High‐Sensitivity LC–MS/MS Method for Simultaneous Determination of Structurally Related Impurities 2‐Isoniazid, 3‐Isoniazid and Isonicotinamide in Isoniazid Active Pharmaceutical Ingredient

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Abstract
A sensitive, selective and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous quantification of trace impurities, namely, 2‐isoniazid, 3‐isoniazid and isonicotinamide, in isoniazid active pharmaceutical ingredient (API) at parts‐per‐million (ppm) levels. Chromatographic separation was achieved within 6 min using a Zorbax SB Aq column maintained at 40°C. The mobile phase consisted of 10 mM ammonium formate buffer (pH 4.0, adjusted with formic acid) and 0.1% (v/v) formic acid in acetonitrile (48:52, v/v), delivered at a flow rate of 1.0 mL/min. The optimized conditions provided efficient separation, reproducible peak responses and stable ionization for all analytes. The method was validated in accordance with ICH Q2 guidelines with respect to specificity, accuracy, precision, linearity, range, detection limit and quantitation limit. The limits of detection and quantification were established at 0.8 and 2.5 ppm, respectively, demonstrating the high sensitivity of the method for trace impurity determination. The validated method was successfully applied to the analysis of isoniazid bulk drug samples and enabled consistent and accurate quantification of the impurities. The developed LC–MS/MS method is rapid, robust and suitable for routine quality control testing and regulatory impurity monitoring of isoniazid API.

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