HPLC测定利格列汀的有关物质

Determination of the Related Substances of Linagliptin by HPLC

关键词:  利格列汀  高效液相色谱  梯度洗脱  有关物质

Key words:  linagliptin  HPLC  gradient elution  related substances

摘要:

目的 建立梯度洗脱高效液相色谱法测定利格列汀原料杂质的方法。方法 色谱柱为Agilent C₁₈柱，流动相A为磷酸盐缓冲液(取磷酸二氢钠2.0 g，加水1 000 mL溶解，并用磷酸调节pH值至2.5±0.1)，流动相B为甲醇-乙腈(55∶45)，以1.0 mL·min^-1流速；检测波长为226 nm；柱温为30 ℃，进样量为10 μL。结果 8-[(3R)-哌啶-3-氨基]-7-(2-丁炔基)-3,7-二氢-3-甲基-1-[(4-甲基-2-喹唑啉基)甲基]-1H-嘌呤-2,6-二酮(杂质A)、8-[(3R)-3-氨基-1-哌啶基]-7-(3-溴-2-丁烯基)-3,7-二氢-3-甲基-1-[(4-甲基-2-喹唑啉基)甲基]-1H-嘌呤-2,6-二酮(杂质B)、8-[(3R)-3-甲酰胺基-1-哌啶基]-(7-(2-丁炔基)-3,7-二氢-3-甲基-1-[(4-甲基-2-喹唑啉基)甲基]-1H-嘌呤-2,6-二酮(杂质C)和利格列汀及其他未知杂质均能达到很好的分离，且杂质A、杂质B、杂质C与利格列汀分别在0.59~5.91 μg·mL^-1(r=0.999 9)，0.59~5.86 μg·mL^-1(r=0.999 8)、0.58~5.79 μg·mL^-1(r=0.999?5)和1.32~13.22 μg·mL^-1(r=0.999 7)内具有良好的线性关系。杂质A、杂质B、杂质C平均回收率(n=9)分别为99.12% (RSD=2.9%)、99.35%(RSD=2.4%)和98.52%(RSD=1.1%)。结论 本方法灵敏快速、准确、可靠，专属性强，可作为利格列汀的杂质检查。

Abstract:

OBJECTIVE To establish an HPLC method for the determination of related substances linagliptin. METHODS The analysis was performed on C₁₈ column by using a gradient method starting with mobile phase composed of phosphate buffer(sodium dihydrogen phosphate 2.0 g dissolved with 1 000 mL waters, adjusted by phosphate to the pH of 2.5±0.1): methanol and acetonitrile(55∶45) at the flow rate of 1.0 mL·min^-1 and the detection wavelength was set at 226 nm. RESULTS The related substances were completely separated from the main peak. The calibration curve were linear, the impurity A in range from 0.59 μg·mL^-1 to 5.91 μg·mL^-1(r=0.999 9), the impurity B in range from 0.59 μg·mL^-1 to 5.86 μg·mL^-1(r=0.999 8), the purity C in range from 0.58 μg·mL^-1 to 5.79 μg·mL^-1(r=0.999 5) and linagliptin from 1.32 μg·mL^-1 to 13.22 μg·mL^-1(r=0.999 7). The average recoveries of the impurity A, the impurity B and the impurity C mentioned above were 99.12%(RSD=2.9%), 99.35%(RSD=2.4%) and 98.52%(RSD=1.1%), respectively. CONCLUSIONS The HPLC method has been developed for quality control, and detection of related substances of linagliptin with high sensitivity and specificity.