Quality-by-Design-Driven Development and Validation of a Sensitive HPLC Method for Trace-Level Determination of Nitrosamine Impurity in a Muscle Relaxant Drug Formulation.

N-Nitroso impurities are very hazardous genotoxic carcinogens that can occur as by-products or breakdown products in pharmaceutical compounds. In this study, a sensitive and quality-by-design-based high-performance liquid chromatography method approach was developed and validated for the trace-level determination of N-nitroso tizanidine in pharmaceutical formulations. Chromatographic separation was achieved using an XSelect HSS T3 column (100Å, 150 × 4.6 mm, 3.5 μm) and an isocratic mobile phase made up of 0.1% formic acid buffer and methanol (50:50 v/v) at a flow rate of 0.6 mL/min to separate the samples. The detection wavelength was set at 230 nm. The temperature of the column and sample was kept at 50°C and 10°C, respectively. A Box Behnken design was employed to evaluate the effects of critical method parameters, flow rate, column temperature, and methanol concentration on critical quality attributes, including retention time, tailing factor, and theoretical plate count. Quadratic models demonstrated good statistical significance with high correlation coefficients and nonsignificant lack-of-fit (p > 0.05). The method was validated in accordance with ICHQ2 (R2) guidelines and exhibited excellent specificity, linearity, precision, accuracy, robustness, and sensitivity. The developed QbD-based method is suitable for routine quality control and regulatory monitoring of N-NTIZ in both active pharmaceutical ingredients and finished dosage forms.