Specific HPLC Method for the Separation of Verapamil and Four Major Metabolites After Oral Dosing

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Abstract
We have developed a high performance liquid chromatographic (HPLC) method which resolves verapamil, norverapamil, D620, D617 and what we believe to be another verapamil metabolite which has been previously unreported. An alkyl-phenyl column is used with a mobile phase of 0.005% sulfuric acid in methanol. The extraction recoveries of verapamil, norverapamil and the internal standard (imipramine) from plasma ranged between 98% and 104%. The day-to-day, and within-day coefficients of variations for verapamil and norverapamil at plasma concentrations of 7.3 and 233 ng/ml ranged between 1.7 and 6.1%. The limit of sensitivity was slightly less than 1 ng for both verapamil and norverapamil. Chromatograms of extracts of serum and urine obtained from five normal subjects who took single oral verapamil doses, indicated the presence of verapamil, norverapamil, and two other known metabolites. Chromatograms of serum extracts also indicated an additional peak which is probably another verapamil metabolite.