HPLC-DAD指纹图谱结合HPLC-Q-TOF-MS/MS成分表征评价胡桃楸枝的质量

Quality Evaluation of Branches of Juglans mandshurica by HPLC-DAD Fingerprint Combined with HPLC-Q-TOF-MS/MS Characterization of Constituents

目的:建立胡桃楸枝的高效液相色谱法(HPLC)指纹图谱,并对不同产地、不同采收期的胡桃楸枝样品进行质量分析。方法:采用Agilent Poroshell 120 SB-C18色谱柱(2.1 mm×100 mm,2.7μm),流动相0.2%甲酸水溶液(A)-0.2%甲酸乙腈溶液(B)梯度洗脱(0~5 min,5%~10%B;5~25 min,10%~16%B;25~40 min,16%~22%B;40~45 min,22%~45%B;45~50 min,45%~65%B;50~52 min,65%~100%B;52~55 min,100%B),流速0.3 mL·min-1,柱温30℃,检测波长270 nm。采用相似度评价、聚类分析、主成分分析和偏最小二乘法-判别分析对指纹图谱数据进行统计分析。采用HPLC串联四极杆飞行时间质谱法(HPLC-Q-TOF-MS/MS)表征胡桃楸枝中的化学成分,选择电喷雾离子源(ESI),在负离子检测模式下进行质谱采集,MS扫描范围m/z 100~1 700,MS/MS扫描范围m/z 50~1 700。结果:40批胡桃楸枝样品中确定了19个共有峰,其相似度处于0.430~0.995,其中春冬季节(4月,5月,12月)采收样品相似度均>0.90,大部分夏季(7~9月)采收样品相似度较低。多元统计分析后发现样品根据采收季节分为两类,而不同产地样品无明显分类规律,说明采收期是影响胡桃楸枝样品质量的主要因素之一。春冬季节采收样品中大部分化学成分的含量高于夏季采收的样品,与夏季采收样品相比,春冬季采收样品质量较好。22个色谱峰是造成不同季节采收样品差异性的主要成分。通过HPLC-Q-TOF-MS/MS表征了胡桃楸枝中的83个化学成分,其中鞣质类成分49个,有机酸类成分7个,萘衍生物14个,黄酮类成分1个,蒽衍生物6个,木脂素类成分2个,二芳基庚烷类成分3个,糖类成分1个。鉴别了13个共有峰和19个不同季节采收样品的差异峰,多为鞣质类化合物。结论:建立的HPLC指纹图谱可用于胡桃楸枝的质量分析,鞣质是胡桃楸枝中的主要成分。采收期对胡桃楸枝质量影响较大。

Objective:To establish a high performance liquid chromatography(HPLC)fingerprint of branches of Juglans mandshurica and to evaluate the quality of the samples from different producing areas and in different harvest periods. Method: Chromatographic separation was performed on an Agilent Poroshell 120 SB-C18 column(2.1 mm×100 mm,2.7 μm)for gradient elution with mobile phase of 0.2% formic acid solution(A)-0.2% formic acid acetonitrile solution(B)(0-5 min,5%-10%B;5-25 min,10%-16%B;25-40 min,16%-22%B;40-45 min,22%-45%B;45-50 min,45%-65%B;50-52 min,65%-100%B;52-55 min,100%B)at a flow rate of 0.3 mL·min-1. The column temperature was 30 ℃ and the detection wavelength was 270 nm. The quality of branches of Juylans mandshurica was evaluated by similarity evaluation,cluster analysis,principal component analysis and partial least squares-discriminant analysis. The chemical constituents of the samples were identified by HPLC coupled with quadrupole time-of-flight mass spectrometry(HPLC-Q-TOF-MS/MS). The mass spectrometry was conducted in negative ion mode with electrospray ionization(ESI). Data were acquired over a range of m/z 100-1 700 for MS and m/z 50-1 700 for MS/MS. Result:A total of 19 common peaks were confirmed in 40 batches of samples,and the similarity ranged from 0.430 to 0.995,of which the similarity of samples collected in spring and winter seasons(April,May and December)was greater than 0.90,while the similarity of most samples collected in summer(July to September)was low. Multivariate statistical analysis showed that the samples were divided into two groups according to the harvest time,but there was no obvious classification rule for the samples from different producing areas. The contents of most constituents in the samples collected in spring and winter were higher than those collected in summer. The result illustrated that different harvest periods had great influence on the quality of branches of J. mandshurica. Compared with the samples collected in summer,the quality of samples collected in spring and winter was better. A total of 22 peaks were proved to be the main constituents that contributed to the difference between samples collected in different seasons. A total of 83 chemical components were identified by HPLC-Q-TOF-MS/MS,including 49 tannins,7 organic acids,14 naphthalene derivatives,1 flavonoid,6 anthracene derivatives,2 lignans,3 diarylheptanoids and 1 saccharide. Totally 13 common peaks were identified. Of the peaks that contributed to discriminate samples collected in different season,19 peaks were identified and most of them were tannins. Conclusion: The established HPLC fingerprint can provide useful information for the quality evaluation of branches of J. mandshurica. Tannin is the main constituents in the samples. Harvest period has great influence on the quality of branches of J. mandshurica.