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1.263~5.050 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、4.503 μg·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。
左利民, 茹仙古丽·依明, 郭鑫, 肖菁, 徐士婕, 赵婷, 连晓芳, 刘惠一, 周怡, 山广志
. HPLC法测定复方氨基酸注射液(3AA)中主成分及有关物质含量*[J]. 药物分析杂志, 2024
, 44(7)
: 1161
-1168
.
DOI: 10.16155/j.0254-1793.2023-0795
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 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) -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 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 positive-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 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 4.344 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(3AA).
[1] UNGER N, HOLZGRABE U. Stability and assessment of amino acids in parenteral nutrition solutions[J]. J Pharm Biomed Anal, 2018, 147: 125
[2] YARANDI SS, ZHAO VM, HEBBAR G, et al. Amino acid composition in parenteral nutrition: what is the evidence?[J]. Curr Opin Clin Nutr Metab Care, 2011, 14(1): 75
[3] 陈鼎雄, 林立. HPLC-ELSD测定复方氨基酸注射液(3AA)中三组分的含量[J]. 药物分析杂志, 2007, 27(10): 1649
CHEN DX, LIN L. HPLC-ELSD determination of three components in compound amino acid injection(3AA)[J]. Chin J Pharm Anal, 2007, 27(10): 1649
[4] 张莹, 黄哲甦. 异硫氰酸苯酯柱前衍生法测定复方氨基酸注射液(3AA)中氨基酸含量[J]. 天津药学, 2013, 25(4): 17
ZHANG Y, HUANG ZS. Determination of compound amion acid injection(3AA) by HPLC with precolumn phenylisothiocyanate derivatization[J]. Tianjin Pharm, 2013, 25(4): 17
[5] 卫生部药品标准. 二部. 第六册[S]. 1996: 78
Drug Specifications Promulgated by the Ministry of Public Health. P R China. Part 2. Vol 6[S]. 1996: 78
[6] 中华人民共和国药典2020年版. 二部[S]. 2020: 1832, 509, 1010
ChP 2020. Vol Ⅱ[S]. 2020: 1832, 509, 1010
[7] EP 11.0 [S]. 2023: 4346, 3136, 3213,109
[8] USP 43-NF 38[S]. 2020: 4571, 2422, 2565
[9] JP 18[S]. 2021: 1897, 1196, 1251
[10] WAHL O, HOLZGRABE U. Amino acid analysis for pharmacopoeial purposes[J]. Talanta, 2016, 154: 150
[11] ZHAO M, MA Y, DAI L, et al. A high-performance liquid chromatographic method for simultaneous determination of 21 free aminoacids in tea[J]. Food Anal Methods, 2013, 6(1): 69
[12] WILSON SF, JAMES CA, ZHU X, et al. Development of a method for the determination of glycine in human cerebrospinal fluid using pre-column derivatization and LC-MS/MS[J]. J Pharm Biomed Anal, 2011, 56(2): 315
[13] 朱影恬, 曾雯瑜, 张中伟, 等. 柱前衍生高效液相色谱测定小儿复方氨基酸注射液中氨基酸[J]. 分析科学学报, 2009, 25(6): 685
ZHU YT, ZENG WY, ZHANG ZW, et al. Determination of amino acids in children compound amino acid injection by pre-column derivatization high performance liquid chromatography[J]. J Anal Sci, 2009, 25(6): 685
[14] 汪媛, 王海龙, 金高娃, 等. PITC柱前衍生HPLC测定复方氨基酸注射液中 18 种氨基酸的含量[J]. 世界科学技术-中医药现代化, 2014, 16(6): 1347
WANG Y, WANG HL, JIN GW, et al. Content determination of 18 amino acids in compound amino acid injection by HPLC with pre-column PITC derivatization[J]. Mod Tradit Chin Med Mater Med-World Sci Technol, 2014, 16(6): 1347
[15] HERBERT P, BARROS P, RATOLA N, et al. HPLC determination of amino acids in musts and port wine using OPA/FMOC derivatives[J]. J Food Sci, 2000, 65(7): 113
[16] 邓红英, 张永文, 李永贵. 柱前衍生高效液相色谱法测定复方氨基酸注射液中氨基酸的含量[J]. 药学研究, 2020, 39(1): 27
DENG HY, ZHANG YW, LI YG. Determination of amino acids in compound amino acids injection by HPLC after pre-column derivatization[J]. J Pharm Res, 2020, 39(1): 27
