多波长HPLC法测定玉屏风散中10种化学成分的含量

Simultaneous determination of ten chemical components in Yupingfeng San by HPLC under multiple UV wavelengths

目的:建立玉屏风散多波长、多成分含量测定的HPLC方法。方法:采用YMC Hydrosphere C_(18)色谱柱(4.6 mm×250 mm,5μm),流动相为乙腈(A)-0.05%磷酸水溶液(B),梯度洗脱,流速为1.0 mL·min^(-1),柱温为30℃,检测波长300 nm(升麻素苷)、257 nm(毛蕊异黄酮葡萄糖苷)、246 nm(升麻素)、294 nm(5-O-甲基维斯阿米醇苷)、256 nm(亥茅酚苷)、248 nm(毛蕊异黄酮)、249 nm(芒柄花素)、221 nm(白术内酯Ⅲ)、220 nm(白术内酯Ⅰ)、278 nm(白术内酯Ⅱ)。结果:在所建立色谱条件下各成分的专属性良好,无干扰峰;线性关系良好,均在0.999 5以上;仪器精密度(n=6)的RSD均小于2%;方法重复性(n=6)的RSD均小于2%;48 h内稳定,RSD均小于2%;回收率在98%~100%,RSD均小于2%,回收率较佳。测得升麻素苷、毛蕊异黄酮葡萄糖苷、升麻素、5-O-甲基维斯阿米醇苷、亥茅酚苷、毛蕊异黄酮、芒柄花素、白术内酯Ⅲ、白术内酯Ⅰ、白术内酯Ⅱ的含量平均值分别为0.032%、0.022%、0.012%、0.038%、0.004%、0.009%、0.006%、0.006%、0.005%、0.004%。结论:经方法学验证,本法可在同一色谱条件下利用不同检测窗口实现多类组分及多个成分的同时测定。

Objective： To develop an HPLC method with multiple wavelength ultraviolet detection for simultaneous deterniination of 10 chemical constituents in Yupingfeng San. Methods： The samples were separated on a YMC Hydrosphere ClS column （ 4.6 mm × 250 mm, 5 p,m ） by a gradient elution using acetonitrile （ A ） and 0.05% phosphoric acid aq. （ B ） as mobile phase at the flow rate of 1.0 mL· min^-1, and the column temperature was 30 ℃.The eluate was detected by multiple wavelength at 300 nm for cimifugin, 257 nm for calycosin-7-O- β -D- glycoside, 246 nm forprim-O-glucosylcimifugin, 294 nm for 4＇-O- β -D-glucosyl-5-O-methyl-visamminol, 256 nm for sec-O-glucosylhamaudol, 248 nm for calycosin, 249 nm for formononetin, 221 nm for atractylenolide HI, 220 nm for atractylenolide Ⅰ , and 278 nm for atraetylenolide Ⅱ. Results： The specificity of the established method was good, and there was no interference peaks. Good linear relation was observed for all the ten compounds.All of the correlation coefficient r were above 0.999 5. The recoveries （ n=9 ） of ten active components were 98%-100%. All of the RSD of precision （ n=6 ） and repeatability （ n=6 ） were below 2%. The average content of cimifugin, calycosin-7-O- β -D-glycoside, prim-O-glucosylcimifugin, 4-0- β -D-glucosyl-5-O-methylvisamminol, sec- O-glucosylhamaudol, calycosin, formononetin, atractylenolide Ⅲ, atractylenolide Ⅰ , atractylenolide Ⅱ were measured, which was 0.032%, 0.022%, 0.012%, 0.038%, 0.004%, 0.009%, 0.006%, 0.006%, 0.005%, and 0.004% in proper order. Conclusion： Though methodology validation, this method can achieve the determination of multi class group and multiple components at the same time under the same chromatographic conditions using different detection window.