A novel liquid chromatography with quadrupole time‐of‐flight‐tandem mass spectroscopy method for ultra‐trace level identification and quantification of the genotoxic impurity 2,6‐diamino‐5‐nitropyrimidin‐4(3H)‐one in valganciclovir hydrochloride

Abstract

In the present study, the main objective is to develop an analytical method for ultra‐trace level measurement of 2,6‐diamino‐5‐nitropyrimidin‐4(3H)‐one (DMNP) in valganciclovir hydrochloride (VAL) using liquid chromatography‐quadrupole time‐of‐flight‐tandem mass spectroscopy (LC–QTOF–MS/MS). In the early stages of guanine synthesis, DMNP is formed, and guanine is known to be the key starting material for the synthesis of VAL. Taking into consideration DMNP potential genotoxicity, this analytical method has been developed. This method is time saving and suitable for confirming the masses of parent and fragment ions by MS and MS/MS further fragmentation. An isocratic program and Acquity UPLC HSS cyano column (100 × 2.1 mm × 1.8 μm) were used to achieve optimal separation between VAL and the DMNP impurity. A 0.1% ammonia solution in Milli‐Q water was used as mobile phase A, and methanol was used as mobile phase B in the ratio 90:10 v/v in isocratic mode. In accordance with the International Conference on Harmonization's requirements, the developed method was validated. The detection and quantification levels were found to be 0.028 and 0.083 ppm respectively. The DMNP impurity is linear from 0.083 to 1.245 ppm levels with correlation coefficient (R2) of 0.9960. The recoveries were found to be 97.0–107.9%.