Abstract

RP-HPLC ANALYTICAL METHOD FOR THE QUANTITATION OF TOLBUTAMIDE IN FORMULATION VEHICLE: APPLICATION TO HOMOGENECITY AND STABILITY EVALUATION IN PRECLINICAL STUDY FORMULATION SAMPLES

Kuldeep Sharma* and N. B. Sridhara murthy

ABSTRACT

A novel, simple, specific, sensitive and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for estimation of tolbutamide. The HPLC method includes use of Diphenhydramine Hydrochloride as an internal standard (IS). The chromatographic analysis was performed using Waters Alliance High Performance Liquid Chromatography system equipped with UV detector and PC based data system with Empower Software. Chromatographic separation was achieved on symmetry sheild, RP 18, 5 μM (Waters) column maintained at 40ºC temperature using gradient mobile phase composition with (0.01 M Ammonium Acetate in Milli- Q Water, pH adjusted to 5 using Acetic Acid) and acetonirile at a flow rate of 1.00 mL/min with a total run time of 10 min. The eluate absorbance was monitored using an UV detector set at 258 nm wavelength. Method validation was performed as per ICH guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 25 to 2508 μg/mL (r2 ≥ 0.997). Precicion & accuracy batches analysed on 3 different days revealed accuracy of tolbutamide in vehicle within the range of 100 to 114%, 91.5 to 107% and 91.2 to 110%, respectively and Inter day (i.e. within batch) accuracy was 107, 98.9 and 98.7 % at Low, Middle and High QC levels, respectively. The validated HPLC method was successfully applied for assay, homogenity and stability determination of preclinical study formulation samples.

Keywords: Tolbutamide; HPLC; Method validation; Homogeneity; Pre-clinical Formulation.