目的:建立一种绿色环保的羊肚菌高效液相指纹图谱分析方法。方法:采用离子液体-水溶液绿色溶剂提取样品,通过单因素考察离子液体种类、离子液体碳链长度、离子液体质量分数、提取时间、料液比对提取量的影响,确立了最终提取条件为40%[OMIM]PF6水溶液(料液比1:10)湿法研磨样品10 min;同时使用绿色高效液相色谱法对羊肚菌进行分析检测:Agilent ZORBAX SB-AQ C18色谱柱(4.6 mm×150 mm,5 μm),0.2%乙酸-乙醇作为绿色流动相体系进行梯度洗脱,流速1.0 mL/min,柱温40℃,检测波长260 nm。采用"中药色谱指纹图谱相似度评价系统(2012版)"对20批试验样品(14批六妹羊肚菌、6批其他食药用菌)进行相似度评价,采用SPSS 22.0软件对所有样品进行了聚类分析和主成分分析。结果:建立了羊肚菌的HPLC指纹图谱,标定10个共有峰,鉴定了5个核苷类成分(尿嘧啶、胞苷、尿苷、鸟苷、腺苷),20批试验样品中14批羊肚菌样品相似度均大于0.9,另外6批常见食药用菌相似度在0.25~0.65之间;聚类和主成分分析结果显示羊肚菌样品与其他食药用菌在本实验条件下化学成分存在显著差异,可有效鉴别羊肚菌和常见食药用菌。结论:本试验建立了简便、绿色、快速的羊肚菌HPLC指纹图谱分析方法,有助于提升羊肚菌的综合质量评价水平。
Objective:To establish a green and environmentally friendly HPLC fingerprint analysis method for Morchella. Methods:An ionic liquid(1-octyl-3-methylimidazolium hexafluorophosphate)-water solution was as extracted-solution. The effects of experimental conditions(the type of ionic liquid,ionic liquid carbon chain length,ionic liquid mass fraction of solution,extraction time,and material-liquid ratio)on the extraction amount were optimized through the single factor test,the final extraction condition was established as 40%[OMIM]PF6 aqueous solution(material-liquid ratio 1:10)wet grinding sample for 10 min. Then green HPLC was used to analyze Morchella samples. The separation was performed on a Agilent ZORBAX SB-AQ C18 column(4.6 mm×150 mm,5 μm)with gradient elution by 0.2% acetic acid-ethanol as a green mobile phase system at 1.0 mL/min,column temperature was 40℃,and detection wavelength was at 260 nm. The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System(2012 Edition)"was used to evaluate the similarity of 20 batches of samples(14 batches of Morchella sextelata,6 batches of various edible medicinal fungi). And the cluster analysis and principal component analysis were performed on the samples by SPSS software. Results:HPLC fingerprint analysis approach of Morchella was established,calibrating 10 common peaks,and 5 nucleoside components(uracil,cytidine,uridine,guanosine,and adenosine)were identified. The similarity of 14 batches Morchella sextelata was above 0.9,and the similarity of the other 6 batches samples was among 0.25~0.65. The clustering and principal component analysis also showed that the chemical compositions between Morchella and other edible medicinal fungi were obviously different under this experimental condition. Conclusion:A convenient,green and rapid HPLC fingerprint analysis approach for Morchella was established,which would help to improve the comprehensive quality evaluation level of Morchella.