指纹图谱结合一测多评法对美洲大蠊氨基酸的质量评价研究

Study on the quality evaluation of amino acids in Periplaneta Americana by fingerprint atlas combined with QAMS method

关键词:  美洲大蠊  氨基酸  指纹图谱  一测多评法  质量评价

Key words:  Periplaneta americana  amino acids  fingerprint  quantitative analysis of multi-components by single marker  quality evaluation

摘要:

目的 建立美洲大蠊的HPLC指纹图谱，并运用一测多评法测定9种氨基酸的含量，综合评价美洲大蠊氨基酸质量差异。方法 样品在6mol?L-1盐酸溶液中120 ℃水解6h，再与异硫氰酸苯酯(phenylisothiocyanate, PITC)40 ℃水浴1h进行衍生化反应。衍生化产物分析采用Kromasil 100-5-C18色谱柱(4.6 mm×250 mmm, 5 μm)，以0.1mol?L-1乙酸钠溶液(pH 6.5±0.05)-乙腈(97﹕3)为流动相A，乙腈-水(4﹕1)为流动相B，梯度洗脱，流速0.8 mL?min-1，检测波长254 nm，柱温40 ℃，进样量2μL。对指纹图谱进行相似度评价、聚类分析(cluster analysis, CA)、主成分分析(principal component analysis, PCA)、正交偏最小二乘法-判别分析(rthogonal partial least squares discriminant analysis, OPLS-DA)。建立内参物丙氨酸与甘氨酸、精氨酸、苏氨酸、酪氨酸、缬氨酸、异亮氨酸、亮氨酸及苯丙氨酸的相对校正因子(f )并对其耐用性考察。采用外标法(external standard method, ESM)与一测多评法(quantitative analysis of multi-components with a single-marker, QAMS)分别测定16批美洲大蠊中9个氨基酸含量，并用独立样本t检验比较验证QAMS方法的准确性。结果 采用中药色谱指纹图谱相似度评价系统(2004A版)、SPSS 27.0与SIMCA 14.1分析16批美洲大蠊的氨基酸指纹图谱：标定17个共有峰，明确15个已知成分；16批次的相似度在0.888至0.982之间，表明其质量一致性较好；CA与PCA结果均分为3大类，意味着不同产地质量存在一定差异；OPLS-DA筛选了脯氨酸、赖氨酸、缬氨酸、异亮氨酸、苏氨酸、亮氨酸、丙氨酸及共有峰9共8个主要的质量差异标志物。含量测定方面，9个成分的专属性、线性关系(r≥0.9999)、精密度、重复性、稳定性均良好，平均加样回收率在98.70%-101.93%，RSD在1.08%-2.24%。QAMS方法学验证内参物丙氨酸与8个氨基酸的f分别为甘氨酸0.8338、精氨酸1.9641、苏氨酸1.4489、酪氨酸1.8306、缬氨酸1.2767、异亮氨酸1.3816、亮氨酸1.4082、苯丙氨酸1.6542。t检验结果ESM法与QAMS法含测结果差异无统计学意义(P>0.05)。结论 本研究所构建的HPLC指纹图谱与一测多评结合方法操作简便、专属性强、重现性好，能够有效评价美洲大蠊氨基酸的组成，为美洲大蠊药材及其提取物中氨基酸成分的质量控制奠定理论基础。

Abstract:

OBJECTIVE To establish an HPLC fingerprint atlas of Periplaneta americana and determine the content of nine amino acids utilizing quantitative analysis of multi-components with a single-marker (QAMS) method, thereby comprehensively evaluating the quality differences of amino acids in Periplaneta americana. METHODS The samples were hydrolyzed in 6 mol?L-1 hydrochloric acid solution at 120 ℃ for 6 hours, and then derivatized with phenylisothiocyanate (PITC) in a 40 ℃ water bath for 1 hour. The analysis of the derivatized products was carried out using a Kromasil 100-5-C18 column (4.6 mm×250 mm, 5 μm), with 0.1 mol?L-1 sodium acetate solution (pH 6.5±0.05)-acetonitrile (97:3) as mobile phase A and acetonitrile-water (4:1) as mobile phase B. The gradient elution was performed at a flow rate of 0.8 mL?min-1, with a detection wavelength of 254 nm and a column temperature of 40 ℃. The injection volume was 2 μL. The fingerprint chromatograms were evaluated for perform similarity evaluation, cluster analysis (CA), principal component analysis (PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA). The relative correction factors (f) of the internal reference alanine to glycine, arginine, threonine, tyrosine, valine, isoleucine, leucine and phenylalanine were established and their durability was investigated. The contents of 9 amino acids in 16 batches of Periplaneta americana were determined by external standard method (ESM) and QAMS, respectively. The accuracy of the QAMS method was verified by independent sample t-test. RESULTS The amino acid fingerprint profiles of 16 batches of Periplaneta americana were analyzed using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2004A edition), SPSS 27.0, and SIMCA 14.1. A total of 17 common peaks were identified, with 15 known components clarified. The similarity among the 16 batches ranged from 0.888 to 0.982, indicating relatively good quality consistency. Both CA and PCA classified the samples into three major groups, suggesting certain differences in quality among samples from different origins. OPLS-DA identified eight major quality-differentiating markers, including proline, lysine, valine, isoleucine, threonine, leucine, alanine and so on. In terms of content determination, the specificity, linearity (r≥0.9999), precision, repeatability, and stability of nine components were all satisfactory. The average recoveries ranged from 98.70% to 101.93%, with Relative Standard Deviations (RSD) between 1.08% and 2.24%. For the validation of QAMS method, the relative correction factors (f) of alanine with respect to eight amino acids were determined as follows: glycine 0.8338, arginine 1.9641, threonine 1.4489, tyrosine 1.8306, valine 1.2767, isoleucine 1.3816, leucine 1.4082, and phenylalanine 1.6542. The t-test results showed no statistically significant difference (P>0.05) between the content determination results obtained by the ESM and the QAMS method. CONCLUSION The HPLC fingerprint combined with QAMS method established in this study is simple to operate, highly specific, and exhibits good reproducibility. It can effectively evaluate the amino acid composition in Periplaneta americana samples, thereby laying a theoretical foundation for the quality control of amino acid components in Periplaneta americana crude drugs and their extracts.