Abstract: A novel, highly selective, sensitive, and fastUHPLC-MS/MSmethodis described and validated for reliable quantification of Ursodeoxycholic acid (UDCA) and their major metabolites, Tauroursodeoxycholic acid (TUDCA) and Glycoursodeoxycholic acid (GUDCA) in human plasma using deuterium-labelled internal standards respectively. Chromatographic separation was achieved through isocratic mode with a reverse-phase C18 Symmetry Shield (50mm*4.6mm, 5.0µm) column and a mobile phase of Acetonitrile: Methanol:2mM Ammonium formate (pH3.5) [48:06:46%v/v] at a flow rate of 0.600mL/min. Electrospray ionization technique with negative ion mode polarity was applied to achieve the best signal intensity and stable response. Solid phase extraction by direct elution method was applied to extract the drugs from the plasma sample. The calibration curve range was validated from a concentration range of 100 to 10000ng/ml for UDCA 90 to15000ng/mL for GUDCA and 9 to 3000ng/mL for TUDCA. Analyte free matrix was obtained through charcoal treatment of plasma. The within-batch and between-batch precision and accuracy were found to be consistent and reproducible for all the analytes across the validation. Extraction recoveries were >85% for all analytes and internal standards. The method did not show any matrix effect or coeluting peaks. All peaks of analytes and respective internal standards (ISTD) were eluted within 4.0min. In this validated method, selective multi-variate analytical approaches were utilized such as best fit linearity range for different strength formulations, shorter analysis time etc.This validated method can be useful for challenging endogenous quantification of Ursodeoxycholic acid and its major metabolites reproducibly and effectively for therapeutic drug monitoring and high throughput clinical studies sample analysis.

Keywords:Ursodeoxycholic acid, Glycoursodeoxycholic acid,Tauroursodeoxycholic acid UHPLC-LC/MS, Bioanalytical, Method Validation.