HPLC法同时测定金线莲中12个核苷类成分的含量

Simultaneous determination of 12 nucleosides in Anoectochilus roxburghii by HPLC

目的:建立金线莲中12个核苷类成分(胞嘧啶、尿嘧啶、胞苷、鸟嘌呤、尿苷、腺嘌呤、肌苷、鸟苷、2′-脱氧鸟苷、β-胸苷、腺苷和2′-脱氧腺苷)含量的HPLC测定方法,并采用聚类分析法比较不同来源金线莲核苷类成分组成和含量差异。方法:采用Uitimate?XB-C18色谱柱(250 mm×4.6 mm,5μm),以甲醇(A)-水(B)为流动相,梯度洗脱,流速1.0 mL·min^-1,检测波长260 nm,柱温30℃,进样量10μL。采用聚类分析法评价不同批次金线莲中核苷类成分的化学模式。结果:12个核苷类成分质量浓度分别在考察的线性范围内与峰面积呈良好的线性关系(r>0.999 4);建立的方法精密度、重复性和稳定性良好;加样回收率为95.0%~114.6%(RSD≤4.8%)。对52批金线莲样品中12个核苷类成分进行测定,其含量较高的主要为腺苷、尿苷、鸟苷、胞苷(均值大于300μg·g^-1),聚类分析发现不同来源金线莲核苷成分具有4类化学表型,含量之间存在差异。结论:建立的金线莲中12个核苷类成分的含量测定方法准确、可靠,可以为金线莲核苷类多指标质量控制体系提供新的技术手段。

Objective:To establish an HPLC method for the simultaneous quantification of the twelve nucleosides including cytosine,uracil,cytidine,guanine,uridine,adenine,nosine,guanosine,2′-deoxyguanosine,β-thymidine,adenosine and 2′-deoxyadenosine in Anoectochilus roxburghii,and compare the differences in compositions and contents by clustering analysis. Methods:The HPLC separation was performed on a Uitimate C18 column(250 mm×4. 6 mm,5 μm) in a gradient elution mode with mobile phase consisting of methanol and water at a flow rate of 1.0 mL·min^-1. The detection wavelength was set at 260 nm and the column temperature was 25 ℃. The injection volume was 10 μL. Furthermore,clustering analysis was performed to evaluate the chemical patterns of nucleosides in different batches of samples. Results:The above-mentioned 12 nucleotides showed good linearity(r> 0.999 4),precision,repeatability and stability. The recovery rates were 95.0%^-114.6%(RSD ≤ 4.8%). The contents of adenosine,uridine,guanosine and cytidine with mean value more than 300 μg·g^-1 were higher than other nucleosides in 52 samples. Clustering analysis indicated 4 chemical phenotypes of nucleoside components with diverse contents. Conclusion:The determination method for 12 nucleosides in A. roxburghii is accurate and reliable,which provides a new technique for muli-index quality control systems of the herb.