DETERMINATION OF DIPHENOXYLATE IN RAT PLASMA BY GRADIENT ELUTION LC-ESI-MS.

A sensitive and simple liquid chromatography/electrospray mass spectrometry (LC-ESI-MS) method for determination of diphenoxylate in rat plasma using one-step protein precipitation was developed and validated. After addition of estazolam as internal standard (IS), protein precipitation by acetonitrile was used as sample preparation. Chromatographically separation was achieved on an SB-C18 (2.1 mm × 50 mm, 3.5 µm) column with acetonitrile-0.1% formic acid as the mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; selected ion monitoring (SIM) mode was used to quantification using target fragment ionsm/z453.1 for diphenoxylate andm/z294.8 for the IS. Calibration plots were linear over the range of 5–1000 ng/mL for diphenoxylate in rat plasma. Lower limit of quantification (LLOQ) for diphenoxylate was 5 ng/mL. Mean recovery of diphenoxylate from plasma was in the range of 96.69–98.27%. RSD of intra-day and inter-day precision were both less than 9%. This method is simple and sensitive enough to be used in pharmacokinetic research for determination of diphenoxylate in rat plasma. [ABSTRACT FROM AUTHOR]