亲水作用色谱串联质谱法分析三七药材中游离氨基酸类成分

Hydrophilic Interaction UPLC Coupled with Tandem Mass Spectrometry Analysis of Free Amino Acids in Notoginseng Radix et Rhizoma

摘要:

目的：建立三七药材中游离氨基酸类成分的分析方法。方法：采用亲水作用色谱串联质谱测定三七药材中22种游离氨基酸，ACQUITY UPLC BEH Amide色谱柱 （2.1 mm×100 mm，1.7μm），流动相A含5 mmol·L^-1甲酸胺、5 mmol·L^-1乙酸铵和0.15%甲酸水溶液，B含1 mmol·L^-1 甲酸铵、1 mmol·L^-1乙酸铵和0.05%甲酸乙腈溶液，梯度洗脱（0~6 min，15%~20%A；6~10 min，20%~30%A；10~11 min，30%~46%A），流速0.4 mL·min^-1，柱温35 ℃；串联质谱正离子多反应模式检测（MRM）。结果：在12 min内22种氨基酸得到较好分离，22种氨基酸浓度与峰面积呈良好的线性关系，相关系数 0.991 7~0.999 9，回收率93.7%~104.7%，日内精密1.13%~4.95%，日间精密度1.84%~5.62%，10批三七药材样品中22种游离氨基酸总含量在4.72~5.96 mg·g^-1。结论：该方法简便、快速、灵敏，适用于三七药材中氨基酸类成分的测定，为三七药材全面质量控制方法的建立提供依据。

Abstract:

Objective: To establish a quantitative method for the free amino acids in Notoginseng Radix et Rhizoma. Method: Hydrophilic interaction chromatography-tandem mass spectrometry was used to determine the 22 amino acids in Notoginseng Radix et Rhizoma. An ACQUITY UPLC BEH amide column (2.1 mm×100 mm, 1.7 μm) was applied for the separation of these amino acids with a gradient mobile phase consisting of solution A (water, 5 mmol·L^-1 ammonium formate, 5 mmol·L^-1 ammonium acetate and 0.15% formic acid) and solution B (acetonitrile, 1 mmol·L^-1 ammonium formate, 1 mmol·L^-1 ammonium acetate and 0.05% formic acid) at a flow rate of 0.4 mL·min^-1. The linear gradient conditions were as follows:0-6 min, 15%-20% A;6-10 min,20%-30% A;10-12 min, 30%-46% A. The column temperature was maintained at 35 ℃. Multiple reaction mode detection (MRM) in positive ion mode was used in this assay. Result: Good separation of the 22 amino acids was obtained within 12 min. Good correlations were found between the investigated compounds concentrations and their peak areas within the test ranges with the correlation coefficient from 0.991 7 to 0.999 9.The average recoveries were from 93.7% to 104.7%. The overall intra-and inter-day variations (RSDs) based on the peak areas of the analyte were in the range of 1.13%-4.95% and 1.84%-5.62% respectively. The total content of 22 free amino acids in 10 batches samples of Notoginseng Radix et Rhizoma was from 4.72 mg·g^-1 to 5.96 mg·g^-1. Conclusion: The proposed method is simple, rapid, sensitive and suitable for the determination of amino acids in Notoginseng Radix et Rhizoma and could provide the basis for Notoginseng Radix et Rhizoma quality control.