Trace-level quantification of N-Nitroso diclofenac in diclofenac sodium by LC–MS/MS using an in-source fragmentation based MRM strategy

This study describes the development and validation of a liquid chromatography- tandem mass spectrometry (LC-MS/MS) method for quantification of N-Nitroso diclofenac in diclofenac sodium drug substance and tablet dosage forms. Chromatographic separation was achieved on a Phenomenex Kinetex C18 column using gradient elution with 5 mM ammonium formate in water containing 0.1% formic acid and methanol at a flow rate of 0.5 mL/min. Detection was carried out in positive electrospray ionization mode using multiple reaction monitoring (MRM) transitions at m/z 294.95→241.98 (quantifier) and m/z 294.95→213.98 (qualifier). To facilitate selective trace-level quantification, the analytical strategy incorporated in-source fragmentation of the labile N-NO bond generating a chemically stable denitrosated precursor ion at m/z 294.95 (loss of NO, 30 Da). The method demonstrated excellent sensitivity with limits of detection and quantification of 0.125 ng/mL and 0.250 ng/mL, respectively. The calibration curve was linear over the concentration range of 0.249–3.735 ng/mL with a correlation coefficient of 0.99997. The validated LC–MS/MS method is suitable for routine quality control and trace-level determination of N-Nitroso diclofenac in pharmaceutical matrices.