Chiral separation and determination of R-(−)- and S-(+)-baclofen in human plasma by high-performance liquid chromatography

The method presented here is a high-performance liquid chromatography (HPLC)–UV detection method for the determination of baclofen R-(−)- and S-(+)-enantiomers in human plasma using a chiral separation technique. Baclofen enantiomers were extracted from human plasma with a reversed-phase solid-phase extraction (SPE) cartridge. The extract was then injected onto a HPLC system with a UV detection system set at 220 nm. The separation was achieved by using a 150×4.6 mm, 5 μm Phenomenex chirex 3216 chiral column with a mobile phase consisting of 0.4 mM CuSO4 in acetonitrile–20 mM sodium acetate (17:83). The calibration curves were linear for both R-(−)- and S-(+)-enantiomers of baclofen in the concentration range of 20–5000 ng/ml. The average regressions were 0.9980 and 0.9991 for R-(−)- and S-(+)-baclofen, respectively. Inter-day precision was 3.3–5.2% for R-(−)-baclofen and 3.5–3.9% for S-(+)-baclofen at a concentration range of 60–4000 ng/ml. Intra-day precisions were 0.6–4.4 and 0.5–3.5% for R-(−)-baclofen and S-(+)-baclofen, respectively. The average extraction recovery was 81.6% for R-(−)-baclofen, 83.0% for S-(+)-baclofen and 94.0% for the internal standard (p-aminobenzoic acid). The limit of quantitation for both R-(−)- and S-(+)-baclofen in human plasma was 20 ng/ml. The method is simple and easy to operate with accuracy and reproducibility and it is suitable for pharmacokinetic studies.