DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING RP-HPLC METHOD FOR QUANTITATIVE DETERMINATION OF NITROXYNIL AND ITS IMPURITIES AS PER ICH GUIDELINES

Objective: An isocratic elution method for the determination of Nitroxynil (NTX), its related substances was developed and validated as per ICH guidelines. Methods: The process impurity4-Hydroxy-3-nitrobenzonitrile (NTX impurity-1) and key starting material 4-cyanophenol (4-CP) along with NTX were separated using a 50: 50%v/v composition of acetonitrile and 1% Orthophosphoric acid solution as mobile phase at a flow rate of 1 ml/min under isocratic mode. Separation was achieved on a Kromasil Eternity XT C18,4.6×250 mm, 5µ within a 30 min run time. The detection was made using a photo diode array detector and quantification was carried out at 244 nm. Forced degradation study was also conducted to confirm the specificity of the method. The method was found to be robust and rugged and complied with ICH guidelines. Results: The calibration curve ranged from 0.5 – 7.5 ppm for NTX and its impurities. The Limit of Detection (LOD) for NTX is 0.09 ppm and Limit Of Quantification (LOQ) was 0.28 ppm. The LOD for NTX impurity-1 and 4-CP were 0.02 and 0.04 ppm, respectively, while the corresponding LOQ values were 0.06 and 0.13 ppm. In samples spiked with NTX and its impurities, the mean recovery was approximately 102.5±2%. Conclusion: The method confirms its stability indicating nature was successfully applied for quality evaluation of NTX in bulk API. The method was validated as per guidelines of the International Conference on Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH).