Rapid and highly sensitive method for the determination of verapamil, [~2H_7]verapamil and metabolites in biological fluids by liquid chromatography-mass spectrometry

摘要

A rapid and highly sensitive method for the determination of verapamil [2,8-bis-(3,4-dimethoxyphenyl)6-methyl-2-isopropyl-6-azaoctanitrile] and [~2H_7]verapamil and their primary metablites D-617 [2-(3,4-dimethoxyphenyl)5-methylamino-2-isopropylvaleronitrile], D-703 [2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dimethoxy-phenyl)-6-methyl-2-iso-propyl-6-azaoctanitrile], D-702 [2-(3,4-dimethoxy-phenyl)-8-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile], norverapamil [2,8-bis-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile] and secondary metabolites D-620 [2-(3,4-dimethoxyphenyl)-5-amino-2-isopropylvaleronitrile], D-717 [2-(4-hydroxy-3-methoxyphenyl)-5amino-2-isopropyl-valeronitrile], and D-715 [2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile] has been developed using high-performance liquid chromatography-electrospray mass spectrometry. D-832, the gallopamil analogue of D-617 and [~2H_3]norverapamil were used as internal standards. The analytes were extracted automatically from plasma and intestinal perfusate using end-capped CN- and C_2 solid-spase extraction cartridges. Separation of the eight analytes was achieved on a LUNA C8 analytical column (150×2 mm I.D, 5 μm particle size) with 5 mM ammonium acetate-acetonitrile as the mobile phase run with a gradient from 70:30 to 40:60 and run times of 15 min. With the mass spectrometer operated in the selected-ion monitoring mode, the limits of quantification in plasma and intestinal perfusate were 1 pmol/ml for D-620, D-617, D-702, D-703, norverapamil, verapamil, and [~2H_7]verapamil and 2.5 pmol/ml for D-717 and D-715 using a sample size of 1 ml plasma and intestinal perfusate. The method described was successfully applied to the determination of verapamil, [~2H_7]verapamil and their metabolites in human plasma and intestinal fluid in pharmacokinetic studies.