Resolution and quantitation of pentazocine enantiomers in human serum by reversed-phase high-performance liquid...

A sensitive and stereospecific HPLC method was developed for the analysis of (-)- and (+)-pentazocine in human serum. The assay involves the use of a phenyl solid-phase extraction column for serum sample clean-up prior to HPLC analysis. Chromatographic resolution of the pentazocine enantiomers was performed on a octadecylsilane column with sulfated-beta-cyclodextrin (S-beta-CD) as the chiral mobile phase additive. The composition of the mobile phase was aqueous 10 mM potassium dihydrogenphosphate buffer pH 5.8 (adjusted with phosphoric acid)-absolute ethanol (80:20, v/v) containing 10 mM S-beta-CD at a flow-rate of 0.7 ml/min. Recoveries of (-)- and (+)-pentazocine were in the range of 91-93%. Linear calibration curves were obtained in the 20-400 ng/ml range for each enantiomer in serum. The detection limit based on S/N=3 was 15 ng/ml for each pentazocine enantiomer in serum with UV detection at 220 nm. The limit of quantitation for each enantiomer was 20 ng/ml. Precision calculated as R.S.D. and accuracy calculated as error were in the range 0.9-7.0% and 1.2-6.2%, respectively, for the (-)-enantiomer and 0.8- 7.6% and 1.2-4.6%, respectively, for the (+)-enantiomer (n=3).