采用柱前衍生化法研究康复新液不同制备环节中氨基酸成分轮廓差异

目的：采用柱前衍生化法及主成分分析（principal component analysis，PCA）、偏最小二乘法-判别分析（partial least square-discriminant analysis，PLS-DA）等多变量数据分析方法动态分析测定不同批次美洲大蠊药材以及由其所制备的提取物和制剂康复新液中12种游离氨基酸含量，筛选出导致差异的特征性氨基酸成分。方法：采用1% FDNB-乙腈溶液进行柱前衍生化；色谱条件：色谱柱为Kromasil C₁₈色谱柱（250 mm&#215;4.6 mm，5.0 μm），流动相A为20 mmol&#183;L^-1乙酸铵-0.4%乙酸水溶液，流动相70%乙腈，梯度洗脱（0~5 min，70% A；5~8 min，70% A→68.5% A；8~12 min，68.5% A→67% A；12~16 min，67% A→65% A；16~17 min，65% A→62% A；17~18 min，62% A→61% A；18~23 min，61% A→59% A；23~24 min，59% A→49% A；24~31 min，49% A→40% A；31~33 min，40~35% A；33~35 min，35% A→30% A；35~37 min，30% A；37~38 min，30% A→70% A；38~40 min，70% A），流速1 mL&#183;min^-1，检测波长360 nm，柱温30℃，进样量10 μL。结果：12种氨基酸在40 min内均能得到较好的分离，r=0.999 1~0.999 9（n=3），平均回收率为90.2%~111.8%，RSD为0.57%~2.6%；制剂中精氨酸（Arg）含量与药材、提取物相较具有统计学差异性（P<0.05）；通过pls-da和各变量重要性因子分析（vip）表明，牛磺酸、甲硫氨酸、异亮氨酸、亮氨酸、谷氨酰胺对药材、提取物、制剂的质量贡献较为明显。结论：通过氨基酸含量的测定可将美洲大蠊药材与提取物、制剂样品区分开，提取物与制剂样品之间含量存在的差异较小，表明康复新液制备工艺较为稳定，其中牛磺酸、甲硫氨酸、异亮氨酸、亮氨酸、谷氨酰胺以及精氨酸为康复新液质量控制标志物。

Objective: To compare the metabolic profiling of twelve free amino acids including Lys,Asp,Arg, Leu,Tau,Val,Gln,Glu,Ile,Thr,Met,and Ser among crude drugs,extracts and preparations of Kangfuxinye by pre-column derivatization and multivariate data analysis methods such as PCA and PLS-DA. Meanwhile,to screen out the characteristic constituents. Methods: The determination method of twelve amino acids established with 1% FDNB-acetonitrile pre-column derivatization was carried out on a Kromasil C₁₈ (250 mm&#215;4.6 mm,5 μm) column. The mobile phase consisted of 20 mmol&#183;L^-1 ammonium acetate-0.4% acetic acid (A) and 70% acetonitrile (B) in gradient elution mode (0-5 min,70%A;5-8 min,70%→68.5%A;8-12 min,68.5%→67%A;12-16 min,67%→65%A;16-17 min,65%→62%A;17-18 min,62%→61%A;18-23 min,61%→59%A;23-24 min,59%→49%A;24-31 min,49%→40%A;31-33 min,40%→35%A;33-35 min,35%→30%A;35-37 min,30%A;37-38 min,30%→70%A;38-40 min,70%A) at the flow rate of 1.0 mL&#183;min^-1. The detection wavelength was 360 nm and the injection volume was 10 μL,while the column temperature was 30. Results: Twelve amino acids had good linearity in the ranges of 0.001 0-1.292 0 mg&#183;mL^-1,correlated coefficient (r) were between 0.999 1 and 0.999 9. The average recovery was 90.2%-111.8%,the value of RSD was 0.57%-2.6%. The content of Arg in preparations was significantly different from that in crude drugs and extracts (P<0.05). It was also found that five amino acids were the main differences among crude drugs,extracts and preparations including Tau,Met,Leu,Ile,Glu by the analysis of PLS-DA and VIP. Conclusion: Extracts and preparation could be distinguished from crude drugs of Periplaneta Americana via the determination the content of 12 amino acids. There was little difference in the content between extracts and preparations,and the results showed that the preparation process of Kangfuxinye was stable. There are six amino acids (Tau,Met,Leu,Ile,Glu,and Arg) that can be used as quality control markers of Kangfuxinye. The above results provide a basis for researches on material foundation of the medicinal effectiveness and mechanism of action.