HPLC–DAD Method for Simultaneous Determination of Dipyrone (Metamizole) and Caffeine in Tablets and Identification of Major Degradation Product by Direct Infusion ESI–MS

A new chromatographic method for simultaneous determination of dipyrone (metamizole) and caffeine in tablets was developed and validated. Excellent resolution and quantification were obtained using a reverse-phase Acclaim® C18 column (250.0 × 4.6 mm; 5.0 μm particles) with acetonitrile/water (65:35 v/v) as mobile phase, adjusted to pH 7.5 using drops of water:triethylamine (99:1 v/v). The elution mode was isocratic with flow rate of 1.0 mL min−1, and ultraviolet (UV) detection was performed at 273 nm. The ranges of linearity for dipyrone and caffeine were 5.0–35.0 and 1.0–7.0 μg mL−1, respectively. Intra- and interday precision showed relative standard deviation (RSD) <2% and analysis of variance (ANOVA) p value >0.05, respectively. The method proved to be precise, sensitive, and exact with low limits of detection and quantification and average recovery of 99.3 and 100.8%. The factorial model of Youden was used to analyze the robustness of the method, while identification of critical factors was conducted by two-tailed t test with 95% confidence interval. The method was shown to be selective, detecting presence of analytes in degradation products originating from study of hydrolytic stability at different pH values (5.0, 6.0, 7.0, and 9.0) at room temperature. Identification of product degradation by direct injection electrospray ionization (ESI) mass spectroscopy (MS) revealed the presence of a fragment ion at m/z 187 and ions corresponding to [M + H]+ and [M + Na]+ at m/z 218 and 240, respectively, confirming the identity of the 4-methylaminoantipyrine product related to dipyrone.