Method development and acid degradation study of doxofylline by rp-hplc and lc-ms/ms

A simple, rapid and accurate RP-HPLC method was developed for the determination of doxofylline and aciddegradation product. The method showed a linear response for concentrations in the range of 1-200 g/ml usingacetonitrile: formic Acid (90: 10); pH-3.0 as the mobile phase with detection at 274 nm and a flow rate of 1 ml/minand retention time 2.9 min. The method was statistically validated for accuracy, precision, linearity, ruggedness,robustness, forced degradation, solution stability and selectivity. Quantitative and recovery studies of the dosage form were also carried out and analyzed; the % RSD from recovery studies was found to be less than 1. Due to simplicity, rapidity and accuracy of the method, we believe that the method will be useful for routine quality control analysis. The acid degradation product as well as pathway was characterized by LC-MS/MS.RESULT AND DISCUSSION:A sensitive, selective, precise and accurate highperformance liquid chromatographic method of analysis ofdoxofylline in both as bulk drug and in formulation wasdeveloped and validated. The mobile phase consisted ofacetonitril: 0.05M formic Acid (90: 10v/v); pH-3.0. Thedetection wavelength was 274nm. This system was found to give the sharp peak for Doxofylline (RT-2.9). The methodwas validated as per ICH guideline (Table 1). Stabilityindicating assay method in which acid stress condition wasused for quantitative estimation of doxofylline in tabletformulation and identification of acid degradation productThe separation of drug from its degradation product wereoptimized by varying the ratio and/or nature of organicmodifier. Finally method was developed using same mobilephase composed of acetonitrile: formic Acid (90: 10); pH-3.0, in that both drug and degradation product showing good elution RT-2.9 (Doxofylline) and RT-4.8 (Aciddegradation product) and m/e-149. The acid degradationproduct and pure drug were identified by LC-MS/MS inorder to establish acid degradation pathway (fig 5 and 6).