一测多评法同时测定蛹虫草中5种核苷类成分的含量

Simultaneous Determination of Five Nucleosides in Cordyceps militaris by the Quantitatire Analysis of Multi-components by Single Marker Method

本研究为建立一测多评法同时测定蛹虫草中尿苷、鸟苷、腺苷、虫草素和N6-(2-羟乙基)腺苷含量,并验证方法准确性采用RPHPLC进行含量测定检测波长260nm进样量5μL流速0.8 mL·min^(-1),采用流动相为乙腈(A)水(B)梯度洗脱:0~10 min,0→10%A;10~30 min,10%→20%A;色谱柱采用岛津Inert Sustain AQ-C_(18),(5μm,4.6 mm×150 mm),柱温30℃。以腺苷为参照物,建立其对尿苷、鸟苷、虫草素和N6-(2-羟乙基)腺苷的相对校正因子,并用该校正因子进行尿苷、鸟苷、虫草素和N6-(2-羟乙基)腺苷的含量计算,实现一测多评;同时采用外标法测定蛹虫草中5种核苷的含量,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。结果显示,尿苷、鸟苷、腺苷、虫草素和N_(6)-(2-羟乙基)腺苷进样量分别在2.460~49.20μg·mL^(-1)、2.375~47.50μg·mL^(-1)、2.570~51.40μg·mL^(-1)、2.515~50.30μg.mL^(-1)和2.490~49.80μg.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98.08%(RSD=1.86%)、99.15%(RSD=1.25%)、97.35%(RSD=1.68%)、98.86%(RSD=1.09%)和101.25%(RSD=1.35%)。蛹虫草中尿苷、鸟苷、腺苷、虫草素和N_(6)-(2-羟乙基)腺苷的含量分别为0.625~1.508 mg·g^(-1)、0.347~1.012 mg·g^(-1)、0.624~1.092 mg·g^(-1)、0.315~1.297 mg·g^(-1)和0.546~1.226mg·g^(-1)。本研究建立的一测多评法可作为蛹虫草中5种核苷类化学成分的含量测定方法。该方法具有良好的准确性、稳定性及重复性;采用一测多评法与外标法实测值之间无显著差异。

A quantitative analysis of multi-components by single marker(QAMS)method was established for the simultaneous determination of uridine,guanosine,adenosine,cordycepin and N^(6)-(2-hydroxyethyl)adenosine in Cordyaps militaris,and the accuracy of the method was verified.A HPLC method was applied for the quality assessment using a SHIMADZU InertSustain AQ-C_(18) column(4.6 mm×150 mm,5μm).The mobile phase consisted of acetonitrile(A)and water(B)with the gradient elution(0-10 min 0→10%A;10~30 min 10%→20%A),and the flow rate was 0.8 mL·min^(-1).The column temperature was set at 30℃.The chromatograms were monitored at 260 nm.The injection volume was 5μL.Adenosine was used as the internal reference substance.The relative correlation factors(RCF)of uridine,guanosine,cordycepin and N;-(2-Hydroxyethyl)to adenosine were calculated.The contents of these five nucleosides were determined by the external standard method and QAMS method respectively.The QAMS method was validated through comparison of the results obtained by the two different methods.Results showed that the linear ranges of uridine,guanosine,adeno sine,cordycepin and N6-(2-hydroxyethyl)were 2.460~49.20 ug·mL^(-1)(r=0.9999),2.375~47.50 ug·mL^(-1)(r=0.9999),2.570-51.40 ug·mL^(-1)(r=0.9999),2.515~50.30 ug·mL^(-1)(r=0.9999)and 2.490~49.80μg·mL^(-1)(r=0.9999),respectively.The average recoveries of them(n=6)were as follows:98.08%(RSD=1.86%),99.15%(RSD=1.25%),97.35%(RSD=1.68%),98.86%(RSD=1.09%)and 101.25%(RSD=1.35%),respectively.The contents of uridine,guanosine,adenosine,cordycepin and N6-(2-hydroxyethyl)in 8 batches of samples of C.militaris were 0.625~1.508 mg·g^(-1),0.347~1.012 mg·g^(-1),0.624~1.092 mg·g^(-1),0.315~1.297 mg·g^(-1);and 0.546~1.226 mg·g^(-1),respectively.The RCF showed good reproducibility.No significant difference was found between the two methods.The QAMS method showed favorable feasibility and economy,which could be used to determine the content of five nucleosides in C.militaris.