复方鱼腥草合剂特征图谱及基于Orbitrap超高分辨质谱结合多元数理统计方法的成分分析

HPLC specific chromatogram of compound Yuxingcao mixture and composition analysis based on Orbitrap ultra-high resolution mass spectrometry combined with multivariate mathematical statistics

目的:建立复方鱼腥草合剂的HPLC特征图谱质量评价方法,并采用超高分辨质谱结合数理统计方法对不同生产企业复方鱼腥草合剂的差异性成分进行分析。方法:特征图谱采用Besil 5 C18-B(250mm×4.6 mm,5μm)色谱柱,流动相为甲醇-0.1%甲酸,梯度洗脱,流速1.0 mL·min-1,柱温35℃,检测波长210 nm。在特征图谱方法的基础上,高分辨质谱以静电场轨道离子阱(Orbitrap)为检测器,在负离子模式下对样品进行分析,利用SIEVE软件对图谱进行预处理和信息筛选,后采用SIMCA-P软件进行数理统计分析。结果:所建立的特征图谱共有16个特征峰,55批样品中均能检出,且相对保留时间也一致,但相对峰面积差别较大,不同生产企业合剂样品存在较大的差别。此结果通过Orbitrap超高分辨质谱得到验证,差异性成分主要为连翘酯苷A、白杨素-6-C-阿拉伯糖-8-C-葡萄糖苷等。结论:建立了复方鱼腥草合剂的特征图谱方法专属性强,重现性好,结合超高分辨质谱并多元数理统计方法,明确了不同生产企业复方鱼腥草合剂的化学成分差异,也为质量标准提升研究提供了参考。

Objective:To establish a quality assessment method for Yuxingcao mixture from different manufacturers based HPLC specific chromatograms and ultra-high resolution mass spectrometry combined with mathematical statistics. Methods: A Besil 5 C18-B(250 mm×4.6 mm, 5 μm) column was used and eluted with methanol-0.1% formic acid in gradient mode at a flow rate of 1.0 mL·min-1. The column temperature was 35 ℃. And the detection wavelength was 210 nm. Then the electrostatic field orbital ion trap was used as the detector of high-resolution mass spectrometry. The samples were analyzed in the negative ion mode. Then the spectra were preprocessed and screened by SIEVE software. And the mathematical statistics analysis was carried out by SIMCA-P software. Results:16 characteristic peaks were detected in 55 batches of samples with consistent relative retention time. But the relative peak areas varied greatly,which indicated significant differences between samples from different manufacturers. These results were verified by Orbitrap ultra-high resolution mass spectrometry. The main differential components were forsythiaside A and chrysin-6-C-arabinose-8-C-glucoside. Conclusion:The established method of specific chromatogram is specific and reproducible. Based on this study,the chemical composition differences between compound Yuxingcao mixture from different manufacturers are clarified,which also provide a reference for the improvement of quality standards.