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

摘要

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 liquidliquid extraction. Separation was achieved on a Chiralcel OD-RH(150 × 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, respectively. A mobile phase consisting of 0.05% trifluoroacetic acid in wateracetonitrile(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 1.0250.0 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 0.961.07. 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.