Abstract

DEVELOPMENT AND VALIDATION OF HIGHLY SENSITIVE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY ELECTRO SPRAY IONIZATION DETECTION METHOD FOR DETERMINATION OF TOLBUTAMIDE IN RAT PLASMA: APPLICATION TO PHARMACOKINETIC STUDY

Kuldeep Sharma*, N. B. Sridhara Murthy

ABSTRACT

A highly sensitive, rapid assay method has been developed and validated for the estimation of tolbutamide 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 tolbutamide and diphenhydramine (internal standard, IS) from Sprague Dawley rat plasma with single step direct dilution using organic solvent. Chromatographic separation was achieved via an isocratic mobile phase (5 mM ammonium formate 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 271.0  155.0 for tolbutamide and 256.1  152.2 for IS. Method validation was performed as per recommendations from USFDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 5 μg/mL and the linearity range extended from 5 to 600 ng/mL. Method was found to be highly sensitive in terms of detection capability with on column load of 29 pg for tolbutamide. The intra- and inter-day precisions were in the range of 0.41-6.59 and 4.28-5.70, respectively. The proposed method was applied for estimation of tolbutamide plasma concentration levels following oral administration at 400, 800 and 1600 mg/kg of test drug suspension formulation over a time period of 24 hours.

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