High Performance Liquid Chromatographic Assay of Chlorpropamide, Stability Study and its Application to Pharmaceuticals and Urine Analysis

Chlorproamide (CLP) is a sulphonyl-urea derivative used in the treatment of type 2 diabetes mellitus. A rapid, sensitive and specific HPLC method with uv detection is described for the determination of CLP in bulk and tablet forms. The assay was performed on an Inertsil ODS 3V (150mm × 4.6mm; 5μm particle size) column using a mixture of phosphate buffer (pH 4.5), methanol and acetonitrile (30:63:7v/v/v) as mobile phase at a flow rate of 1mLmin-1 and with uv detection at 254nm. The column temperature was 30?C and injection volume was 20μL. The retention behaviour of CLP as a function of mobile phase pH, composition and flow rate was carefully studied, and chromatographic conditions, yielding a symmetric peak with highest number of theoretical plates, were optimized. The calibration curve was linear (r = 0.9999) over the concentration range 0.5 – 300 μgmL-1. The limits of detection (LOD) and quantification (LOQ) were found to be 0.1 and 0.3 μgmL-1, respectively. Both intra-day and inter-day precisions determined at three concentration levels were below 1.0% and the respective accuracies expressed as %RE were =1.10%. Assays were performed under slightly altered chromatographic conditions, and the results were not significantly different from those obtained under optimum conditions, reflecting the robustness of the method. Inter-equipment and interanalysts deviations were insignificant testifying the ruggedness of the method. The method was validated for selectivity via placebo blank and synthetic mixture analyses. The method was applied to determine CLP in tablets and the results were comparable with those obtained by a reference method with respect to accuracy and precision. As part of stress testing, CLP was subjected to forced degradation under acid and base-induced hydrolysis, oxidation, heat and light, followed by assay using the developed method, and the results indicated that the drug was prone to oxidative degradation, but inert to other stress-conditions. The method was sensitive and selective enough to determine the drug content in spiked urine, and the results were satisfactory.