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目的:采用高效液相-蒸发光散射检测(HPLC-ELSD)玉屏风散颗粒中黄芪甲苷和甘露醇的含量,为玉屏风散的质量控制提供实验基础。方法:采用色谱柱出自Waters公司,型号为XBridge-Amide,规格:4.6 mm×150 mm,色谱柱填料颗粒直径3.5 μm;流动相:乙腈(A)-水溶液(B,其中含少量甲醇),流速1 mL/min;梯度洗脱流程见表1;柱温30 ℃;蒸发光检测器:以氮气为载气,流速202.7 kPa,漂移管温度110 ℃,撞击器为“on”。常规对高效液相色谱仪进行方法学的严谨考察,包括专属性试验、精密度试验、稳定性试验、重复性试验、线性回归试验和加样回收试验。在高效液相色谱仪方法学严谨的前提下,对玉屏风散颗粒进行样品分析研究,并测定其中黄芪甲苷和甘露醇的含量。结果:1)专属性试验结果显示:阴性对照品溶液1在与甘露醇对照品溶液和玉屏分散供试品溶液色谱图相同时间处未见相同色谱峰;阴性对照品2在与黄芪甲苷对照品溶液和供试品溶液色谱图相同时间处未见相同色谱峰。提示甘露醇和黄芪甲苷均具有良好的专属性。2)线性回归结果显示:黄芪甲苷的线性范围为1.1~5.1 mg,其回归方程为:Y=0.732X-4.136(r=0.999 7);同黄芪甲苷线性回归方法得,甘露醇在1.967~19.674 μg范围内具有良好的线性关系,得到甘露醇回归方程为Y＝1.587X+4.983(r＝0.999 6)。3)精密度试验结果显示,黄芪甲苷RSD＝0.58%,甘露醇RSD＝0.36%表明高效液相仪器精密度良好。4)本研究稳定性结果显示黄芩甲苷峰面积的RSD＝0.84%,甘露醇峰面积的RSD=0.70%,符合色谱峰相对峰面积的RSD<3%则说明稳定性良好,提示玉屏风散供试品溶液24 h内稳定性良好。5)本研究结果显示色谱峰相对峰面积RSD平均值1.76%,各色谱峰相对峰面积的RSD<3%表示重复性良好,提示玉屏风散供试品溶液的重复性良好。6)结果显示黄芪甲苷对照品溶液、甘露醇对照品溶液平均回收率分别为100.608%、103.487%,RSD分别为0.93%、0.47%,提示高相液相检测方法所测加样回收率结果可靠。7)3个不同批次的玉屏风散中黄芪甲苷成分和甘露醇成分的含量未见明显差异,黄芪甲苷和甘露醇含量平均值分别为0.961 mg/g和0.099 mg/g。结论:高效液相-蒸发光散射检测方法操作简便准确,重复性和稳定性均良好,可作为测定玉屏风散颗粒中黄芪甲苷和甘露醇含量的测定方法。

English Summary:

To determine the contents of astragaloside A and mannitol in Yupingfeng powder granules by high performance liquid Chromatography-Evaporative Light Scattering (HPLC-ELSD)，and to provide experimental basis for the quality control of Yupingfeng Powder.Methods:The chromatographic column of XBridge-Amide was produced by Waters Company with specifications of 4.6 mm×150 mm and packing particle diameter of 3.5 um.The mobile phase was acetonitrile (A)-aqueous solution (B，containing a small amount of methanol) with flow rate of 1 mL/min.The gradient elution process was shown in Table 1.Column temperature was 30 C.The evaporative light detector was based on nitrogen.The carrier gas flow rate is 202.7 kPa，the drift tube temperature was 110 degrees，and the impactor was “on”.Routine rigorous methodological investigation of high performance liquid chromatography (HPLC) instrument included specificity test，precision test，stability test，repeatability test，linear regression test and sample addition recovery test.On the premise of rigorous methodology of high performance liquid chromatography，Yupingfeng Powder granules were analyzed，and the contents of astragaloside A and mannitol were determined.Results:1) Specific test results showed that the negative reference solution 1 did not have the same chromatographic peak at the same time as mannitol reference solution and Yuping Powder sample solution chromatogram; the negative reference solution 2 did not have the same chromatographic peak at the same time as astragaloside reference solution and sample solution chromatogram.It was suggested that mannitol and astragaloside had good specificity.2) The linear regression results showed that the linear range of astragaloside A was 1.1-5.1 mg，and the regression equation was Y=0.732X-4.136 (r=0.9997).The linear regression method with astragaloside A showed that mannitol had a good linear relationship in the range of 1.967～19.674 ug，and the regression equation of mannitol was Y=1.587X+4.983 (r=0.999 6).3) The precision test results showed that astragaloside RSD=0.58%，mannitol RSD=0.36%，indicating that the precision of high performance liquid chromatography instrument was good.4) The results of this study showed that the RSD of baicalin peak area was 0.84%，and that of mannitol peak area was 0.70%.The RSD<3% which accorded with the relative peak area of chromatographic peak indicated that the stability of Yupingfeng Powder solution was good within 24 h.5) The results showed that the average RSD of relative peak area was 1.76%.RSD<3% of relative peak area indicated good repeatability，suggesting that the solution of Yupingfeng Powder had good repeatability.6) The results showed that the average recoveries of astragaloside A reference solution and mannitol reference solution were 100.608% and 103.487% respectively，and RSD were 0.93% and 0.47% respectively，suggesting that the recoveries of samples determined by high-phase liquid chromatography were reliable.7) There was no significant difference in the contents of astragaloside IV and mannitol in 3 batches of Yupingfeng powder.The average contents of astragaloside IV and mannitol were 0.961 mg/g and 0.099 mg/g，respectively.Conclusion:The method is simple，accurate，reproducible and stable.It can be used for the determination of astragaloside A and mannitol in Yupingfeng Powder Granules.

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