Challenges in the HPLC analysis of hydrolytically unstable compounds: Metamizole and 4-methylaminoantipyrin case...

The on-column hydrolytic degradation of metamizole to impurity C in aqueous mobile phases represents a major source of analytical errors and false-positive results. Accurate and precise determination of impurity C is critical because this analyte is a direct precursor to a monitored genotoxic nitrosamine impurity. To resolve this issue, a novel isocratic non-aqueous hydrophilic interaction liquid chromatography method was developed on a triazole-bonded stationary phase. The separation was performed on Cosmosil HILIC column column (150 × 3.0 mm, 2.5 µm). The mobile phase was composed of 10 mM ammonium formate and 5 mM formic acid in a mixture of methanol and acetonitrile (3:97, v/v). An isocratic flow rate of 0.8 mL/min was utilized at a column temperature of 10 °C. A short analysis time of 3 min was achieved under optimized conditions. Impurity C exhibited minimal retention (k = 0.4), whereas metamizole was strongly retained on the column (k > 100). Due to this distinct selectivity, on-column artifact formation was completely prevented throughout the chromatographic run. To manage metamizole accumulation on the stationary phase, a periodic column regeneration step was introduced after every 30 sample injections. The method was coupled with mass spectrometric detection in positive electrospray ionization mode. Quadrupole mass analyser was employed in selected ion monitoring mode using a target ion at m/z = 218 as [M+H]⁺. A low limit of quantification of 2 ng/mL (2 ppm) and excellent linearity (r = 0.9991) were obtained. The calculated back-fit bias remained between -2.6% and 2.3% across the entire calibrated range. Critical verification parameters demonstrated full compliance with International Council for Harmonisation guidelines. The practical utility of the procedure was confirmed through the analysis of two production batches of metamizole.