Heleno Ferreira Raquel Balestri, Duarte Jonathaline Apollo, Ferreira Flávio Dias, de Oliveira Luis Flávio Souza, Machado Michel Mansur, Malesuik Marcelo Donadel, Paula Fávero Reisdorfer, Steppe Martin, Schapoval Elfrides Eva Shermann, Paim Clésio Soldateli
Laboratório de Pesquisa em Desenvolvimento e Controle de Qualidade, Universidade Federal do Pampa, Uruguaiana, RS, Brazil.
Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Pampa, Uruguaiana, RS, Brazil.
J Anal Methods Chem. 2019 Mar 3;2019:7534609. doi: 10.1155/2019/7534609. eCollection 2019.
A stability-indicating LC method was developed for quantification of linagliptin (LGT) and three synthetic impurities. The method utilizes a Thermo Scientific® RP-8 column (100 mm × 4.6 mm; 5 m) with the PDA detector for quantitation of impurities. A mixture of 0.1% formic acid with pH 3.5 (A) and acetonitrile (B) was used as the mobile phase at a flow rate of 0.6 mL·min with gradient elution. The percentage of mobile phase B increases from 30% to 70% over 5 min and decreases from 70% to 30% between 5 and 8 min. The method was validated according to International Council for Harmonization (ICH) guidelines. The LOD values obtained were 0.0171 g·mL and 0.015 g·mL for LGT and impurities, respectively. The LOQ values were 0.06 g·mL for LGT and impurities. In all cases, the correlation coefficients of LGT and impurities were >0.999, showing the linearity of the method. The % recovery of the LGT and added impurity were in the range of 92.92-99.79%. The precision of the method showed values less than 1.47% for LGT and less than 4.63% for impurities. The robustness was also demonstrated by small modifications in the chromatographic conditions. The selectivity was evidenced because the degradation products formed in stress conditions did not interfere in the determination of LGT and impurities. Toxicity prediction studies suggested toxicity potential of the impurities, which was confirmed using biological safety studies in vitro.
开发了一种用于定量利格列汀（LGT）和三种合成杂质的稳定性指示液相色谱法。该方法采用赛默飞世尔科技®RP - 8柱（100 mm×4.6 mm；5 µm），配备PDA检测器用于杂质定量。以0.1%甲酸（pH 3.5）与乙腈的混合物（A）作为流动相，流速为0.6 mL·min，采用梯度洗脱。流动相B的百分比在5分钟内从30%增加到70%，在5至8分钟之间从70%降至30%。该方法根据国际协调理事会（ICH）指南进行了验证。LGT和杂质的检测限（LOD）值分别为0.0171 μg·mL和0.015 μg·mL。LGT和杂质的定量限（LOQ）值均为0.06 μg·mL。在所有情况下，LGT和杂质的相关系数均>0.999，表明该方法具有线性。LGT和添加杂质的回收率在92.92 - 99.79%范围内。该方法的精密度显示，LGT的值小于1.47%，杂质的值小于4.63%。通过对色谱条件的微小改变也证明了该方法的稳健性。选择性得到了证明，因为在强制条件下形成的降解产物不会干扰LGT和杂质的测定。毒性预测研究表明杂质具有潜在毒性，这在体外生物安全性研究中得到了证实。
