DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR THE QUANTITATIVE DETERMINATION OF NITAZOXANIDE AND IT’S RELATED SUBSTANCES IN ACTIVE PHARMACEUTICAL INGREDIENTS

Objective: Nitazoxanide (NAT) is a veterinary antibiotic used for the treatment of protozoal infections in livestock and sheep. The estimation of NAT, its process impurities, and degradation products have not been reported till date. In this study, we aimed to develop and validate a chromatographic separation method for the determination of NAT, its process impurities, and related substances as per International Conference on Harmonization (ICH) guidelines. Methods: Chromatographic separation of process impurities such as 2-amino-5-nitro thiazole also called NAT-related substance A, aspirin, and degradation impurities such as salicylic acid and tizoxanide (TIZ) were separated by reverse-phase high-performance liquid chromatography using gradient elution. The separation of NAT and TIZ was most critical as they are structurally similar. The mobile phase consisted of a gradient elution containing a composition of acetonitrile and 2% orthophosphoric acid solution adjusted to pH 2.5 at a flow rate of 1 mL/min. Separation was achieved on a YMC Pack C8 L7 column with a run time of 40 min. The detection was carried out using a photodiode array detector and quantification was carried out at 210 nm. Forced degradation study was also conducted to confirm the specificity. The robustness and ruggedness of the method were evaluated. Results: The relative retention times (RRT) for aspirin, salicylic acid, NAT, and TIZ were 6.33, 6.52, 22.43, and 6.45, respectively, indicating good separation. The asymmetry factor for all the peaks is ranged from 1.1 to 1.2 indicating acceptable chromatography. The % recovery from spiked studies ranged from 90% to 110% for all the impurities when spiked in the range of 50–150% of their nominal concentrations. For all the known impurities, the limit of detection ranged from 0.06 to 0.20 parts per million (ppm) and the limit of quantification ranged from 0.19 to 0.61 ppm. Conclusion: The method was validated as per ICH guidelines and further was successfully applied for the quality evaluation of NAT in bulk active pharmaceutical ingredients.