HPLC法测定复方氨基酸注射液(3AA)中主成分及有关物质含量

HPLC determination of the content and the related substances of compound amino acid injection(3AA)

目的:建立复方氨基酸注射液(3AA)的主成分和有关物质测定的高效液相色谱方法。方法:采用反相高效液相色谱法并结合二维柱切换LC/MSn法对制剂中主要杂质进行分离和结构鉴定,建立复方氨基酸注射液(3AA)中缬氨酸、异亮氨酸和亮氨酸及其有关物质含量检测方法。采用Capcell PAK AQ C_(18)(250 mm×4.6 mm,3μm)色谱柱,以0.2 mol·L^(-1)磷酸二氢钠溶液(用磷酸调pH至2.8)-乙腈(98∶2)为流动相,流速1.0 mL·min^(-1),柱温40℃,检测波长210 nm,进样量20μL。采用Thermo Accucore AQ C18(100 mm×4.6 mm,2.6μm)色谱柱,以0.1%甲酸溶液为流动相A,以0.1%甲酸溶液-乙腈为流动相B,流速0.4 mL·min^(-1),柱温40℃;质谱条件采用ESI电离源,正离子扫描模式,扫描范围为m/z 100~1000,二级质谱采用数据依赖型扫描开展。结果:主峰和相邻杂质峰分离度良好,缬氨酸的线性范围为1.263~5.050 mg·mL^(-1),平均回收率(n=9)为99.0%;异亮氨酸的线性范围为1.350~5.402 mg·mL^(-1),平均回收率(n=9)为99.4%;亮氨酸的线性范围为1.647~6.588 mg·mL^(-1),平均回收率(n=9)为99.5%。3批样品中主要杂质均为原料引入的工艺杂质,其中甲硫氨酸含量分别为4.344、3.751、4.503μg·mL^(-1),苯丙氨酸含量分别为4.636、4.889、4.753μg·mL^(-1)。其他单个未知杂质分别为0.01%、0.02%、0.01%。结论:经方法学验证,本方法可用于复方氨基酸注射液(3AA)的质量控制。

Objective:To establish an HPLC method of the content and related substances of compound amino acid injection(3AA).Methods:RP-HPLC was adopted to determine compound amino acid injection(3AA),combining with the use of two-dimensional column switching-LC/MSn method was applied to separate and identify the impurities.The determination was performed on Capcell PAK AQ C_(18)(250 mm×4.6 mm,3μm)column with 0.2 mol·L^(-1)sodium dihydrogen phosphate solution(adjusted pH to 2.8 with phosphoric acid)-acetonitrile(98∶2)as mobile phase at the flow rate of 1.0 mL·min^(-1).The column temperature was 40℃,and the detection wavelength was 210 nm.And the injection volume was 20μL.The LC/MSn method was performed on a Thermo Accucore AQ C18(100 mm×4.6 mm,2.6μm)column with 0.1%formic acid solution as mobile phase A,0.1%formic acid solution-acetonitrile as mobile phase B,at a flow rate of 0.4 mL·min^(-1),and at a column temperature of 40℃.The mass spectrometry conditions were performed using an ESI ionisation source in the positive-ion scanning mode with a scan range of m/z 100-1000,and the secondary mass spectrum was carried out by data-dependent scanning.Results:The related substances were completely separated from the main constituents in RP-HPLC.The standard curve of valine was linear over the range of 1.263-5.050 mg·mL^(-1),with the average recovery of 99.0%(n=9).The standard curve of isoleucine was linear over the range of 1.350-5.402 mg·mL^(-1),with the average recovery of 99.4%(n=9).The standard curve of leucine was linear over the range of 1.647-6.588 mg·mL^(-1),with the average recovery of 99.5%(n=9).The main impurities in the three batches of samples were all process impurities introduced from the raw materials,with methionine content of 4.344μg·mL^(-1),3.751μg·mL^(-1),4.503μg·mL^(-1),respectively,phenylalanine content of 4.636μg·mL^(-1),4.889μg·mL^(-1),4.753μg·mL^(-1),respectively.The maximum single impurity contents were 0.01%,0.02%and 0.01%,respectively.Conclusion:The method is proved by the methodology validation that it can be used for the quality control of compound amino acid injection(3AA).