基于HS-SPME/GC-QQQ-MS/MS的冬虫夏草“腥气”辨识方法建立与应用

Establishment and Application of Identification Method for Fishy Odor of Cordyceps Based on HS-SPME/GC-QQQ-MS/MS

目的:建立和应用一种基于顶空-固相微萃取-气相色谱-三重四极杆质谱联用技术的冬虫夏草"腥气"分析方法。方法:采用Inert Cap Pure-WAX毛细管柱(0.25 mm×30 m,0.25μm),进样口温度250℃,分流比5∶1,载气为高纯氦气,色谱柱流量1.43 mL·min^(-1),线速度43.3 cm·s^(-1),吹扫流量3.0 mL·min^(-1);程序升温(初始温度50℃,保持5 min,以10℃·min^(-1)升温至250℃,保持10 min;柱平衡时间2.0 min)。离子源为电子轰击离子源(EI),离子源温度200℃,质谱监测模式为多反应监测。结果:收集到7批冬虫夏草正品,其中四川3批,青海3批,西藏1批;6批伪品,其中四川3批,贵州2批,新疆1批。在冬虫夏草中共筛查出81种挥发性成分,按化学结构可分为13类(酯类、酮类、醛类、烯类、酚类、酸类、醇类、苯类、醚类、吡嗪类、烃类、含氮杂环类、含氧杂环类),表明冬虫夏草的"腥气"是复合气味。不同产地冬虫夏草中挥发性成分种类无明显差异,提示西藏(那曲),青海(玉树、果洛)和四川(理塘、壤塘、色达)的冬虫夏草中挥发性物质较为一致,即组成"腥气"的化学成分没有质(种类)的差异。但不同产区冬虫夏草中部分挥发性物质的含量存在较大差异,即组成"腥气"的化学成分有量(含量)的差异。筛选出了16个含量差异较大的化学成分,包括丙二醇甲醚乙酸酯,乙酸己酯,丙位辛内酯,2-辛酮,正辛醛,(E)-2-庚烯醛,癸醛,(E)-壬烯醛,(E,E)-2,4-壬二烯醛,异戊酸,正戊酸,正己酸,庚酸,壬酸,正辛醇,2-乙基吡嗪,可作为该药材产地鉴别的标志物进行研究。冬虫夏草正品与伪品之间挥发性成分存在较大的差异,筛查出了34个化学成分,包括乙酸乙酯,苯乙酮,2-乙基己醇,乙酸己酯,2,3-丁二酮,2-辛酮,2-壬酮,2-莰酮,异佛尔酮,甲基壬基甲酮,2-苯基-1-丙烯,4-乙基-2-甲氧基苯酚,芳樟醇,2-异丙基-5-甲基环己醇,3-烯-2-酮,2-茨醇,二甲基二硫,二甲基三硫,正辛醛,苯甲醛,苯乙醛,香草醛,α-蒎烯,β-蒎烯,双戊烯,苯乙烯,4-甲基苯酚,桉叶油醇,乙二醇单丁醚,2-甲基吡嗪,2-甲基萘,1-甲基萘,丙位癸内酯和5-乙基-2-甲基-吡啶,这些化合物可能是该药材真伪鉴别的标志物。结论:建立的冬虫夏草"腥气"辨识方法具有高灵敏度、准确和简便的特点,可为其他中药材的挥发性成分分析提供参考。

Objective:To establish and apply a new practical analytical method for identifying the fishy odor of Cordyceps based on headspace-solid phase microextraction-gas chromatography-triple quadrupole mass spectrometry (HS-SPME/GC-QQQ-MS/MS) technique.Method:The Inert Cap Pure-WAX capillary column(0.25 mm×30 m,0.25μm)was used for chromatographic separation.The injection port temperature was set at 250℃.The injection mode was split injection with a ratio of 5∶1.High purity helium was used as the carrier gas and control mode was set to constant pressure.The column flow rate was 1.43 mL·min^(-1),the linear velocity was 43.3 cm·s^(-1),and the purge flow rate was 3.0 mL·min^(-1).The chromatographic column temperature program as follows:maintained the initial temperature at 50℃for 5 min,and increased the temperature at a rate of 10℃·min^(-1) to 250℃,held for 10 min.The column equilibrium time was 2.0 min.The ion source of mass spectrographic analysis was electron ionization with ion source temperature of 200℃,and the monitoring mode was set to multiple reaction monitoring.Result:Seven batches of Cordyceps samples were collected,including3 batches from Sichuan,3 batches from Qinghai and 1 batch from Tibet.There were six batches of counterfeits,including 3 batches from Sichuan,2 batches from Guizhou and 1 batch in Xinjiang.A total of 81 volatile compounds were screened out in Cordyceps,which could be divided into 13 types(esters,ketones,aldehydes and others)according to the compound structure,indicating that the fishy odor of Cordyceps was a complex odor.There was no significant difference in the types of volatile compounds of Cordyceps from different regions,which suggested that these volatile compounds in Cordyceps produced in Tibet(Naqu),Qinghai(Yushu and Guoluo)and Sichuan(Litang,Rangtang and Seda)were relatively consistent.However,the contents of some volatile compounds in Cordyceps produced in different regions were quite different,and 16 volatile compounds with significant difference were screened out,including 1-methoxy-2-propyl acetate,γ-octalactone,hexyl acetate and others,those compounds maybe could been used as the quality markers for identification of regions of Cordyceps.There was a large difference in volatile compounds between Cordyceps and its counterfeits,and 34 volatile compounds were screened out,including ethyl acetate,acetophenone,2-ethyl-1-hexanol and others,those compounds maybe could been used as the quality markers for authenticity identification of Cordyceps.Conclusion:In summary,the established method for identifying the fishy odor of Cordyceps in this paper has the characteristics of high sensitivity,accuracy and simplicity,which can provide reference for the analysis of volatile compounds in other Chinese herbal medicines.