固相萃取-液相色谱-串联质谱法同时测定葛根中11种黄酮苷元的含量

Simultaneous Determination of 11 Flavonoid Aglycones in Pueraria Lobata by Solid Phase Extraction-Liquid Chromatography-Tandem Mass Spectrometry

建立了固相萃取-液相色谱-串联质谱法(LC-MS/MS)同时测定葛根中葛根素、甘草素、大豆素、野黄芩素、大豆黄素、槲皮素、鸢尾黄素、染料木素、山萘酚、黄芩素和刺芒柄花素11种黄酮苷元含量的方法。在0.10 g葛根样品中加入0.5 g叔丁基对苯二酚,36 mL 50%(体积分数)乙醇溶液和4 mL盐酸,在氮气保护下水解120 min,提取其中的11种黄酮苷元。采用Waters HLB固相萃取柱,以20%(体积分数)甲醇溶液为淋洗液,以甲醇为洗脱液对样品进行净化。以Mightysil RP-18色谱柱为固定相,以甲醇-水体系(大豆苷、大豆黄苷、刺芒柄花苷)或甲醇-0.15%(体积分数,下同)甲酸溶液体系(其他8种黄酮苷和11种黄酮苷元)为流动相进行梯度洗脱,采用LC-MS/MS测定其中11种黄酮苷元的含量。用体积比为4∶6的甲醇-0.15%甲酸溶液的混合液配制的混合标准溶液系列绘制标准曲线,外标法定量。结果显示:11种黄酮苷元的质量分数在0.05%~2.50%内与其对应的峰面积呈线性关系,葛根素、大豆素和黄芩素的检出限(3S/N)均为0.04%,甘草素、野黄芩素、大豆黄素、槲皮素、鸢尾黄素、染料木素、山萘酚和刺芒柄花素的检出限均为0.02%;对实际样品进行3个浓度水平的加标回收试验,11种黄酮苷元的回收率为75.0%~94.0%,测定值的相对标准偏差(RSD,n=6)为3.5%~12%;方法用于实际葛根样品的测定,葛根素、大豆素、大豆黄素、染料木素和刺芒柄花素均被不同程度地检出。

A method for simultaneous determination of 11 flavonoid aglycones(puerarin,liquiritigenin,daidzein,scutellarein,glycitein,quercetin,tectorigenin,genistein,kaempferol,baicalein and formononetin)in Pueraria lobata by liquid chromatography-tandem mass spectrometry(LC-MS/MS)with solid phase extraction was developed.Pueraria lobata sample of 0.10 g were hydrolyzed with 0.5 g of tert-butylhydroquinone,36 mL of 50%(φ)ethanol solution and 4 mL of hydrochloric acid for 120 min under the protection of nitrogen to extract 11 flavonoid aglycones.The sample solution was purified by Waters HLB solid phase extraction column with 20%(φ)methanol solution as leacheate and methanol as eluent.Mightysil RP-18 chromatographic column was used as the stationary phase,and the methanol-water system(daidzein,daidzein and formononein)or methanol-0.15%(φ,the below same)formic acid solution system(other 8 flavonoid glycosides and 11 flavonoid aglycones)was used as the mobile phase for gradient elution.11 flavonoid aglycones were determined by LC-MS/MS.The mixed standard solution series prepared with a mixture of methanol and 0.15%formic acid solution at volume ratio of 4∶6 were used to draw the standard curves,and external standard method was used for quantitative analysis.As shown by the results,linear relationships between values of mass fraction of 11 flavonoid aglycones and their peak area were kept in the range of 0.05%-2.50%,with detection limits(3 S/N)of 0.04%for puerarin,daidzein and baicalein,and 0.02%for liquiritigenin,scutellarein,glycitein,quercetin,tectorigenin,genistein,kaempferol and formononetin.Recovery test was made on actual samples at 3 concentration levels by stardand addition method,giving results in the range of 75.0%-94.0%,with RSDs(n=6)of the determined values in the range of 3.5%-12%.This method was applied to Pueraria lobata sample,and puerarin,daidzein,glycitein,genistein and formononetin were detected in varying degrees.