丙氨酰谷氨酰胺9个有关物质的UPLC法测定

Simultaneous Determination of Nine Related Substances in Alanyl Glutamine by UPLC

建立超高效液相色谱法测定丙氨酰谷氨酰胺(1)原料药中有关物质环-(L-丙氨酰-L-谷氨酰胺)(2)、环-(L-丙氨酰-L-谷氨酸)(3)、L-焦谷氨酰-L-丙氨酸(4)、L-焦谷氨酸(5)、D-丙氨酰-L-谷氨酰胺(6)、L-丙氨酰-L-谷氨酸(7)、L-丙氨酸(8)、L-谷氨酰胺(9)和丙氨酰丙氨酰谷氨酰胺(10)。采用Inertsil酰胺柱,流动相为乙腈-0.05mol/L磷酸二氢钾缓冲液(用磷酸调至pH4.5)(72∶28),检测波长205nm。结果1~10分别在0.20~2.00、0.50~5.00、0.02~0.20、0.25~2.50、0.30~3.00、0.10~1.00、0.40~4.00、0.10~1.00、0.05~0.50和0.10~1.00μg/ml范围内线性关系良好(r>0.999)。有关物质2~10的平均回收率(n=9)分别为99.5%、97.3%、98.1%、97.9%、98.8%、97.7%、96.8%、96.5%和97.7%,RSD分别为2.30%、2.47%、2.01%、2.13%、1.92%、1.83%、2.27%、2.63%和2.23%。2~10的检出限分别为0.01、0.005、0.02、0.01、0.01、0.02、0.02、0.01和0.01μg/ml,定量限分别为0.03、0.015、0.06、0.03、0.03、0.06、0.06、0.03和0.03μg/ml。方法准确可靠、重现性好、精密度高,可同时检测1中9种有关物质,为其质量控制提供依据。

An UPLC method was established for the determination of the following related substances: Cyclo-(Ala-Glu)(2), Cyclo-(Ala-Gln)(3), Py-Ala-OH(4), L-Pyroglutamic Acid(5), D-Alanyl-L-Glutamine(6), Ala-Gln(7), L-Alanine(8), L-Glutamine(9) and Ala-Ala-Glu(10) in alanyl glutamine(1). An Inertsil amide column was used, with the mobile phase of acetonitrile-0.05 mol/L potassium dihydrogen phosphate buffer(previously adjusted to pH 4.5 with phosphoric acid)(72∶28) at the detection wavelength of 205 nm. It was linear for 1-10 in the range of 0.20-2.00, 0.50- 5.00, 0.02-0.20, 0.25-2.50, 0.30-3.00, 0.10-1.00, 0.40-4.00, 0.10-1.00, 0.05-0.50 and 0.10-1.00 μg/ml(r>0.999). The average recoveries of 2-10 were 99.5%, 97.3%, 98.1%, 97.9%, 98.8%, 97.7%, 96.8%, 96.5% and 97.7%, with RSDs of 2.30%, 2.47%, 2.01%, 2.13%, 1.92%, 1.83%, 2.27%, 2.63% and 2.23%, respectively. The detection limit of 2-10 were 0.01, 0.005, 0.02, 0.01, 0.01, 0.02, 0.02, 0.01 and 0.01 μg/ml, and the quantitation limit were 0.03, 0.015, 0.06, 0.03, 0.03, 0.06, 0.06, 0.03 and 0.03 μg/ml. The method is accurate, reliable, reproducible, and suitable for the simultaneous determination of nine related substances in alanyl glutamine, which provides a reliable method for the quality control of alanyl glutamine.