高效液相色谱法测定阴地蕨中山奈酚和槲皮素含量∗

摘要

Objective To establish a method for simultaneous determination of kaempferol and quercetin in Botrychium ternatum ( a kind of Ethnic Chinese herbal medicine) by HPLC and HPLC-MS/MS. Methods Agilent 1200 HPLC, API3200 QTRAP MS/MS, Ecilipse XDB-C18 column (15 mm×4.6 mm, 5μm) and the mobile phase consisting of methanol-0.1%formic-water (85��15) were applied to identify kaempferol and quercetin in Botrychium ternatum;Agilent 1260 HPLC and Eclipse plus C18(100 mm×4.6 mm, 5 μm) column were used for determination of the two.The mobile phase was a mixture of methanol and 0.4% phosphoric acid solution (45��55) with a flow rate of 0.8 mL��min-1 and the detection wavelength was 360 nm. Results In mass spectrogram of Botrychium ternatum, characteristic peaks of m/z 287. 1 [ M+H]+ and m/z 301.0 [ M+H]+ were consistent with those of standard sample of kaempferol and quercetin, which confirmed their existence in Botrychium ternatum.The two flavonoids both had a good linearity in the range of 2.500–25.00μg��mL-1(r = 0.999 9 and 0.999 8, respectively).RSD of precision and reproducibility of kaempferol and quercetin were 0.98%,1.89% and 0.79%,2.70%, respectively. Conclusion The method is simple, accurate with high sensitivity and suitable for the determination of kaempferol and quercetin in Botrychium ternatum.%目的：通过高效液相色谱-质谱串联( HPLC-MS/MS)法鉴定阴地蕨中山奈酚成分，并建立HPLC法同时测定阴地蕨中山奈酚和槲皮素含量。方法采用 Agilent 1200 LC高效液相色谱仪及 API3200 QTRAP MS/MS质谱仪， Ecilipse XDB-C18(15 mm×4.6 mm，5μm)色谱柱，甲醇-0.1％甲酸溶液(85��15)为流动相，鉴定阴地蕨中山奈酚及槲皮素；用Agilent 1260 LC高效液相色谱仪，Eclipse plus C18色谱柱(100 mm×4.6 mm，5μm)，甲醇-0.4％磷酸溶液(45��55)为流动相，流速0.8 mL��min－1，检测波长360 nm，测定阴地蕨中的山奈酚及槲皮素含量。结果出现m/z 287.1[M＋H]＋及m/z 301.0[ M＋H]＋峰为质谱的特征峰，分别与山奈酚及槲皮素对照品具有高相似度，证明样品中含有山奈酚和槲皮素；山奈酚和槲皮素的线性范围均为2.500~25.000μg��mL－1，r分别为0.9999和0.9998；精密度实验RSD分别为0.98％和0.79％( n＝5)；加样回收率RSD分别为1.89％，2.70％( n＝5)。结论阴地蕨中含有丰富的山奈酚及槲皮素，HPLC法适于同时测定阴地蕨中的山奈酚及槲皮素含量，方法简便可行，重复性好，精密度高，结果可靠。