Linagliptina: desenvolvimento de método indicativo de estabilidade por cromatografia líquida de alta eficiência e estudos de segurança biológica

Abstract

Linagliptin (LGT) is an inhibitor of DPP4 used for glycemic control of type 2 mellitus diabetes. Since the drugs used in the production process of the pharmaceutical formulations are not considered totally pure, the Regulatory Guides describing the need of the drug quantification and impurities potentials. Therefore, the aim of this study was to develop a method by highperformance liquid chromatography to quantify the drug and three potential synthetic impurities and biological safety studies. The development and validation of the method for LGT and the three impurities were carried out by HPLC coupled to a photodiode array detector. The chromatographic separation was performed in a RP8 column (150 x 4.6 mm, 5 μm), at 30°C. The separation was performed by means of a linear gradient elution (eluent A: formic acid 0.1% pH 3.5; eluent B: acetonitrile). The gradient obeys the following sequence: 3070% of B (0 - 4 minutes) and 7030 of B (4,01 - 8 minutes) at a flowrate of 0.6 mL min1 and run time of 10 minutes. The injection volume was 20 μL and detection at 294, 278, 268 e 280 nm for LGT, impurity 1, 2 and 3, respectively. The method showed selectivity and specific for the LGT and synthetic impurities, therefore no interference of degradation products (UV-A, basic hydrolysis (sodium hydroxide 1.0 mol L1) and acid (hydrochloric acid 1.0 mol L1), peroxide (30%), temperature (60 ° C) and acidic and basic hydrolysis under thermal conditions (80° C)) and the excipients in the quantification of the drug. The response was linear from 2.41 to 144.0 μg mL1 (r = 0.9996) for the LGT and in the range of 0.06 to 3.6 μg mL1 (r = 0.9963, 0.9994 and 0.9991 for impurity 1, 2 and 3, respectively ). The method showed accuracy, with recovery levels of 99.79, 93.12, 92.92 and 93.99% for LGT and impurity 1, 2 and 3, respectively. The intra and inter-day precision was suitable (RSD 1.36, 4.14, 3.50 and 4.08% for LGT and impurity 1, 2 and 3, respectively) and the robustness showed no significant differences with small changes in the analytical conditions (pH of the mobile phase, temperature, flow-rate and detector). The kinetics of degradation in alkaline conditions associated with temperature (80° C), showed results of the first order. The evaluation of the biological was performed by cytotoxicity, mutagenicity and genotoxicity through the cell viability test, micronucleus and comet, respectively. In biological safety testing LGT had only cytotoxic activity at a concentration of 10 times the maximum plasma concentration, while the impurity 1 and 3 set out cytotoxic, mutagenic and genotoxic in a concentration equivalent to 10% maximum plasma concentration of the drug and impurity 2 showed cytotoxicity and genotoxicity in concentration equivalent to 10% of the maximum plasma concentration of the drug. According to the results, it can be seen that the proposed method by HPLC is stability-indicating and suitable for quantitative analysis of the drug and synthetic impurities. Furthermore, it was observed that the LGT and synthetic impurities are in accordance with biological safety studies, if they are used in appropriate concentration.