Determination of obeticholic acid and its glycine conjugate in human plasma using liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatography/tandem mass spectrometry method was recommended by the authors for the determination orally bioavailable farnesoid X receptor agonist obeticholic acid along with its glycine conjugate glycoobeticholic acid human plasma. Obeticholic acid_d5 and glycoobeticholic acid_d5 were used as internal standards, respectively. After extraction with a mixture of tert-butyl methyl ether and dichloromethane, samples were separated on a phenyl column with a mobile phase of 2mM ammonium acetate with 0.01% formic acid and methanol (15:85, v/v). Analysis was performed on an AB Sciex 4500 triple quadrupole mass spectrometer and data acquisition was performed by multiple reaction monitoring (MRM) in the negative ionization mode. Obeticholic acid and its conjugate were quantified in the linearity range of 0.50-100.00 ng/mL and the correction coefficients were≥0.99 during the validation. Precision and accuracy in different days (inter-day) and single day (intra-day) were meeting the acceptance criteria specified in the recent US FDA bioanalytical method validation guidelines. A variety of stability experiments in neat samples and plasma samples were conducted and the results are meeting the acceptance criteria. A run time of 2.5 min for each sample made it possible to analyze a greater number of samples in a short time, thus increasing productivity. This method could be useful to quantitate obeticholic acid and glycoobeticholic acid in real clinical samples.