A Validated LC-MS/MS Method for Determination at Trace Level of Nitrosamine Impurities in Doxofylline API

Abstract

Background: The FDA looked at popular amine-based API synthesis processes and found that these processes might additionally introduce nitrosamine-hazardous substances. The current method describes trace-level quantification of Nitrosamine impurities (NDEA, NDIPA, NDIPA, NEIPA, NMPA, NDBA, NDMA and NMBA) in the Doxofylline API. Materials and Methods: With the help of Waters® ACQUITY® UPLC BEH C18 (150x4.6 mm, 5 μm) and a 0.8 mL/min flow, a gradient programme (0.1%v/v formic acid and methanol) continuing for 14 min was used to achieve separation. Using Multiple Reaction Monitoring (MRM), all nitrosamine impurities were ionized and measured in the positive polarity mode of APCI. Results: Protonated molecular ions (M+H)+ were acquainted at: m/z 75 (parent), m/z 58 (product), m/z 103 (parent), m/z 47 (product), m/z 131 (parent), m/z 89 (product), m/z 117.1 (parent), m/z 74.8 (product), m/z 147.1 (parent), m/z 117 (product), m/z 137 (parent), m/z 66 (product), m/z 159 (parent), m/z 103 (product), m/z 267.2 (parent), m/z 181.1 (product), respectively, for NDMA, NDEA, NDIPA, NEIPA, NMBA, NMPA, NDBA and Doxofylline, with their retention times observed as 4.04, 6.83, 8.97, 7.98, 4.92, 9.25, 11.06 and 7.15 min, respectively. Conclusion: The coefficient of determination (r2) for individual impurities was found to be between 0.996 and 1.000. S/N method was used to establish limits of detection and quantification, which turned out as 0.0040 μg/mL-0.0174 μg/mL and 0.0060 μg/mL-0.0262 μg/mL, respectively. Each of the validation parameters falls within the allowed ranges as per USP<1225>.