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

Lívia Maronesi Bueno; Joanna Wittckind Manoel; Mariana Koetz; Amélia Teresinha Henriques; Martin Steppe; Yasmin Vendruscolo Piton; Marcelo Arbo; Solange Cristina Garcia; Elfrides Eva Scherman Schapoval

doi:10.22456/2527-2616.128661

Authors

Lívia Maronesi Bueno Programa de Pós-Graduação em Ciências Farmacêuticas - Universidade Federal do Rio Grande do Sul (UFRGS)
Joanna Wittckind Manoel Programa de Pós-Graduação em Ciências Farmacêuticas - Universidade Federal do Rio Grande do Sul (UFRGS)
Mariana Koetz Programa de Pós-Graduação em Ciências Farmacêuticas - Universidade Federal do Rio Grande do Sul (UFRGS)
Amélia Teresinha Henriques Programa de Pós-Graduação em Ciências Farmacêuticas - Universidade Federal do Rio Grande do Sul (UFRGS)
Martin Steppe Programa de Pós-Graduação em Ciências Farmacêuticas - Universidade Federal do Rio Grande do Sul (UFRGS)
Yasmin Vendruscolo Piton Programa de Pós-Graduação em Ciências Farmacêuticas - Universidade Federal do Rio Grande do Sul (UFRGS)
Marcelo Arbo Programa de Pós-Graduação em Ciências Farmacêuticas - Universidade Federal do Rio Grande do Sul (UFRGS)
Solange Cristina Garcia Programa de Pós-Graduação em Ciências Farmacêuticas - Universidade Federal do Rio Grande do Sul (UFRGS)
Elfrides Eva Scherman Schapoval Programa de Pós-Graduação em Ciências Farmacêuticas - Universidade Federal do Rio Grande do Sul (UFRGS)

DOI:

https://doi.org/10.22456/2527-2616.128661

Keywords:

dapagliflozin, UPLC, analytical validation, drug impurities, toxicity assay

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.

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