UPLC-MS/MS法测定不同温度定向炮制黄芪中8种苷类和4种苷元成分的含量
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篇名: UPLC-MS/MS法测定不同温度定向炮制黄芪中8种苷类和4种苷元成分的含量
TITLE: Determination of 8 Glycosides and 4 Aglycones in Directionally Processed Astragalus membranaceus under Different Temperatures by UPLC-MS
摘要: 目的:建立测定黄芪中8种苷类(黄芪皂苷Ⅰ、Ⅱ、Ⅲ、Ⅳ和毛蕊异黄酮苷、芒柄花苷、黄芪异黄烷苷、黄芪紫檀烷苷)及4种苷元(毛蕊异黄酮、芒柄花素、黄芪异黄烷、黄芪紫檀素)成分含量的方法,并考察不同炮制温度对上述12种成分含量的影响。方法:分别以生黄芪和不同温度(120、140、160、180、200℃)烘制后的黄芪为样品,采用超高效液相色谱-串联质谱(UPLC-MS/MS)法测定其中12种成分的含量。色谱柱为ACQUITYUPLCHSST3,流动相为0.1%甲酸水溶液-0.1%甲酸乙腈溶液(梯度洗脱),流速为0.5mL/min,柱温为30℃,检测波长为260nm,进样量为2μL。采用电喷雾离子源,在正离子模式下进行监测扫描,扫描范围为质菏比50~1500,毛细管电压为2000V,离子源温度为100℃,脱溶剂温度为400℃,雾化气(N2)流速为40L/h,脱溶剂气(N2)流速为800L/h,碰撞能量为20~30V,数据采集速率为0.5s/scan。结果:黄芪皂苷Ⅰ、黄芪皂苷Ⅱ、黄芪皂苷Ⅲ、黄芪皂苷Ⅳ、毛蕊异黄酮苷、毛蕊异黄酮、芒柄花苷、芒柄花素、黄芪异黄烷苷、黄芪异黄烷、黄芪紫檀烷苷、黄芪紫檀素的检测质量线性范围分别为0.00116~0.2320、0.000276~0.0552、0.00022~0.0440、0.000225~0.0450、0.000734~0.5870、0.00117~0.2340、0.000742~0.1480、0.00130~0.260、0.00398~0.7950、0.000476~0.4760、0.00189~0.3780、0.000336~0.3360μg(R2均不低于0.9992);检测限分别为6.2×10-6、4.8×10-6、3.8×10-6、3.4×10-6、5.8×10-6、4.8×10-6、4.2×10-6、3.2×10-6、5.8×10-6、2.6×10-6、4.2×10-6、6.4×10-6μg;定量限分别为12.6×10-6、16.2×10-6、14.4×10-6、14.8×10-6、18.8×10-6、16.4×10-6、15.4×10-6、10.8×10-6、20.2×10-6、12.4×10-6、14.6×10-6、23.4×10-6μg;精密度、稳定性(24h)、重复性试验的RSD均小于3.0%(n=6);平均加样回收率分别为99.1%、100.2%、98.7%、101.9%、98.6%、102.1%、99.2%、100.3%、98.7%、99.2%、99.3%、100.8%,RSD分别为1.9%、2.2%、2.4%、1.8%、2.1%、1.7%、2.3%、1.9%、2.4%、1.8%、2.2%、1.9%(n=6)。含量测定结果显示,黄芪皂苷Ⅰ、Ⅱ、Ⅲ的含量随着烘制温度的升高而逐渐降低,黄芪皂苷Ⅳ的含量随着温度的升高而逐渐升高;黄酮苷类成分毛蕊异黄酮苷、芒柄花苷、黄芪异黄烷苷、黄芪紫檀烷苷的含量均随着烘制温度的升高而降低,而对应的苷元成分毛蕊异黄酮、芒柄花素、黄芪紫檀素的含量均随着烘制温度的升高而先升高后降低;黄芪异黄烷的含量随着烘制温度的升高而降低。结论:所建立的UPLC-MS/MS法可用于黄芪中12种成分含量的测定;黄芪经不同温度炮制后,苷类成分含量总体呈降低趋势,苷元成分含量总体呈升高趋势。
ABSTRACT: OBJECTIVE:To establish a method for the determination of 8 glycosides(astragaloside Ⅰ,Ⅱ,Ⅲ,Ⅳ and calycosin glucopyranoside ,2′-hydroxy-3′,4′-dimethoxy-isoflavan-glucoside and 9,10-dimethoxy-pterocarpan-glucoside) and 4 aglycones(calycosin,formononetin,7,2′-dihydroxy-3′,4′-dimethoxy-isoflavan and 3-hydroxy-9,10-dimethoxy-pterocarpan) in Astragalus membranaceus ,and to investigate the effects of different processing temperatures on the contents of above 12 components. METHODS :The contents of 12 components in A. membranaceus and samples processed under different temperatures(120,140,160,180,200 ℃)were determined by UPLC-MS/MS. The determination was performed on ACQUITY UPLC HSS T 3 column with mobile phase consisted of 0.1 mol/L formic acid water solution -0.1 mol/L formic acid acetonitrile solution (gradient elution )at the flow rate of 0.5 mL/min. The column temperature was 30 ℃. The detection wavel ength was 260 nm,and sample size was 2 μL. Electrospray ion source(ESI)was used under positive ion mode (ESI+). The mass scanning range was mass ratio (m/z)of 50-1 500,with capillary voltage of 2 000 V and ion source temperature of 100 ℃. The desolvation temperature was 400 ℃;flow rate of atomizing gas (N2) was 40 L/h,and that of desolvation was 800 L/h;collision energy (CE)was 20-30 V;data acquisition rate was 0.5 s/scan. RESULTS:The linear range of astragaloside Ⅰ,astragaloside Ⅱ,astragaloside Ⅲ,astragaloside Ⅳ,calycosin-glucopyranoside, calycosin,ononin,formononetin,2′-hydroxy-3′,4′-dimethoxy-isoflavan-glucoside,7,2′-dihydroxy-3′,4′-dimethoxy-isoflavan,9, 10-dimethoxy-pterocarpan-glucoside and 3-hydroxy-9,10-dimethoxy-pterocarpan were 0.001 16-0.232 0,0.000 276-0.055 2, 0.000 22-0.044 0,0.000 225-0.045 0,0.000 734-0.587 0,0.001 17-0.234 0,0.000 742- 0.148 0,0.001 30-0.260,0.003 98-0.795 0, 0.000 476-0.476 0,0.001 89-0.378 0,0.000 336-0.336 0 μg(all R2≥0.999 2),respectively. The limits of detection were 6.2×10-6, 4.8×10-6,3.8×10-6,3.4×10-6,5.8×10-6,4.8×10-6,4.2×10-6,3.2×10-6,5.8×10-6,2.6×10-6,4.2×10-6,6.4×10-6 μg,respectively. The limits of quantitation were 12.6×10-6,16.2×10-6,14.4×10-6,14.8×10-6,18.8×10-6,16.4×10-6,15.4×10-6,10.8×10-6,20.2×10-6, 12.4×10-6,14.6×10-6,23.4×10-6 μg,respectively. RSDs of precision ,stability(24 h)and repetition tests were all lower than 3.0%(n=6). The average recoveries were 99.1%,100.2%,98.7%,101.9%,98.6%,102.1%,99.2%,100.3%,98.7%, 99.2%,99.3% and 100.8%,with the RSDs of 1.9%,2.2%,2.4%,1.8%,2.1%,1.7%,2.3%,1.9%,2.4%,1.8%,2.2% and 1.9%(n=6),respectively. The results showed that the contents of astragaloside Ⅰ,Ⅱ and Ⅲ decreased gradually with the increase of processing temperature ;the content of astragaloside Ⅳ increased gradually with the increase of temperature. The content of flavonoid glycosides ,such as calycosin glucopyranoside ,ononin,2′-hydroxy-3′,4′-dimethoxy-isoflavan-glucoside and 9, 10-dimethoxy-pterocarpan-glucoside decreased with the increase of temperature ;the corresponding aglycone components as flavonoid glycosides ,formononetin,3-hydroxy-9,10-dimethoxy- pterocarpan increased firstly and then decreased with the increase ; the content of 7,2′-dihydroxy-3′,4′- dimethoxy-isoflavan decreased with the increase of temperature. CONCLUSIONS :Established UPLC-MS/MS method can be used for determination of 12 components in A. membranaceus . After processed under different temperature,the contents of glycosides decreased in general ,while the contents of aglycones increased in general.
期刊: 2020年第31卷第03期
作者: 刘蓬蓬,张凡,史辑,单国顺,贾天柱
AUTHORS: LIU Pengpeng ,ZHANG Fan,SHI Ji,SHAN Guoshun ,JIA Tianzhu
关键字: 黄芪;超高效液相色谱-串联质谱法;苷类;苷元;含量测定;定向炮制
KEYWORDS: Astragalus membranaceus ;UPLC-MS/MS;Glycosides;Aglycones;Content det ermination;Directional processing
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