Enantiomeric Separation of Verapamil and its Active Metabolite, Norverapamil, and Simultaneous Quantification in Human Plasma by LC–ESI-MS-MS

A simple, selective and high-throughput liquid chromatography– tandem mass spectrometry method has been developed and validated for the chromatographic separation and quantification of (R)- and (S)-enantiomers of verapamil and its active metabolite, nor-verapamil, in human plasma. All four analytes along with deuter-ated internal standards (D6-verapamil and D6-norverapamil) were extracted from 50 mL human plasma by liquid–liquid extraction. Separation was achieved on a Chiralcel OD-RH (1503 4.6 mm, 5 mm) analytical column with resolution factors of 1.4 and 1.9 for (R)- and (S)-enantiomers of verapamil and norverapamil, respective-ly. A mobile phase consisting of 0.05 % trifluoroacetic acid in water–acetonitrile (70:30, v/v) afforded capacity factors of 2.45, 3.05, 2.27 and 3.13 for (R)- and (S)-enantiomers of verapamil and norverapamil, respectively. Detection was carried out on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion modes. The method was validated over the concentration range of 1.0–250.0 ng/mL for all four analytes. Absolute recovery for the analytes ranged from 91.1 to 108.1%. Matrix factors calculated at three quality control levels varied from 0.96–1.07. The method was successfully applied to a pharmacoki-netic study in 18 healthy Indian males after oral administration of a 240-mg verapamil tablet formulation under fasting conditions