High-performance thin-layer chromatography-based method development for the analysis of 4-methoxy-2-nitroaniline as potential genotoxic impurity

Omeprazole contains benzimidazole substitute, and it acts as a proton-pump inhibitor used for gastroduodenal ulcers. 4-Methoxy-2-nitroaniline is a potent genotoxic impurity present in omeprazole which may affect drug efficacy and safety. Our objective was to develop a high-performance thin-layer chromatography (HPTLC) method for the identification and determination of 4-methoxy-2-nitroaniline impurity present in omeprazole. For separation of impurity and drug substance, various experimental conditions were studied, by using the suitable mobile phase methanol‒n-hexane‒ethyl acetate (0.5:5:5.5, V/V) based on its polarity for drug and impurity. The detection wavelengths used for omeprazole and its impurity (4-methoxy-2-nitroaniline) were 291 nm and 223 nm, and the retention factor (RF) values obtained were 0.25 ± 0.02 and 0.89 ± 0.02, respectively. The method showed accurate linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ) results. Linearity range was taken 100‒500 ng per band for omeprazole and 10–50 ng per band for the impurity. The percent relative standard deviation (%RSD) values were obtained below 2%. The recovery study indicated that the method was accurate to trace impurity in drug. LOD and LOQ values of omeprazole were 3.2 ng per band and 9.8 ng per band and for impurity 0.41 ng per band and 1.25 ng per band. These values indicate that the method is highly sensitive.