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 C18(250 mm ×4.6 mm,3 μm)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20 µL.采用Thermo Accucore AQ C18(100 mm ×4.6 mm,2.6 μm)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 100～1 000,二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1.2635.050 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、4.753 μg·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.
HPLC determination of the content and the related substances of compound amino acid injection(3AA)
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 iden-tify the impurities.The determination was performed on Capcell PAK AQ C18(250 mm ×4.6 mm,3 μm)column with 0.2 mol·L-1 sodium dihydrogen phosphate solution(adjusted pH to 2.8 with phosphoric acid)-acetoni-trile(98∶2)as mobile phase at the flow rate of 1.0 mL·min-1.The column temperature was 40 ℃,and the de-tection wavelength was 210 nm.And the injection volume was 20 µL.The LC/MSn method was performed on a Thermo Accucore AQ C18(100 mm ×4.6 mm,2.6 μm)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 posi-tive-ion scanning mode with a scan range of m/z 100-1 000,and the secondary mass spectrum was carried out by data-dependent scanning.Results:The related substances were completely separated from the main constitu-ents 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 bat-ches of samples were all process impurities introduced from the raw materials,with methionine content of 4.344 µg·mL-1,3.751 μg·mL-1,4.503 µg·mL-1,respectively,phenylalanine content of 4.636 μg·mL-1,4.889 μg·mL-1,4.753 µg·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(3 AA).
compound amino acid injection(3AA)content determinationtwo-dimensional column switchingtandem mass spectrometryrelated substancesstructural identificationcontrol of drug quality
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