目的：建立一测多评法同时测定冬虫夏草中尿苷、肌苷、鸟苷、腺苷、2'-脱氧腺苷含量，并验证方法准确性。方法：采用RP-HPLC进行含量测定，使用Agilent ZOBAX SB-AQ C18色谱柱（150 mm×4.6 mm，5 μm），以乙腈（A）-水（B）为流动相，梯度洗脱（0~5 min，0% A；5~15 min，0% A→10% A；15~30 min，10% A），流速1.0 mL·min-1，柱温30℃，检测波长260 nm，进样量5 μL。以腺苷为参照物，建立其对尿苷、肌苷、鸟苷、2'-脱氧腺苷的相对校正因子，并用该校正因子进行尿苷、肌苷、鸟苷、2'-脱氧腺苷的含量计算，实现一测多评；同时采用外标法测定冬虫夏草中5个核苷的含量，并比较计算值与实测值的差异，以验证一测多评法的准确性和可行性。结果：尿苷、肌苷、鸟苷、腺苷、2'-脱氧腺苷进样量分别在4.945~98.90、1.852~37.04、4.795~95.90、5.175~103.5和1.892~37.84 μg·mL-1范围内呈现良好线性关系；平均加样回收率（n=6）分别为98.3%（RSD=1.6%）、101.3%（RSD=1.3%）、100.6%（RSD=1.1%）、98.8%（RSD=1.4%）和101.1%（RSD=1.7%）。尿苷、肌苷、鸟苷、2'-脱氧腺苷相对于腺苷的相对校正因子分别为1.41、1.90、1.74和0.964；且在不同实验条件下重现性良好（RSD<3.0%）；一测多评法的计算结果与外标法实测值之间无显著差异。20批冬虫夏草中尿苷、肌苷、鸟苷、腺苷和2'-脱氧腺苷的含量范围分别为0.101%~0.210%、0.037%~0.135%、0.066%~0.203%、0.015%~0.102%和0.001 8%~0.013%。结论：建立的一测多评法可作为冬虫夏草中5个核苷类化学成分的含量测定方法。
Objective: To establish a quantitative analysis of multi-components by single-marker(QAMS)for the determination of uridine,inosine,guanosine,adenosine and 2'-deoxyadenosine in Cordyceps sinensis,and validate its feasibility for quality evaluation.Methods: An HPLC method was applied to quality assessment using a ZOBAX SB-AQ C18 column(150 mm×4.6 mm,5 μm).The mobile phase consisted of acetonitrile(A)and water(B)with the gradient elution(0-5 min,0%A;5-15 min,0%A→10%A;15-30 min,10%A),and the flow rate was 1.0 mL·min-1.The column temperature was set at 30℃.The chromatograms were monitored at 260 nm.The injection volume was 5 μL.Five nucleosides(uridine,inosine,guanosine and adenosine)and 2'-deoxyadenosine were selected as analytes to evaluate the quality of Cordyceps sinensis.The relative correction factors(RCFs)of adenosine to the other four ingredients were calculated.The method was evaluated by the comparison of the quantitative results between external standard method and QAMS method.Results: The linear ranges of uridine,inosine,guanosine,adenosine and 2'-deoxyadenosine were 4.945-98.90 μg·mL-1(r=0.999 9),1.852-37.04 μg·mL-1 (r=0.999 9),4.795-95.90 μg·mL-1(r=0.999 9),5.175-103.5 μg·mL-1(r=0.999 9)and 1.892-37.84 μg·mL-1 (r=0.999 9),respectively;the average recoveries(n=6)were 98.3%(RSD=1.6%),101.3%(RSD=1.3%),100.6%(RSD=1.1%),98.8%(RSD=1.4%)and 101.1%(RSD=1.7%),respectively.RCFs of uridine,inosine,guanosine,and 2'-deoxyadenosine with reference to adenosine were 1.41,1.90,1.74 and 0.964,respectively,and the repeatability was good in different experimental conditions(RSD<3.0%).There were no significant differences between the quantitative results of the QAMS method and external standard method(ESM).The contents of uridine,inosine,guanosine,adenosine and 2'-deoxyadenosine in 20 batches of samples of Cordyceps sinensis were 0.101%-0.210%,0.037%-0.135%,0.066%-0.203%,0.015%-0.102% and 0.001 8%-0.013%,respectively.Conclusion: The established QAMS method could be used for the assay of 5 nucleosides in Cordyceps sinensis.
