Simultaneous Determination of Two Potential Genotoxic Impurities in L-Malic Acid by Liquid Chromatography-Tandem Mass Spectrometry

Background: Genotoxic impurities (GTIs) are produced during the synthesis of active pharmaceutical ingredients and pharmaceutical excipients. L-malic acid, an important active pharmaceutical ingredient and excipient, is widely used in the pharmaceutical industry. However, the detection of potential GTIs in L-malic acid has not been reported. Objective: This study aims to establish a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine the concentration of potential GTIs in L-malic acid, including N-nitroso-aspartic (NASP) and 2-chlorosuccinic acid (CSA). Methods: In this work, GTIs were separated by a reverse-phase Accucore C18 column (100 mm × 2.1 mm, 2.6 μm), with gradient elution using methanol and 0.05% ammonia. The multiple reaction monitoring (MRM) negative mode was used to detect GTIs, with transitional ion pairs of m/z from 131.6 to 88.0 for NASP, and from 150.9 to 70.9 for CSA. Results: The limit of detections (LODs) of NASP and CSA were 2 ng/mL (0.02 ppm) and 5 ng/mL (0.05 ppm), respectively. Both the limit of quantifications (LOQs) of NASP and CSA were 20 ng /mL (0.2 ppm). Good linearity of calibration curves in the concentration ranging from 10 to 500 ng/mL was obtained. The precision was less than 5%, and the intermediate precision was less than 10%. The accuracy ranged from 95.4% to 102.4%, with a relative standard deviation (RSD) of less than 5%. Also, the solution's stability and robustness were acceptable. Conclusion: Compliant with requirements from (International Council for Harmonization) ICH guidelines, this method can be used for routine analysis and stability studies for GTIs’ levels in pharmaceutical quality control.