NaH2PO4·H2O (3.4 g/L) and pentane-1-sulphonic acid sodium salt (0.4 g/L) as a buffer (pH 2.5, 3, 3.5, 4) in combination with acetonitrile. It is clear from the molecular structure (Fig. 1), that all compounds do not possess a functional group which can readily ionize indicating polar in nature. Hence we started the development activity with C8 stationary phase of various manufacturers using different mobile phases. The poor resolution between Metoclopramide and ACETYLMETO and broad peak shape for Metoclopramide implies that

C8 stationary phase is not suitable for this application. Hence C18 stationary phase was chosen to improve resolution among MEK inhibitor the peaks and peak shape for Metoclopramide. The peak shape for Metoclopramide PI3K inhibitor and resolution among all components improved with Waters X-terra RP18, 150 mm × 4.6 mm, 3.5 μ columns. The resolution among related impurities and Metoclopramide was found poor using mobile phase with octane-1-sulfonic acid sodium salt. Mobile phase containing pentane-1-sulfonic acid sodium salt with ammonium phosphate instead of octane-1-sulfonic acid sodium salt gives the better resolution.

However, one unknown impurity is merging with ACETYLMETO. Ammonium phosphate is replaced with sodium phosphate buffer keeping pentane-1-sulfonic acid sodium salt as such, gives the better separation among the impurities. Initially methanol was used as an organic modifier which gives the poor baseline with baseline drift. The retention for all impurities was increased leading to inadequate resolution among the peaks. To improve the resolution among the peaks and response, acetonitrile was tried as an organic modifier. The baseline was found to be good and response for all components was improved. The peak shape for all components was also improved and hence acetonitrile the was selected as the organic modifier. The mobile phase was buffered because of the existence of ionizable groups in the chemical structure of the drug, which could

ionize at different pH values. The pH values tested were 2.5, 3.0 and 3.5. Finally, the best results were obtained at pH 3.0 ± 0.1 by adjusting with orthophosphoric acid solution. The choice of this mobile phase is justified by the excellent symmetry of the peaks and adequate retention times of Metoclopramide and its degradents. Based on the spectra of Metoclopramide and its related substances 273 nm was selected as detection wavelength for the method. The UV spectrum of Metoclopramide and its impurities were shown in Fig. 2. Different mobile phase flow rates (1.0, 1.2 and 1.4 mL/min) were investigated. The optimum flow rate for which the column plate number was maximum, with the best resolution between all compounds and a short runtime (18 min) observed was 1.2 mL/min. Column thermostat temperatures were used at 30 °C, 35 °C and 40 °C for better peak shapes, baseline and resolution.