Stereoselective determination of apomorphine enantiomers in serum with a cellulose-based high-performance liquid chromatographic chiral column using solid-phase extraction and ultraviolet detection.

A novel and rapid method for the separation and determination of R-(-)- and S-(+)-enantiomers of apomorphine in serum by high-performance liquid chromatography with UV detection is reported. The method involved a solid-phase extraction of the R-(-)- and S-(+)-enantiomers of apomorphine and the internal standard R-(-)-propylnorapomorphine from serum using a C8 Bond-Elut column. The HPLC system consisted of a reversed-phase cellulose-based chiral column (Chiralcel OD-R, 250 x 4.6 mm I.D.) with a mobile phase of 35:65 (v/v) acetonitrile-0.05 M sodium perchlorate (pH 2.0, adjusted with 60-62% perchloric acid) at a flow-rate of 0.5 ml/min with UV detection at 273 nm. The detection and quantitation limits were 10 ng/ml for each enantiomer using 1 ml of serum. Linear calibration curves from 10 to 1000 ng/ml for both R-(-)- and S-(+)-enantiomers show coefficient of determination of more than 0.9995. Precision calculated as %R.S.D. and accuracy calculated as % error were 0.2-4.7 and 3.1-6.9%, respectively, for the R-(-)-enantiomer and 1.3-4.2 and 0.3-6.8%, respectively, for the S-(+)-enantiomer.