Development and Validation of Bioanalytical Method for Orlistat through the Integration of High-Performance Liquid Chromatography (HPLC) And Mass Spectrometry (MS)

Abstract

An analytical method was successfully developed and validated for the quantification of tetrahydrolipostatin (Orlistat) in rat plasma to support pharmacokinetic studies. The analysis utilized liquid chromatography coupled with ion spray mass spectrometry (LC-MS) in multiple reaction monitoring (MRM) mode. Separation was achieved using a Luna Phenyl-Hexyl column and a mobile phase of acetonitrile and ammonium acetate (20:80). The analyte's retention time was 2.182 minutes, with a total run time under 5 minutes. No interference of other biological materials or plasma elements were observed. The method demonstrated a lower limit of quantification (LLOQ) of 1200 ng/mL and a lower limit of detection (LOD) of 250 ng/mL with 1 mL plasma aliquots, while also showing the capability to detect concentrations as low as 1250 ng/mL. Calibration curves exhibited linearity between 1250 and 100000 ng/mL. Intra- and inter-assay precision studies showed variability below 10%. The method's recovery, precision, and accuracy adhered to bioanalytical standards, and orlistat stability was confirmed under conditions likely to occur during clinical trial sample analysis. This LC-MS method is robust, sensitive, specific, accurate, and reproducible for quantifying orlistat in rat plasma.