A sensitive and rapid UFLC-APCI-MS/MS bioanalytical method for quantification of endogenous and exogenous Vitamin K1 isomers in human plasma: Development, validation and first application to a pharmacokinetic study

Due to lack of suitable bioanalytical methods in previous literature, for simultaneous estimation of Vitamin K1 isomers, in compliance with the current regulatory expectation, we aimed to develop a sensitive and rapid method with UFLC-APCI-MS/MS (ultrafast liquid chromatography - tandem mass spectrometry) using human plasma. A simple and cost effective procedure was implemented with the combination of protein precipitation and liquid extraction, to isolate the targets from plasma sample, while achieving an insignificant matrix effects and high recovery (≥88.2%). A short 9.0min run time per sample was accomplished by using water in methanol (1.0% v/v) and acetonitrile, which pumped at 0.8mL/min, on to the COSMOSIL® packed column, for separating the trans and cis isomers of Vitamin K1 along with the corresponding stable labeled D7 internal standards (ISs). The analytes and ISs were quantified, at their parent to product ion mass transitions of 451.3 →187.1m/z and 458.1→194.3m/z respectively, using an APCI (atmospheric pressure chemical ionization) source of the tandem mass, in MRM (multiple reaction monitoring) mode. Performance of the method over the calibration range: 0.1–150.0ng/mL, while using a low sample volume (0.3mL), was successfully evaluated through full method validation in compliance with the latest regulations. Fully validated method with significant results was applied to human pharmacokinetic study, and had a potential to further advance the clinical research programs and generic drug development of Vitamin K1, intended for the regulatory submission. [Display omitted] •Establishment of an optimized method with the new chromatographic conditions.•Method comply most recent validation guidelines of US-FDA, EMA and ANVISA.•New method with significant results in comparison with related literature.•Method to differentiate the therapeutic and endogenous levels of the analytes.•First highly selective method with successful application to PK study samples.