在线二维液相色谱法测定发酵虫草菌粉(Cs-4)中6种活性物质

Determination of 6 Active Ingredients in Fermentative Cordycepic Fungal Powder(Cs-4) by Online Two-Dimensional Liquid Chromatography

目的:建立发酵虫草菌粉(Cs-4)中尿嘧啶、尿苷、腺苷、腺嘌呤、鸟苷、麦角甾醇6种活性物质的二维液相色谱测定方法。方法:采用二维液相色谱测定法,第一维色谱系统采用亲水相互作用色谱柱,第二维系统采用C18色谱柱。样品经乙腈溶液提取,过滤后进样,5种核苷和碱基在第一维液相系统分离测定,麦角甾醇在第二维液相系统测定。结果:方法中各物质线性范围:尿嘧啶,0.3~30μg·mL-1;尿苷,0.4~40μg·mL-1;腺苷,0.8~80μg·mL-1;腺嘌呤,1.0~100μg·mL-1;鸟苷,2.5~250μg·mL-1;麦角甾醇1.5~150μg·mL-1。方法中各物质加样回收率范围与精密度:尿嘧啶,96.7%~103.4%,1.8%~3.1%;尿苷,98.9%~101.4%,1.7%~2.6%;腺苷,95.9%~104.7%,1.5%~3.5%;腺嘌呤,97.0%~104.0%,1.4%~4.0%;鸟苷,98.2%~101.4%,1.9%~3.1%;麦角甾醇,96.0%~102.0%,2.5%~3.7%。结论:本研究方法显著提高了分析效率,准确度高,可满足药物评价、质量监督等工作的需求;对发展发酵虫草菌粉(Cs-4)等中药复杂成分分离方法,指导药物临床使用具有重要意义。

Objective:To establish a two-dimensional liquid chromatography method for the determination of six active substances including uracil, uridine, adenosine, adenine, guanosine and ergosterol in fermentative cordycepic fungal powder(Cs-4). Methods:The samples were extracted with acetonitrile solution and filtered. After injection, 5 nucleosides and bases were separated and determined in the first dimensional liquid phase system, and ergosterol was determined in the second dimensional liquid phase system. Results: In the method the Linear range of uracil, uridine, adenosine, adenine, guanosine, and ergosterol is 0.3-30 μg·mL-1, 0.4-40 μg·mL-1, 0.8-80 μg·mL-1, 1.0-100 μg·mL-1, 2.5-250 μg·mL-11.5-150 μg·mL-1. The recovery and RSD of uracil, uridine, adenosine, adenine, guanosine and ergosterol are 96.7%-103.4% and 1.8%-3.1%,98.9%-101.4% and 1.7%-2.6%,95.9%-104.7% and 1.5%-3.5%, 97.0%-104.0% and 1.4%-4.0%, 98.2%-101.4% and 1.9%-3.1%, 96.0%-102.0% and 2.5%-3.7%. Conclusion: The method significantly improved the analysis efficiency and accuracy, and could meet the needs of drug evaluation and quality supervision.The method is of great significance for the development of separation method for complex components of fermentative cordycepic fungal powder(Cs-4) and guiding clinical use.