高效液相色谱法定量分析虫草类药材的16个核苷类成分

Simultaneous Determination of Sixteen Nucleotides,Nucleosides and Nucleobases in Cordyceps by HPLC

目的定量分析虫草类药材(冬虫夏草及虫草花)中16个核苷类成分。方法以热水为溶剂,超声提取制备样品;采用HPLC-DAD测定冬虫夏草、虫草花中核苷类成分的含量,色谱柱为Agilent Zobax SB-Aq(250 mm×4.6 mm,5μm),预柱为Zobax SB-Aq(12.5 mm×4.6 mm,5μm),流动相A为1 mmol·L^(-1)乙酸铵和1.5 mmol·L^(-1)NaH_2PO_4-Na_2HPO_4缓冲液,用乙酸调节p H至4.9,流动相B为甲醇,梯度洗脱,检测波长为260 nm。结果 16个核苷类成分具有良好的线性关系,在线性范围内相关系数均大于0.999;平均加样回收率为97.7%～105.2%。冬虫夏草总核苷含量为0.46%～0.76%,虫草花总核苷含量为0.26%～0.37%;且通过聚类分析可有效区分冬虫夏草与虫草花。结论本研究建立测定16个核苷类物质的方法准确可靠,可用于虫草类产品的品质评价。

OBJECTIVE To simultaneous quantification of sixteen nucleosides and their related compounds in Cordyceps（C. sinensis and C. militaris）. METHODS The samples were prepared by sonicated extraction with hot water as solvent,and the contents of sixteen analytes in C. sinensis and C. militaris were determined by HPLC-DAD. Agilent Zobax SB-Aq column（ 150 mm ×4.6 mm,5 μm） and a guard column Zobax SB-Aq（12.5 mm × 4.6 mm,5 μm） were used for the separation. Gradient elution buffer consisted of A buffer solution（1 mmol·L^-1 ammonium acetate and 1.5 mmol·L^-1 NaH2PO4-Na2HPO4,p H was adjusted to 4.9 by acetic acid） and B solution（methanol）. Detection wavelength was set at 260 nm. RESULTS The 16 analytes have good linearity（R20.999）within the tested ranges. The average recoveries of standard addition for the compounds were between 97. 7% and 105. 2%. The nucleoside compounds were the main components in Cordyceps samples,and the total contents of 16 nucleoside compounds in C. sinensis samples were 0.46% to 0. 76%,and were 0. 26% to 0. 37% in C. militaris samples. Furthermore,the cluster analysis can effectively distinguish these two Cordyceps samples. CONCLUSION The HPLC method for determination of 16 nucleoside substances developed in this paper is accurate and reliable,which can be used for quality evaluation of Cordyceps.