Development and validation of an UPLC-MS/MS method for the quantification of columbin in biological matrices: Applications to absorption, metabolism, and pharmacokinetic studies.

The aim of this study is to develop a sensitive UPLC-MS/MS method to quantify columbin in biological sample. Chromatographic separation was accomplished using Waters UPLC BEH C18 column with acetonitrile and 0.1% of formic acid in water as the mobile phases. The mass analysis was performed on an API 5500 Qtrap mass spectrometer via multiple reaction monitoring (MRM) with positive scan mood. The one-step protein precipitation by methanol was used to extract the analyte from blood samples. The results showed that the linear response range for columbin was 1.22-2,500nM. The intra and inter day variances were less than 15% and the accuracy was in acceptable range (85-115%). The analysis was done within 3.0min, and only 50μL of blood was needed. The validated method was used to determine the pharmacokinetic profile of columbin in Wistar rats, and its transport characteristics in the Caco-2 cell culture model. The results showed that columbin was poorly bioavailable (2.8% p.o. and 14% i.p.) in rats, but its transport was rapid across the Caco-2 cell monolayers, suggesting that extensive first-pass metabolism in the liver was the likely reason for its poor bioavailability. The results revealed that the validated method can be used for columbin analysis in both bioequivalent buffer and blood.