Development and validation of highly sensitive method for determination of misoprostol free acid in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry: Application to a clinical pharmacokinetic study

Abstract

A highly sensitive, selective and evaporation free SPE extraction, ESI-LC–MS/MS method has been developed for estimation of misoprostol free acid in human plasma using misoprostol acid-d 5 as an internal standard (IS). The analyte was separated using isocratic mobile phase on reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M−H] anions, m/ z 367–249 for misoprostol acid and m/ z 372–249 for the IS. The total run time was 5.0 min and the elution of misoprostol acid and misoprostol acid-d 5 (IS) occurred at 3.6 min. The developed method was validated in human plasma with a lower limit of quantification of 2.5 pg/mL. A linear response function was established for the range of concentrations 2.5–1200 pg/mL ( r > 0.998) for misoprostol acid in human plasma. The intra and inter-day precision values for misoprostol acid met the acceptance as per FDA guidelines. Misoprostol acid was stable in the battery of stability studies viz., bench-top, auto-sampler and freeze/thaw cycles. The developed assay method was applied to an oral pharmacokinetic study in humans.