A liquid chromatography/electrospray ionization tandem mass spectrometric method for the quantification of calcitriol in human plasma: application to pharmacokinetic study in human subjects

Calcitriol is a synthetic vitamin D analog which is active in the regulation of the absorption of calcium from the gastrointestinal tract and its utilization in the body. Circulating concentrations of endogenous calcitriol complicate the analysis of pharmacokinetic parameters and the determination of bioequivalence (BE) when the drug is administered exogenously. The main objective of this study is to evaluate the significance of endogenous concentration and baseline correction in the bioequivalence assessment of drug Calcitriol. An ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method has been developed for the determination of Calcitriol in human plasma using Calcitriol d6 as the internal standard by means of baseline correction approach. The plasma samples were prepared by solid phase extraction on Phenomenex Strata-X cartridges using 500µL human plasma sample. Chromatography was carried out on Waters Acquity UPLC BEH C18 (100mm × 2.1mm, 1.7µ) analytical column under gradient conditions using a mobile phase consisting of acetonitrile–4.0 mM ammonium trifluoroacetate. The precursor to product ion transition for the analyte and IS was monitored on Waters Xevo TQ-S triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ionization mode. The method was validated over a wide dynamic concentration range of 5–200 pg/mL for Calcitriol. Stability was evaluated under different conditions including bench top, processed sample, freeze and thaw and long term. The method was applied to support a bioequivalence study of 0.5 mcg fixed dose formulation in 10 healthy Indian subjects. Assay reproducibility was demonstrated by reanalysis of 120 incurred samples.