液相色谱-串联质谱法测定花椒中的6种山椒素及其产地鉴别

Determination of 6 kinds of sanshools in Zanthoxylum bungeanum by liquid chromatography tandem mass spectrometry and identification of their origins

中文关键词:  液相色谱-串联质谱法  山椒素  汉源花椒  聚类分析  PCA分析

英文关键词:liquid chromatography tandem mass spectrometry  sanshool  Hanyuan Zanthoxylum bungeanum  cluster analysis  principal component analysis

中文摘要:

      目的 建立液相色谱-串联质谱法(liquid chromatography tandem mass spectrometry, LC-MS/MS)快速测定花椒中6种山椒素的方法, 并进行产地分析。方法 样品通过甲醇超声提取, LC-MS/MS外标法测定, 对提取方式、仪器条件进行了优化, 与高效液相色谱法(high performance liquid chromatography, HPLC)检测进行比较, 对方法正确度、精密度、检出限和定量限进行了验证。利用该方法对不同产地的10个青花椒、33个红花椒进行测定, 通过聚类分析、主成分分析(principal component analysis, PCA)进行产地分析鉴别。结果 6种山椒素在LC-MS/MS上分离良好, 在0.10~5.00 μg/mL范围内线性关系良好, 相关系数(r2)≥0.996, 检出限为0.10×10–3~0.18×10–3 mg/g, 定量限为0.33×10–3~0.61×10–3 mg/g; 在低、中、高3个水平下6种山椒素的平均回收率在86.67%~100.67%之间, 相对标准偏差为0.72%~9.11%, 正确度和精密度均符合方法验证要求, 检出限、定量限均低于HPLC方法, 灵敏度、准确度更高。通过对不同产地花椒的检测结果显示, 羟基-α-山椒素为含量最高的麻味物质, 且两类花椒测定结果均表现出汉源花椒中6种山椒素总量高于其他产地花椒的特点。通过聚类分析、PCA分析表明, 汉源青花椒中羟基-β-山椒素, 红花椒中羟基-α-山椒素、羟基-γ-山椒素、γ-山椒素含量均高于所测其他产地同种花椒中麻味物质含量, 是区别于其他产地花椒的主要麻味物质。结论 该方法前处理简单, 灵敏度、准确度高, 可用于花椒中6种山椒素的检测, 且通过对花椒中6种山椒素的检测及聚类、PCA分析能有效区分汉源花椒与其他产地花椒, 为鉴别真假汉源花椒提供了可靠的方法。

英文摘要:

      Objective To establish a rapid and efficient method for the determination of 6 types of sanshools in Zanthoxylum bungeanum by liquid chromatography tandem mass spectrometry (LC-MS/MS) and perform origin analysis. Methods The extract was extracted by methanol ultrasonication and determined by LC-MS/MS external standard method. The extraction method and instrumental conditions were optimized and compared with high performance liquid chromatography (HPLC) detection. Methodological validation of accuracy, precision, detection limit and quantification limit were performed. Ten green Zanthoxylum bungeanum and 33 red Zanthoxylum bungeanum from different origins were determined by this method and the origin analysis and identification were performed by cluster analysis and principal component analysis (PCA). Results The 6 kinds of sanshools were well separated on LC-MS/MS, with a good linearity relationship in the range of 0.10–5.00 μg/mL, with the correlation coefficients (r2) ≥0.996. The limits of detection ranged from 0.10×10–3 to 0.18×10–3 mg/g and the limits of quantification were from 0.33×10–3 to 0.61×10–3 mg/g. The average recoveries of the 6 kinds of sanshools at the low, medium and high levels ranged from 86.67% to 100.67%, with the relative standard deviations of 0.72% to 9.11%. The accuracy and precision met the requirements of method validation. The limit of detection and limit of quantification were lower than those of HPLC method, which showed higher sensitivity and accuracy. The results of Zanthoxylum bungeanum of different origins showed that hydroxy-α-sorcinol was the most abundant pungent substance, and both types of Zanthoxylum bungeanum showed that the total amount of the 6 kinds of sanshools in Hanyuan Zanthoxylum bungeanum was higher than in other origins. Cluster and PCA showed that the content of hydroxy-β-sanshool in Hanyuan green Zanthoxylum bungeanum and hydroxy-α-sanshool, hydroxy-γ-sanshool and γ-sanshool in red Zanthoxylum bungeanum were higher than those species from other origins, and were the main pimgent substances that distinguished them from other peppercorns. Conclusion The method is simple in pre-treatment, with high sensitivity and accuracy, and can be used for the detection of 6 types of capsainoids in Zanthoxylum bungeanum. Moreover, through the detection of 6 types of capsainoids in Zanthoxylum bungeanum and the analysis of cluster and PCA, it can effectively distinguish Hanyuan Zanthoxylum bungeanum from other origins, which provides a reliable method for the identification of genuine and fake Hanyuan Zanthoxylum bungeanum.