Simultaneous analysis of dapagliflozin and its three related impurities by stability-indicating UPLC method and in vitro toxicity evaluation

Abstract

A new stability- indicating analytical method by UPLC was developed for the simultaneous determination of dapagliflozin and three of its synthesis impurities. A Waters® Acquity UPLC H- Class model was used for method development and validation. The separation was achieved in a Zorbax phenyl column (50 x 3.0 mm, 1.8 μm), using a mixture of acetonitrile: water (70:30, v/v) as mobile phase in isocratic mode. All the peaks were well detected by a photodiode array detector (PDA) at 230 nm. The method was properly validated according ICH guidelines with respect to linearity, specificity, precision, accuracy and robustness. The calibration curves of each analyte showed determination coefficients (r2) > 0.99 and the method was linear at the concentrations range 30-70 μg/mL for dapagliflozin and 1-10 μg/mL for the impurities. Lastly, this UPLC method presented low limits of detection (LOD) and quantification (LOQ) for both dapagliflozin and impurities, being a technique with high sensitivity. The toxicity evaluation of dapagliflozina and its related impurities were evaluated using 3T3 cells. MTT reduction and neutral red uptake assays were performed in cytotoxicity tests. In addition, mitochondrial membrane potential (ΔψM), measurement of intracellular reactive oxygen and DNA damage (measured by comet assay) were evaluated. The impurity 3 showed significant damage in cytotoxicity tests at a concentration of 0.5 µM, being even more expressive at higher concentrations. On the other hand, under the conditions tested, DNA damage was not detected and the compounds tested do not induce significant cell death.