A Novel LC–MS/MS Approach for Ultra‐Trace Analysis of Nitroso Drug‐Related Impurities in Valaciclovir HCl: Method Development and Validation

ABSTRACT The primary research gap lies in the absence of a validated, and robust LC–MS/MS method capable of simultaneously quantifying N ‐nitroso‐ N ‐methyl valaciclovir and N ‐nitroso‐ N ‐ethyl valaciclovir within the same analytical run. Thus, a rapid, sensitive, and selective LC–MS/MS method using multiple reaction monitoring acquisition mode was developed and validated for the simultaneous N ‐nitroso‐ N ‐methyl valaciclovir and N ‐nitroso‐ N ‐ethyl valaciclovir impurities in valaciclovir hydrochloride active pharmaceutical ingredient (API). The method was established through systematic mass‐based identification and chromatographic optimization, followed by full validation in accordance with ICH guidelines. Chromatographic separation was achieved on a Shim‐pack Scepter C18 column using Formic acid buffer and acidified methanol under gradient conditions, with MRM detection in positive ESI mode. The method was linear ( r ≥ 0.99), precise (%RSD < 6%), accurate (recoveries 81.9%–113.1%), and robust, with low ppm limits of detection and quantification. The method is suitable for routine quality control and regulatory applications.