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目的建立超高效液相色谱-质谱联用法(ultra performance liquid chromatography-mass spectrometry,UPLC-MS)及高效液相色谱法(high performance liquid chromatography,HPLC)对人工虫草菌丝体中虫草素进行快速筛查及定量测定分析方法。方法样品经水-甲醇(85:15,V:V)提取;超高效液相色谱-质谱联用法进行定性筛查,0.3%甲酸水溶液-甲醇梯度洗脱,正离子多反应监测(multiple reaction monitoring,MRM)模式扫描;高效液相色谱法进行定量测定,水-甲醇(85:15,V:V)等度洗脱后利用紫外检测器检测。结果确定252.0/136.0及252.0/69.0 2对离子对用于MRM定性分析,定性准确性高;定量测定方法中,虫草素在167~1337μg/mL浓度范围内线性关系良好(r~2=0.9997);最低定量限为2.64μg/g;3个不同浓度加标回收率为98.1%~101.0%,相对标准偏差为0.06%~1.24%;精密度、重复性良好。结论该研究结合了UPLC-MS和HPLC各自的分析优势,快速、高效、准确地对人工虫草菌丝体中虫草素进行定性和定量分析,提高了工作效率,大大缩短了分析时间,在样品量较大及探究人工菌丝体培育条件方面具有优势。
OBJECTIVE To establish a rapid screening method for cordycepin in the mycelium of Cordyceps sinensis using ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and high performance liquid chromatography (HPLC) Check and quantitative determination of analytical methods. Methods The samples were extracted with water-methanol (85:15, V: V), qualitatively analyzed by ultra performance liquid chromatography-mass spectrometry, and eluted with 0.3% formic acid in water and methanol. Multiple reaction monitoring , MRM) mode of detection; high performance liquid chromatography quantitative determination of water - methanol (85: 15, V: V) isocratic elution by UV detector detection. The results showed that 252.0 / 136.0 and 252.0 / 69.0 2 pairs were suitable for qualitative analysis of MRM. The linearity was good (r 2 = 0.9997) in the quantitative range of 167 ~ 1337μg / mL ; The lowest limit of quantification was 2.64μg / g; the recoveries of three different concentrations were 98.1% -101.0% with relative standard deviations of 0.06% -1.24%; the precision and reproducibility were good. Conclusion This study combines the advantages of UPLC-MS and HPLC respectively, and can qualitatively and quantitatively analyze cordycepin in the mycelium of Cordyceps sinensis rapidly, efficiently and accurately, which improves the working efficiency, greatly shortens the analysis time, Larger and to explore artificial mycelial growth conditions have advantages.