Rapid and highly sensitive method for the determination of verapamil, [H-2(7)]verapamil and metabolites in biological fluids by liquid chromatography-mass spectrometry

A rapid and highly sensitive method for the determination of verapamil [2,8 -bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] and [H-2( 7)]verapamil and their primary metabolites 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-isopropyl-6-azaoctanitrile], D-702 [2 -(3,4-dimethoxy-phenyl)-8-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-isopropyl- azaoctanitrile], norverapamil [2,8-bis-(3,4-dimethoxy-phenyl)-2-isopropyl-6 -azaoctanitrile] and secondary metabolites D-620 [2-(3,4-dimethoxyphenyl)-5 -amino-2-isopropylvaleronitrile], D-717 [2-(4-hydroxy-3-methoxyphenyl)-5-am ino-2-isopropyl-valeronitrile], and D-715 [2-(4-hydroxy-3-methoxyphenyl)-8- (3,4-dimethoxy-phenyl)-2-isopropyl-6-azaoctanitrile] has been developed usi ng high-performance liquid chromatography-electrospray mass spectrometry. D -832, the gallopamil analogue of D-617 and [H-2(3)]norverapamil were used a s internal standards. The analytes were extracted automatically from plasma and intestinal perfusate using end-capped CN- and C-2 solid-phase extracti on cartridges. Separation of the eight analytes was achieved on a LUNA C8 a nalytical column (150x2 mm I.D., 5 mu m particle size) with 5 mM ammonium a cetate-acetonitrile as the mobile phase run with a gradient from 70:30 to 4 0:60 and run times of 15 min. With the mass spectrometer operated in the se lected-ion monitoring mode, the limits of quantification in plasma and inte stinal perfusate were 1 pmol/ml for D-620, D-617, D-702, D-703, norverapami l, verapamil, and [H-2(7)]verapamil and 2.5 pmol/ml for D-717 and D-715 usi ng a sample size of 1 ml plasma and intestinal perfusate. The method descri bed was successfully applied to the determination of verapamil, [H-2(7)]ver apamil and their metabolites in human plasma and intestinal fluid in pharma cokinetic studies. (C) 2000 Elsevier Science B.V. All rights reserved.