Validated liquid chromatography–tandem mass spectrometry method for simultaneous determination of clopamide, reserpine and dihydroergotoxine: Application to pharmacokinetics in human plasma

Abstract

A simple, sensitive and rapid high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed and validated for the simultaneous quantitation of clopamide, reserpine and dihydroergotoxine (ergoloid mesylates) in human plasma. Under basic conditions, liquid–liquid extraction using ethyl acetate was efficiently used for extraction of the analytes from plasma samples in presence of indapamide as internal standard (IS). The analytes were separated with isocratic elution on Phenomenex® Synergi Fusion-RP 80A column (50×4.6mm, 4μm). With positive ion electrospray ionization (ESI), the analytes were quantified and monitored on a triple quadrupole mass spectrometer using Multiple Reaction Monitoring (MRM) scanning mode. Satisfactory results regarding linearity, recovery, stability, accuracy and precision of the analytes were obtained. The method was linear in the concentration range of 0.04–30.00ng/mL for reserpine, 1–96.00ng/mL for clopamide, and 0.05–40.00ng/mL for dihydroergotoxine alkaloids, respectively. For all analytes, the high sensitivity of HPLC–MS/MS method revealed sufficient lower limit of quantification (LLOQ) ranged from 0.04–1ng/mL using 1mL of plasma. The recoveries from spiked control samples were ≥86.16% for all analytes and IS. The intra- and inter-day precision variations were lower than 13.03% while the accuracy values ranged from 91.76% to 111.50%. The developed method was successfully applied to pharmacokinetic study of fixed dose combination of clopamide, reserpine and dihydroergotoxine in healthy male volunteers