基质固相分散-高效液相色谱法同时测定冬虫夏草和蛹虫草中的9种核苷及碱基

Simultaneous determination of nine nucleosides and nucleobases in Cordyceps sinensis and Cordyceps militarris by matrix solid-phase dispersion coupled with high performance liquid chromatography

目的：建立基质固相分散一高效液相色谱法（MSPD-HPLC）同时测定野生冬虫夏草、深层发酵培养的蛹虫草菌丝体和固态培养的蛹虫草子实体中的腺嘌呤、腺苷、胸腺嘧啶、胸苷、尿嘧啶、尿苷、胞嘧啶、乌苷和虫草素。方法：采用MSPD—HPLC法同时测定上述9种核苷及碱基。结果：MSPD提取条件为：弗罗里硅土为分散剂，分散剂与样品质量比为4：1，15mL石油醚为淋洗剂，15mL甲醇为洗脱剂。标准曲线在线性范围内具有较好的线性关系（r〉0．9997），化合物的检出限和定量限分别为12．4—79．4ng·mL^-1和41．2～264．6ng·mL^-1。日内和目间精密度均低于7．28％。样品加标回收率在81．54％-95．77％之间。结论：本法简单快速，样品用量少，可广泛应用于中草药和生物药品中核苷和碱基的同时测定。

Objective： To develop a matrix solid-phase dispersion（MSPD）-high performance liquid chromatography （HPLC） for simultaneous determination of uracil, adenine, thymine, uridine , adenosine, thymidine, cytidine, guanosine and cordycepin in natural Cordyceps sinensis, cultured Cordyceps militarris mycelia and Cordyceps militarris fruiting bodies. Methods： Nine nucleosides and nucleobases were extracted by MSPD and determined by HPLC. Results： The experimental conditions for the MSPD were optimized. Florisil was used as dispersant, petroleum ether as washing solvent and methanol as elution solvent. The ratio of Florisil to sample was selected to be 4： 1. The calibration curve had good linear relationship （r〉0.9997）. The limits of detection and quantification were in the range of 12.4-79.4ng·mL-1 and 41.2-264.6ng·mL-1,respectively. Intra- and interday precision were lower than 7.28%. The recoveries were between 81.54% and 95.77%. Conclusion： The present method was simple,and consumed less sample. The present MSPD-HPLC method should be applied for simultaneous extraction of nucleosides and nucleobases in herbal materials, pharmaceutical products and biological drugs.