Enantiomeric separation of verapamil and its active metabolite, norverapamil, and simultaneous quantification in human plasma by LC-ESI-MS-MS.

ABSTRACT

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, norverapamil, in human plasma. All four analytes along with deuterated internal standards (D(6)-verapamil and D(6)-norverapamil) were extracted from 50 µL human plasma by liquid-liquid extraction. Separation was achieved on a Chiralcel OD-RH (150 × 4.6 mm, 5 µm) analytical column with resolution factors of 1.4 and 1.9 for (R)- and (S)-enantiomers of verapamil and norverapamil, respectively. A mobile phase consisting of 0.05% trifluoroacetic acid in water-acetonitrile (7030, 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 pharmacokinetic study in 18 healthy Indian males after oral administration of a 240-mg verapamil tablet formulation under fasting conditions.