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建立了高效液相色谱-串联质谱(HPLC-ESI/MS)法对芦丁、金丝桃苷、槲皮素和槲皮苷的抗氧化活性进行定量研究。样品用甲醇溶解,采用Hewlett Packard ODS Hypersil C_(18)色谱柱(100 mm×4.6 mm,5μm)、0.1%甲酸水-甲醇为流动相梯度洗脱,流速0.6 m L·min~(-1),柱温25℃;电喷雾源,负离子模式多反应选择离子检测(MRM),用于定性、定量的母离子和子离子分别为m/z 609→300(芦丁)、m/z 463→300(金丝桃苷)、m/z 301→151(槲皮素)、m/z 447→300(槲皮苷),采用外标法进行定量分析。结果表明:芦丁、金丝桃苷、槲皮素和槲皮苷在0.5~1 600 ng·mL~(-1)范围内线性关系良好(R>0.998),定量限为0.5 ng·m L~(-1),仪器精密度良好(RSD<3.0%),样品反应液在24 h内稳定性良好。本方法具有灵敏度高,分析时间短等特点,相对于其他检测方法,提高了检测的准确性,适用于对贯叶连翘中黄酮醇类化合物抗氧化活性的定量研究。经计算,芦丁、金丝桃苷、槲皮素和槲皮苷对ABTS~(.+)的清除率分别为18.35%、36.28%、41.50%和37.18%。
A high performance liquid chromatography-tandem mass spectrometry (HPLC-ESI / MS) method was developed for the quantitative study of the antioxidant activity of rutin, hyperoside, quercetin and quercitrin. The samples were dissolved in methanol and eluted with a gradient of 0.1% formic acid in water and methanol on a Hewlett Packard ODS Hypersil C 18 column (100 mm × 4.6 mm, 5 μm) with a flow rate of 0.6 m L · min -1 , Column temperature 25 ℃; electrospray source, negative ion mode multiple reaction selective ion detection (MRM), qualitative and quantitative precursor and daughter ions were m / z 609 → 300 (rutin), m / z 463 → 300 (Hyperoside), m / z 301 → 151 (quercetin), m / z 447 → 300 (quercitrin) were determined by external standard method. The results showed that there was a good linear relationship between rutin, hyperoside, quercetin and quercitrin in the range of 0.5-1 600 ng · mL -1 (R> 0.998), and the limit of quantification was 0.5 ng · m L ~ (-1). The precision of the instrument was good (RSD <3.0%). The stability of the sample reaction solution was good within 24 h. Compared with other detection methods, this method has the advantages of high sensitivity and short analysis time, which improves the accuracy of detection and is suitable for the quantitative study of the antioxidant activity of flavonol in Hypericum perforatum. The clearance rates of rutin, hyperoside, quercetin and quercitrin to ABTS ~ (+) were calculated to be 18.35%, 36.28%, 41.50% and 37.18% respectively.