HPLC法测定复方氨基酸(15)双肽(2)注射液中有关物质的含量

Determination of related substances in compound amino acids (15) and dipeptides (2) injection

目的:建立测定复方氨基酸(15)双肽(2)注射液中有关物质含量的高效液相色谱法。方法:采用Ultimate AQ-C_(18)(4.6 mm×250 mm,5μm)色谱柱,以0.01 mol·L^(-1)磷酸氢二铵溶液(pH 2.0)-甲醇(98∶2)为流动相,流速为0.7 mL·min^(-1),检测波长为205 nm,进样量为50μL。结果:在选定的色谱条件下,环-(甘氨酰谷氨酰胺)峰、焦谷氨酸峰和环-(甘氨酰酪氨酸)峰与各自相邻色谱峰可以有效分离;环-(甘氨酰谷氨酰胺)、焦谷氨酸、环-(甘氨酰酪氨酸)分别在7.863～52.42μg·mL^(-1)、5.256～35.04μg·mL^(-1)、0.939 3～6.262μg·mL^(-1)的范围内线性关系良好(r=0.999 9);精密度试验的RSD值均小于0.2%;重复性试验的RSD值均小于2.0%;环-(甘氨酰谷氨酰胺)、焦谷氨酸和环-(甘氨酰酪氨酸)定量限分别为3.15、14.0和7.5 ng,检测限分别为0.95、7.0和2.5 ng;加样回收率分别为103.0%、101.5%和103.3%(n=9),RSD值分别为1.0%、0.9%和0.9%。9批样品环-(甘氨酰谷氨酰胺)的量为383～492 mg·L^(-1),焦谷氨酸的量为191～315 mg·L^(-1),环-(甘氨酰酪氨酸)的量为39～49 mg·L^(-1)。结论:该方法专属性强,结果准确,可用于复方氨基酸(15)双肽(2)注射液中有关物质的测定,能有效地反应药品质量。

Objective:To establish an HPLC method for the determination of related substances in compound amino acids (15)and dipeptides (2)injection.Methods :The test was performed in Ultimate AQ-C18 column (4.6 min× 250 mm,5 μm)under the elution of 0.01 mol·L^-1 diammonium hydrogen phosphate buffer solution (pH 2.0)-methanol (98:2).The flow rate was 0.7 mL ~rain-1,the detection wavelength was set at 205 nmand the injection volume was 50 μL.Results:The peaks of cyclo-(Gly-Gln),pyroglutamic acid and cyclo-(Gly-Tyr)were effectively separated from their adjacent chromatographic peaks under the developed chromatographic condition.The linear ranges of cyclo-(Gly-Gln ),pyroglutamic acid and cyclo-(Gly-Tyr)fell into 7.863 -52.42,5.256 - 35.04,0.939 3 -6.262 μg·mL^-1 with r =0.999 9,respectively.The RSDs of precision test were less than 0.2%and the RSDs of repeatability test were less than 2.0%.The LOQ values of cyclo-(Gly-Gln),pyroglutamic acid and cyclo-(Gly-Tyr)were 3.15,14.0 and 7.5 ng,respectively,LOD values of cyclo-(Gly-Gln),pyroglutam- ic acid and cyclo-(Gly-Tyr)were 0.95,7.0 and 2.5 ng,respectively.The average spiked recoveries of cyclo-(Gly- Gln),pyroglutamic acid and cyclo-(Gly-Tyr)were 103.0%,101.5 % and 103.3 %(n =9)with RSDs as 1.0%, 0.9%and 0.9%,respectively.The contents of cyclo-(Gly-Gln),pyroglutamic acid and cyclo-(Gly-Tyr)in 9 batches of samples ranged from 383 -492,191 -315,and 39 -49 μg·mL^-1,respectively.Conclusion:The established method has good specificity and accuracy.It can be used for the determination of the related substances in compound amino acids (15)and dipeptides (2)injection.