超高效液相色谱-质谱联用法及高效液相色谱法在人工虫草菌丝体中虫草素筛查及含量测定中的应用

摘要

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167～1337 μg/mL浓度范围内线性关系良好(r2=0.9997);最低定量限为2.64 μg/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 method for rapid screening and quantitative determination of cordycepin in mycelium of Cordyceps sinensis with ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and high performance liquid chromatography (HPLC).Methods Cordycepin were extracted from mycelium of Cordyceps sinensis with water-methanol (85∶15,V∶V).UPLC-MS was used for qualitative screening and a gradient elution of 0.3％ formic acid aqueous solution with methanol was performed.The positive ion mode and multiple reaction monitoring (MRM) model was used for scanning.HPLC was adopted for quantitative determination with a mixture of water and methanol (85∶15,V∶ V) as mobile phase for isocratic elution,and samples were detected by UV detector.Results Determination of two ion pairs of 252.0/136.0 and 252.0/69.0 for qualitative analysis with MRM,the accuracy of results was high.In the quantitative determination,cordycepin had a good relationship with the concentration of 167～1337 μg/mL (r2=0.9997),the limit of quantitation (LOQ) was 2.64 μg/g,the recovery rates of 3 spiked levels were 98.1％～101.0％,and the RSD was 0.06％～1.24％.The method also had good precision and repeatability.Conclusion On the basis of analysis advantages of UPLC-MS and HPLC,the qualitative and quantitative analysis of cordycepin in the mycelium of Cordyceps sinensis are performed quickly,efficiently and accurately,which improve work efficiency and shorten the analysis time.The established method has advantages in the detection of large amount of samples and exploration of artificial mycelium cultivation conditions.