Liquid Chromatography Tandem Mass Spectrometric Method Development and Validation for the Quantification of Orlistat in Biological Matrices

Abstract

A specific, linear and precise liquid chromatographic-tandem mass spectrometric method was established and validated for the quantitation of orlistat in sample plasma. Zorbax C18 (4.6 mm i.d.× 50.0 mm; 5.0 μm) stationary phase was utilized to achieve chromatography elution, through a flowing rate of 0.90 mL/min. Isocratic elution was done using methanol, acetonitrile and 0.10% v/v HCOOH in a fraction of 80:10: 10 v/v/v as the mobile phasic system. For drug and internal standard separation, the precipitation extraction technique used acetonitrile as solvent. A triple quadrupole mass detector was employed for the quantification of ions. Electrospray ionization in a positive ionizing method, which was executed in multiple reaction monitorings (MRM) with parent/product ion transitions of m/z 496.4→337.31 for orlistat and 506.23→57.07 for amprenavir internal standard. The calibration graph was executed between the concentrations of 4.75–190.0 ng/mL and the resulting equation was y = 0.0058x + 0.0022 with r2 value of more than 0.99. Orlistat recovery values were found to be more than 93.65%, and its accuracy, measured in relative error, was in the range of -4.48 to 3.49%. Accuracy findings, sensitivity and recovery values of orlistat in the sample plasma for the established technique evidences its importance in pharmacokinetic and bioequivalence study.