高效液相色谱-串联质谱法同时测定沙棘中的11种黄酮类物质

Simultaneous Determination of Eleven Flavonoids in Sea Buckthorn Using High Performance Liquid Chromatography-Tandem Mass Spectrometry

建立了同时测定沙棘种子和叶片中异鼠李素、槲皮素、山奈酚、表没食子儿茶素、芦丁、没食子儿茶素没食子酸酯、柚皮素、表儿茶素没食子酸酯、木犀草素、柚皮苷和表儿茶素11种黄酮类物质的高效液相色谱-串联质谱(HPLC-MS/MS)分析方法。色谱采用C18柱分离,流动相为0.1%甲酸溶液-甲醇,等度洗脱,流速0.2mL/min;质谱采用电喷雾电离、负离子(ESI-)扫描,以多反应监测(MRM)模式检测。11种黄酮类物质在各自的线性范围内具有良好的线性关系(r>0.99),回收率>90.9%,相对标准偏差在10%以内。对11份种质种子及3份种质叶片的检测结果表明,不同种质种子和种质叶片中的不同黄酮类化合物间存在明显差异,沙棘叶片检测出的10种黄酮总含量(169.676~215.818μg/g)明显高于种子中检测出的10种黄酮总含量(37.060~134.419μg/g);种子中含量较高的黄酮类化合物分别为表儿茶素没食子酸酯(13.802~103.318μg/g)、表没食子儿茶素(2.113~22.456μg/g)、芦丁(9.684~21.367μg/g)和槲皮素(4.152~10.133μg/g);叶片中含量较高的黄酮类化合物分别为芦丁(80.694~143.169μg/g)、表儿茶素没食子酸酯(15.301~92.029μg/g)、表儿茶素(19.180~56.712μg/g)、山奈酚(8.455~16.393μg/g)和表没食子儿茶素(6.995~15.539μg/g)。本方法简便快速、准确可靠,可作为一种高通量快速测定沙棘种子和叶片中黄酮类化合物的方法。

A method of high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)was developed for determination of 11 flavonoids in seeds and leaves of sea buckthorn.Elven flavonoids,including isorhamnetin,quercetin,kaempferol,(-)-epigallocatechin,rutin,(-)-gallocatechin gallate,naringenin,(-)-epicatechin gallate,luteolin,naringin and L-epicatechin were identified.The optimized conditions for HPLC-MS/MS were that the separation were done using C18 column,with mobile phase gradient elution with 0.1%formic acid-methanol,flow rate of 0.2 mL/min,and MS/MS detection by electrospray ion source negative ion multiple reaction monitoring(MRM)mode.Eleven flavonoids had a good linear relationship in their respective linear ranges,with the correlation coefficients of over 0.99.The recoveries the method were over 90.9%,and the relative standard deviations were less than 10%.The analytical results for seeds of 11 cultivars and leaves of three cultivars showed that there were significant differences in different flavonoids among seeds or leaves of different cultivars;total content of10 flavonoids in leaves of sea buckthorn was higher than that of seeds(169.676-215.818μg/g vs.37.060-134.419μg/g).Major flavonoids in seeds were(-)-epicatechin gallate(13.802-103.318μg/g),(-)-epigallocatechin(2.113-22.456μg/g),rutin(9.684-21.367μg/g)and quercetin(4.152-10.133μg/g),respectively;and it was rutin(80.694-143.169μg/g),(-)-epicatechin gallate(15.301-92.029μg/g)、Lepicatechin((19.180-56.712μg/g),kaempferol(8.455-16.393μg/g)and(-)-epigallocatechin 6.995-15.539μg/g)in leaves of sea buckthorn,respectively.This method is simple,fast,accurate and reliable.It can be used as a high throughput method for rapid determination of flavonoids in sea buckthorn seeds and leaves.This is important for selection and utilization of sea buckthorn resources.