Achiral and chiral high-performance liquid chromatography of verapamil and its metabolites in serum samples

Rapid and simple achiral and chiral HPLC assays have been developed for the determination of verapamil and its metabolites in serum samples. Two achiral reversed-phase columns, Hisep C₁₈ (150×4.6 mm) and NovaPak C₁₈ (150×3.9 mm) were used for the simultaneous separation of all analyzed compounds. An α₁-AGP column (100×4.0 mm) was recommended for successful chiral separations of verapamil and its seven metabolites. All analyses were realised with fluorescence detection at λ_ex=276 nm and λ_em=310 nm. Limits of quantitation were in the range 1.0 to 5 ng/ml for all compounds. Both off-line SPE (SepPak C₁₈ cartridges) and the on-line SPE with a semipermeable surface SDS C₈ pre-column, (10×4.6 mm) were used for the clean-up and sample preconcentration. Extraction recoveries for all analyzed compounds were 87.7±5.8 to 92.7±4.0% for off-line SPE and 94.3±4.2 to 98.2±5.1% for on-line SPE. The complete assay could be applied for achiral and chiral monitoring verapamil and all its metabolites in serum samples.