Abstract HPLC is the dominant separation technique for drug detection, separation and quantification. To optimize the method, a number of chromatographic parameters were analyzed, such as sample pre-treatment, selection of mobile phase, columns, detector selection. The aim of this article is to review the development, optimization and validation of the method. HPLC method development depends on the chemical structure of molecules, synthetic route, solubility, polarity, pH and pKa values and activity of functional groups, etc. Validation of HPLC method according to ICH guidelines provides information on various phases and known characteristics such as accuracy, specificity, linearity limit of detection, limit of quantification.
Keywords: Chromatography, HPLC, Reversed Phase, Validation.