SIMULTANEOUS ESTIMATION AND QUANTIFICATION OF OXYBUTYNIN AND ITS ACTIVE METABOLITE N-DESMETHYL OXYBUTYNINE WITH SENSITIVE HPLCMS/ MS VALIDATED METHODS IN HUMAN PLASMA

Sheeba Nair*, Bhavesh Dasandi, Dharmesh Parmar, Shivprakash and Dr. Denish Karia

ABSTRACT

A simple and specific method for simultaneous determination of Oxybutynin and N-Desmethyl Oxybutynine in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. The analytes and the deuterated internal standard were extracted from 200 μL plasma by liquid phase extraction. Detection and quantitation was done by multiple reaction monitoring in positive ionization with Q3 LCMS-8050, Shimadzu. Mass parameters 358.11/142.10 and 329.78/95.98 and 369.48/142.08 m/z on a triple quadrupole mass spectrometer were chosen for analysis of Oxybutynin and N-Desmethyl Oxybutynine. Linearity was established in human plasma covering the concentration range 75.051 pg/mL to 7400.849 pg/mL for Oxybutynine and 75.163 pg/mL to 7411.923 pg/mL for N-Desmethyl Oxybutynine. Correlation coefficient was consistently greater than 0.98 for Oxybutynin and N-Desmethyl Oxybutynine using (Trientine-D4 and N1-Acetyl trientine trihydrochloride D4) as internal standards. Chromatographic analysis was carried out on column Synergi Hydro-RP 80 A (4.6 x 50 mm) 4S μ with a flow rate of 0.4 mL/min, at 40˚C temperature. A Gradient elution method was applied using (A) Methanol 58% and (B) 0.1% Formic acid buffer (42%). The method was applied to support a bioequivalence study with reference scaled study design of 5 mg tablet formulation in 32 healthy Indian subjects.

Keywords: Oxybutynine and N-Desmethyl Oxybutynine, LC-MS/MS, Validation, ICH.