基于超高效液相色谱-四极杆/静电场轨道阱高分辨质谱的妇可靖胶囊中11种成分定量研究

Quantitative research of multiple active components in Fukejing Capsule based on ultra performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry

目的建立基于超高效液相色谱-四极杆/静电场轨道阱高分辨质谱(ultra performance liquid chromatographyquadrupole/orbitrap high resolution mass spectrometry,UPLC-Q-OrbitrapHRMS)的妇可靖胶囊中多成分(没食子酸、丹参素、阿魏酸、迷迭香酸、丹酚酸B、木犀草素、芹菜素、芦荟大黄素、大黄素、丁烯基苯酞、欧当归内酯A)的定量分析方法。方法液相采用Acquity UPLC?BEH C18色谱柱(50 mm×2.1 mm,1.7μm),以乙腈-0.1%甲酸水溶液为流动相,梯度洗脱,体积流量为0.2 m L/min;质谱采用HESI离子源,利用其高分辨的特点采用一级Full mass扫描的方式进行定量;并将定量测定结果导入多元数据处理软件SIMCA14.0中进行质量评价分析。结果在优化的色谱质谱条件下,没食子酸、丹参素、阿魏酸、迷迭香酸、丹酚酸B、木犀草素、芹菜素、芦荟大黄素、大黄素、丁烯基苯酞、欧当归内酯A分别在0.15~1.50、0.80~8.00、1.50~15.00、0.04~0.40、0.015~0.150、0.10~1.00、0.004~0.040、0.004~0.040、0.02~0.20、0.01~0.10、0.04~0.40μg/m L线性关系良好(r≥0.999 5);精密度、重复性及稳定性良好(RSD≤4%);加样回收率在98%~102%,RSD均小于3%;所测成分在各批次样品中的质量分数依次为没食子酸26.36~31.03μg/g、丹参素178.85~210.79μg/g、阿魏酸320.91~343.16μg/g、迷迭香酸2.84~3.09μg/g、丹酚酸B 3.55~4.25μg/g、木犀草素27.33~32.36μg/g、芹菜素1.89~2.13μg/g、芦荟大黄素0.47~0.60μg/g、大黄素3.17~3.57μg/g、丁烯基苯酞1.99~2.54μg/g、欧当归内酯A 7.51~8.53μg/g;分析结果表明大多数批次药物质量较为稳定,其中丹参素和阿魏酸对药物质量具有较大影响,可对其进行重点监控以保证药物批次质量。结论建立的定量方法灵敏度高且准确性好,方法学考察结果符合测定要求,可用于妇可靖中多种活性成分的快速测定;并为其质量评价提供新的科学依据和参考。

Objective To establish a quantitative analysis method of multiple active components （gallic acid, tanshinol, ferulic acid, rosmarinic acid, salvianolic acid B, luteolin, apigenin, aloe-emodin, emodin, butylidenephthalide, and levistilide A） in Fukejing Capsule （FC） based on ultra performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry （UPLC-Q- Orbitrap HRMS）. Methods The column was BEH C18 （50 mm × 2.1 mm, 1.7 μm） and the mobile phase was consisted ofacetonitrile-water （containing 0.1% formic acid） at a flow rate of 0.2 mL/min with gradient elution; Mass spectrometer conditions： Heated electrospray ionization source （HESI） and full mass quantify methods were used to perform the determination, and the results of the contents were imported into the muti-data processing soft ware SIMCA14.0 to make a quality evaluation. Results Under the optimized conditions, gallic acid, tanshinol, ferulic acid, rosmarinic acid, salvianolic acid B, luteolin, apigenin, aloe-emodin, emodin, butylidenephthalide, and levistilide A all showed good liner relationship （r 〉 0.999 5） in the range of 0.15-I .50, 0.80-8.00, 1.50- 15.00, 0.04-0.40, 0.015-0.150, 0.10-1.00, 0.004-0.040, 0.004-0.040, 0.02-0.20, 0.01-0.10, and 0.04-0.40 gg/mL, respectively; The results of the accuracy, the repeatability and the stability all reached the standards （RSD 〈 4%）; The recoveries ranged from 98%-102% and RSDs were below 3%; The result of the content ranges in different batches were 26.36-31.03 gg/g （gallic acid）, 178.85-210.79 pg/g （tanshinol）, 320.91-343.16 μg/g （ferulic acid）, 2.84-3.09 μg/g （rosmarinic acid）, 3.55-4.25 μg/g （salvianolic acid B）, 27.33-32.36 μg/g （luteolin）, 1.89-2.13 μg/g （apigenin） A, 0.47-0.60 μg/g （aloe-emodin）, 3.17-3.57 μg/g （emodin）, 1.99-2.54 μg/g （butylidenephthalide）, and 7.51-8.53 μg/g （levistilide A）. The analysis results showed that the quality of the most batches was stable, the tanShinol and ferulic acid had a great influence on the quality of the medicine, and could be monitored to ensure the quality of different batches. Conclusion The methods established in this paper have a high sensitivity and accuracy; The results of the methodology conform to the relevant requirements and the methods can rapidly determinate the multiple active components in FC; The research also provides a new scientific basis and reference for the quality assessment at the same time.