超声波提取HPLC-UV法测定人工蛹虫草中8种核苷类物质

Simultaneous determination of eight kinds of nucleotides from cultured Cordyceps militaris by ultrasonic extraction and HPLC-UV

蛹虫草中核苷及其代谢化合物的分析检测对于蛹虫草的生理和药理研究是非常重要的。该文采用超声波辅助提取HPLC-UV法，以反相DIAMONSILC18（250mm×460mm，5μm）为色谱柱，以水（含O．1％乙酸）和甲醇混合液为流动相进行梯度洗脱，同时测定了人工蛹虫草中的尿嘧啶、次黄嘌呤、鸟嘌呤、胸腺嘧啶、肌苷、鸟苷、腺苷和虫草素等8种化合物。结果表明，超声波提取的最优条件为提取溶剂为甲醇，提取温度25℃，提取时间15min，超声功率IOOW，料液比1：100，在最优的超声提取和色谱分离条件下，8种核苷类化合物在检测范围内的线性关系较好（r^2〉O．9995），检出限和定量限最低可达到16．9ng／mL和56．4ng／mL，日内和日间精密度（RSD）分别小于4．1％和4．6％，加样回收率为81．9％～93．7％。此法可用于冬虫夏草及其代用品中核苷类化合物的分析检测。

Determination ofnucleosides and their metabolic compounds is important for the study on physiological and pharmacological properties of Cordyceps militafis. Herein, a rapid Ultrasonic extraction and HPLC method was developed for the simultaneous determination of eight nucleosides and nucleobases, namely uracil, hypoxanthine, guanine, thymine, inosine, guanosine, adenosine and cordycepin. The separation was performed on Japan SHIMADZU HPLC system with ultraviolet detector, DIAMONSIL C18 column and gradient elution with water（0.1% acetic acid）and methanol. The results showed that the optimal ultrasonic extraction conditions were extraction with methanol at 25℃ for 15min, with ultrasonic power 100W, ratios of solid to liquid by 1：100. And under the conditions, the correlation coefficients of 8 analytes were high （r^2 〉 0.9995） within the test ranges. The LOD and LOQ were lower to 16.9ng/ml and 56.4ng/ml with 5 μL of injection volume, respectively. The overall RSD for intra- and inter-day of 8 analyres were less than 4.1% and 4.6%, the recovery of the method was in the range of 81.9%-93.7%. The developed method was suitable for analysis of nucleosides and nucleobases in Cordyceps and its substitutes.