Abstract

DEVELOPMENT AND VALIDATION OF A HIGHLY SENSITIVE LC-MS/MS-ESI METHOD FOR THE DETERMINATION OF DIPHENHYDRAMINE IN RAT PLASMA: APPLICATION TO PHARMACOKINETIC STUDY

Vinay Dhiman* and N. B. Sridhara Murthy

ABSTRACT

A highly sensitive, rapid assay method has been developed and validated for the estimation of diphenhydramine in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electro spray ionization in the positive-ion mode. The assay procedure involves extraction of diphenhydramine and tolbutamide (internal standard, IS) from Sprague Dawley rat plasma with single step direct dilution with organic solvent. Chromatographic separation was achieved using an isocratic mobile phase (5 mM Ammonium acetate with 0.05% formic acid:acetonitrile :: 30:70, v/v) at a flow gradient of 0.4 to 0.7 mL/min on an Atlantis dC18 column (maintained at 40 ± 1°C) with a total run time of 3.0 min. The MS/MS ion transitions monitored were 256.1  152.2. for diphenhydramine and 271.0  155.0 for for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 2.01 ng/mL and the linearity range extended from 2.01 ng/mL to 1759 ng/mL. Method was highly sensitive with on column load of 29 pg/mL detection capability. The intra- and inter-day precisions were in the range of 1.06 to 8.82%and 3.02 to 10.1%, respectively.

Keywords: Diphenhydramine; LC-MS/MS; method validation; rat plasma; pharmacokinetics.