromatography B, 865 (2008) 48–54 Development and validation of a stereoselective liquid chromatography–tandem mass spectrometry assay for quantification of S- and R-metoprolol in human plasma Berit P. Jensen a,∗ , Caroline F. Sharp b , Sharon J. Gardiner a , Evan J. Begg a a Clinical Pharmacology, Department of Medicine, University of Otago, Christchurch, New Zealand b Department of Pharmacy, Christchurch Hospital, Christchurch, New Zealand Received 15 November 2007; accepted 14 February 2008 Available online 20 February 2008 Abstract A stereoselective liquid chromatography–tandem mass spectrometry assay was developed and validated for quantification of S- and R-metoprolol at concentrations of 0.5–50 g/L in human plasma. Metoprolol was extracted from plasma by liquid–liquid extraction with ethyl acetate (82% recovery). Chromatographic separation of the enantiomers was achieved on a chiral Chirobiotic T column using an isocratic mobile phase consisting of methanol/acetic acid/ammonia (100/0.15/0.15, v/v/v). An ion trap mass spectrometer with an electrospray interface was used for detection in the positive mode, monitoring the m/z transition 268 → 191 for metoprolol. Standard curves for S- and R-metoprolol fitted quadratic functions (r 2 ≥ 0.9995) over the range 0.5–50 g/L in plasma, with 0.5 g/L representing the limit of quantification. In this range, relative standard deviations were <6% for intra-day precision and <10% for inter-day precision. The accuracy was within the range of 92–105%. © 2008 Elsevier B.V. All rights reserved. Keywords: Metoprolol; Enantiomers; LC–MS/MS; Plasma 1. Introduction Metoprolol, (±)1-(isopropylamino)-3-[p-(-methoxyethyl) phenoxy]-2-propanol (Fig. 1), is a 1-selective adrenoreceptor blocking drug. It is widely used in the management of hyperten- sion, ischaemic heart disease and heart failure [1,2]. Metoprolol is administered as the tartrate, succinate or fumarate salt in a racemic mixture. The -blocking acti- vity resides primarily in the S-enantiomer [2]. Metoprolol is extensively metabolised by phase I processes in the liver. The metabolic pathways are stereoselective, with the S-enantiomer being the predominant form in humans, reflected by a plasma ratio of S/R-metoprolol >1 in most people [3–5]. The metabo- lism of metoprolol is subject to genetic polymorphism since a large proportion involves the Cytochrome P450 2D6 (CYP2D6) enzyme [2,4]. CYP2D6 extensive metabolisers have been shown ∗ Corresponding author. Present address: Clinical Pharmacology, Department of Medicine, University of Otago, Christchurch, P.O. Box 4345, Christchurch 8140, New Zealand. Tel.: +64 3 364 0640; fax: +64 3 364 1003. E-mail address: berit.jensen@cdhb.govt.nz (B.P. Jensen). to have a greater S/R-metoprolol ratio in plasma than poor metabolisers [4]. For clinical studies of metoprolol, it is thus important to be able to selectively monitor the individual enan- tiomers. Several analytical methods have been developed for the stereoselective analysis of metoprolol in biofluids. Direct methods of analysis, which require no derivatisation with chiral reagents to form diastereomers prior to separation, are preferred for clinical studies. Such methods have used chiral stationary phases that were based on either proteins [6–10] or polysaccha- ride derivatives [3,11–13] applied in the normal phase mode. In a single case, a stationary phase based on macrocyclic gly- copeptides was used [14]. Following extraction of metoprolol from plasma or urine by solid-phase or liquid–liquid extraction, fluorescence detection was used in all methods, with limits of quantification in the ranges of 0.5–25 g/L. While fluorescence detection is generally a sensitive and rela- tively selective detection technique for HPLC, a higher degree of selectivity can be achieved using tandem mass spectrometry (MS). Metabolites, other drugs, or endogenous compounds will usually have a different mass to charge ratio (m/z) from the ana- 1570-0232/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2008.02.006