Abstract

HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE ANALYSIS OF CLOBAZAM IN RABBIT PLASMA

Rajni Bala*, Sushil Khanna, Pravin Pawar and Sandeep Arora

ABSTRACT

In the present investigation a simple, accurate and precise HPLC method for the determination of clobazam, in rabbit plasma was developed. Mobile phase used was water and acetonitrile with orthophosphoric acid added to adjust the pH to 3.5 and was binary eluted at flow rate of 1.0 mL min-1. The chromatogram was run with UV detection at wavelength of 220 nm using Ibuprofen as internal standard. Stock solution of clobazam 1mg/ml was prepared in acetonitrile; standards were prepared using rabbit drug free plasma obtained from healthy rabbit as diluent. The calibration curve of clobazam was prepared with standards of the final concentrations of 5, 10, 25, 50 and 100 ng/ml in rabbit plasma. Working solution of ibuprofen (3000 ng/ml in acetonitrile) was prepared by dilution of stock solution on daily bases (1mg/ml in acetonitrile).The retention time for Clobazam was 4.31 min. A linear response was observed in the range of 10- 100 ng/ml with a regression coefficient of 0.996. It was concluded that the proposed method is precise, accurate and rapid and could be used for routine analysis of clobazam in plasma for bioanalytical studies.

Keywords: clobazam, rabbit plasma, HPLC, biovailability, benzodiazepines.