Highly Sensitive LC–QTOF‐MS Method Development and Validation for Trace Level Determination of N ‐Nitroso Tizanidine in Tizanidine Drug Substance and Formulations

Abstract

Nitrosamine drug substance–related impurities have been a central focus of regulatory scrutiny due to their potential mutagenic and carcinogenic effects. Among these, N ‐nitroso tizanidine ( N ‐NTZD) is a possible impurity that may form during the manufacturing, processing, or storage of tizanidine (TZD) drug substance and drug products. Given the mutagenic nature of these impurities, ensuring that patient safety requires the development of highly sensitive and reliable analytical methods capable of detecting them at trace levels. To address this need, a high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was developed and validated for trace‐level detection. Chromatographic separation was achieved using a Poroshell EC C18 column with gradient elution, employing 0.1% formic acid in water and methanol as mobile phase components. Detection was performed using positive electrospray ionization to enable accurate mass measurement. The method was validated in accordance with regulatory guidelines, covering specificity, sensitivity, linearity, precision, accuracy, and solution stability. Results demonstrated excellent linearity and accuracy across the range from the limit of detection up to 150% of the specification level. Sample testing confirmed the necessity of such a highly sensitive quantification method to strengthen control measures and enhance the safety of pharmaceutical products.