基于UPLC-QE-Orbitrap-MS法测定补肺益肾方中10个入血成分的含量

目的：应用UPLC-QE-Orbitrap-MS法建立补肺益肾方中10个成分（人参皂苷Re、人参皂苷Rb₁、人参皂苷Rg₁、黄芪甲苷、贝母素甲、五味子乙素、淫羊藿苷、橙皮苷、川陈皮素、芍药苷）的含量测定方法。方法：应用Phenomenex kinetex C₁₈色谱柱（100 mm×2.1 mm，2.6 μm），以0.1%甲酸水（A）和乙腈（B）为流动相，0.3 mL·min^-1流速梯度洗脱（0~2 min，5% B；2~12 min，5% B → 100% B；12~15 min，100% B），柱温30℃，电喷雾离子源（ESI），Full mass模式进行正负切换全扫测定。结果：10个成分线性范围内线性关系良好（r>0.998 3），加样回收率和RSD分别在93.7%~104.0%和2.1%~4.1%范围内，精密度、稳定性考察符合定量分析的要求。3批样品中各成分测定结果分别为0.555~0.579、0.534~0.550、0.439~0.472、0.020~0.025、0.264~0.294、0.277~0.295、0.321~0.362、6.285~6.510、0.106~0.112、4.061~4.327mg·g^-1。结论：本研究建立的测定方法高效、可靠、重复性好，为补肺益肾方有效成分的定量研究提供参考。

Objective: To establish an UPLC-MS/MS method for simultaneous determination of 10 absorbed active components (ginsenoside Re,ginsenoside Rb₁,ginsenoside Rg₁,astragaloside Ⅳ,peimine,schisandrin B, icariin, hesperidin, nobiletin and paeoniflorin) in Bufei Yishen recipe by UPLC-QE-Orbitrap-MS. Methods: The chromatographic separation was performed on a Phenomenex kinetex C₁₈ column (100 mm×2.1 mm,2.6 μm) with a mobile phase consisting of acetonitrile (B) -0.1% formic acid water (A) at a flow rate of 0.3 mL·min^-1 (0-2 min, 5%B; 2-12 min, 5%B → 100%B; 12-15 min, 100% B). The column temperature was 30℃. Electrospray ionization source (ESI) was used combined with full mass mode for positive and negative switching full sweep measurement. Results: Good linearity was obtained (r>0.998 3) for 10 potential active components. The recoveries of 10 potential active components were within the range of 93.7% -104.0% with the RSDs within 2.1%-4.1%. Precision and stability investigation met the requirements of quantitative analysis. Contents of the analytes in the samples were 0.555-0.579 mg·g^-1, 0.534-0.550 mg·g^-1, 0.439-0.472 mg·g^-1, 0.020-0.025 mg·g^-1, 0.264-0.294 mg·g^-1,0.277-0.295 mg·g^-1,0.321-0.362 mg·g^-1,6.285-6.510 mg·g^-1,0.106-0.112 mg·g^-1 and 4.061-4.327 mg·g^-1, respectively. Conclusion: The method is efficient, reliable and repeatable, which provides reference for quantitative study of the active component in Bufei Yishen recipe.

[1] 田燕歌,董浩然,刘学芳,等.补肺益肾方对慢性阻塞性肺疾病模型大鼠肺泡表面活性蛋白A、D及肺泡结构的影响[J].中医学报, 2018, 33(1):99 TIAN YG,DONG HR,LIU XF,et al. Effects of Bufei Yishen recipe on alveolar surfactant protein ANAD and alveolar structure in rats with chronic obstructive pulmonary disease[J]. J Chin Med,2018, 33(1):99
[2] 田燕歌,崔红新,李建生,等.调补肺肾三法调控慢性阻塞性肺疾病稳定期大鼠炎症反应的R值综合评价[J].中医杂志,2015, 56(15):1330 TIAN YG, CUI HX, LI JS, et al. Comprehensive evaluation of R value of regulating inflammatory response in rats with chronic obstructive pulmonary disease(COPD)at stable stage by three methods of regulating lung and kidney[J]. J Tradit Chin Med, 2015, 56(15):1330
[3] 王明明.补肺益肾方对幼龄哮喘豚鼠模型IL-5信号通路的调控机制[J].长春中医药大学学报, 2016, 32(2):234 WANG MM. Regulation mechanism of Bufei Yishen recipe on IL-5 signaling pathway in young asthma guinea pig model[J]. J Changchun Univ Tradit Chin Med,2016, 32(2):234
[4] 崔红新,田燕歌,李建生,等.调补肺肾3法调控COPD大鼠肺脏炎症信号通路的R值综合评价研究[J].中国中药杂志, 2015, 40(8):1570 CUI HX, TIAN YG, LI JS, et al. The comprehensive evaluation of the R value of regulating lung and kidney by 3 methods of regulating lung and kidney in COPD rats[J]. China J Chin Mater Med,2015, 40(8):1570
[5] 郝丽丽.基于肺泡上皮细胞A549中NF-κB信号通路调控的调补肺肾三方对炎症反应机制的影响[D].郑州:河南中医药大学,2014 HAO LL. The Effect of Regulating Lung and Kidney by Regulating NF-κB Signaling Pathway in Alveolar Epithelial Cell A549 on the Inflammatory Response Mechanism[D]. Zhengzhou:Henan University of Chinese Medicine,2014
[6] 王洋.补肺益肾方调控NF-κB通路抑制MRC-5细胞炎症反应的机制[D].郑州:河南中医药大学, 2017 WANG Y. Mechanism of Bufei Yishen Fang Regulating NF-κB Pathway in Inhibiting Inflammatory Response of MRC-5 Cells[D]. Zhengzhou:Henan University of Chinese Medicine,2017
[7] 田燕歌.补肺益肾方联合舒肺贴抑制炎症反应治疗COPD大鼠的机制研究[D].北京:北京中医药大学,2015 TIAN YG. Study on the Mechanism of Bufei Yishen Decoction Combined with Shufei Pian to Inhibit Inflammatory Response in COPD Rats[D]. Beijing:Beijing University of Chinese Medicine, 2015
[8] 王明明.补肺益肾方对幼龄哮喘豚鼠模型气道重塑中嗜酸粒细胞凋亡的影响[J].辽宁中医杂志, 2014, 41(11):2488 WANG MM. Effect of Bufei Yishen recipe on apoptosis of eosinophils in airway remodeling of guinea pigs with asthma[J]. Liaoning J Tradit Chin Med,2014,41(11):2488
[9] 李卫卫.调补肺肾三方调控NFκB/TGF-β1/Smad2信号通路改善COPD大鼠气道重塑的远后效应[D].郑州:河南中医药大学, 2013 LI WW. Regulation of NF κB/TGF-β 1/Smad2 Signaling Pathway by Regulating the Lung and Kidney in Rats with COPD[D]. Zhengzhou:Henan University of Chinese Medicine,2013
[10] 李建生,崔红新,田燕歌,等.调补肺肾三法调节慢性阻塞性肺疾病稳定期大鼠T淋巴细胞亚群的R值综合评价[J].中医杂志, 2015, 56(12):1054 LI JS, CUI HX, TIAN YG, et al. Comprehensive evaluation of R value of regulating T lymphocyte subsets in stable rats with chronic obstructive pulmonary disease by regulating lung and kidney therapy[J]. J Tradit Chin Med,2015, 56(12):1054
[11] 李琳琳.补肺益肾方联合舒肺贴对COPD大鼠免疫球蛋白及T淋巴细胞亚群的影响[D].郑州:河南中医药大学, 2015. LI LL. Effects of Bufei Yishen Recipe Combined with Shufei Paste on Immunoglobulin and T Lymphocyte Subsets in COPD Rats[D]. Zhengzhou:Henan University of Chinese Medicine,2015
[12] 李亚,田燕歌,李琳琳,等.补肺益肾方联合舒肺贴对慢性阻塞性肺疾病大鼠T淋巴细胞亚群的影响[J].辽宁中医杂志,2016, 43(8):1748 LI Y, TIAN YG, LI LL, et al. Effect of Bufei Yishen recipe combined with Shufei paste on T lymphocyte subsets in rats with chronic obstructive pulmonary disease[J]. Liaoning J Tradit Chin Med, 2016, 43(8):1748
[13] 姚凤佳.补肺益肾方调节树突状细胞治疗COPD大鼠的机制研究[D].郑州:河南中医药大学, 2014 YAO FJ. Study on the Mechanism of Bufei Yishen Recipe Regulating Dendritic Cells in Treating COPD Rats[D]. Zhengzhou:Henan University of Chinese Medicine,2014
[14] 田燕歌,李亚,李建生,等.调补肺肾三法对慢性阻塞性肺疾病大鼠肺组织氧化应激的影响和远后效应[J].中华中医药杂志, 2014, 29(2):621 TIAN YG,LI Y, LI JS, et al. Effects of three methods of regulating lung and kidney on oxidative stress and long-term effect of lung tissue in rats with chronic obstructive pulmonary disease[J]. China J Tradit Chin Med Pharm,2014, 29(2):621
[15] LI SY, LI JS, WANG MH, et al. Effects of comprehensive therapy based on traditional Chinese medicine patterns in stable chronic obstructive pμlmonary disease:a four center, open-label, randomized, controlled study[J]. BMC Compl Altern Med, 2012(12):197
[16] 李竹英,郭玮,刘紫君.补肺益肾方对慢性阻塞性肺疾病稳定期患者生活质量的影响[J].中国中医急症, 2017, 26(4):690 LI ZY,GUO W,LIU ZJ. Effect of BFYS on quality of life in patients with chronic obstructive pulmonary disease at stable stage[J]. J Tradit Chin Med,2017, 26(4):690
[17] 王国力,邓虎,徐艳玲.补肺益肾方提高慢性阻塞性肺疾病患者活动耐力的临床研究[J].世界中西医结合杂志,2017,12(1):66 WANG GL, DENG H, XU YL. Clinical study on improving activity endurance of patients with chronic obstructive pulmonary disease by Bu fei Yishen recipe[J]. World J Integr Tradit West Med,2017, 12(1):66
[18] 闫广利,孙晖,张爱华,等.基于中医方证代谢组学的中药质量标志物发现研究[J].中草药,2018, 49(16):3729 YAN GL, SUN H, ZHANG AH, et al. Discovery of quality markers of Chinese materia medica based on chinmedomics[J]. Chin Tradit Herb Drugs,2018, 49(16):3729
[19] WANG X,ZHANG A,ZHOU X,et al. An integrated chinmedomics strategy for discovery of effective constituents from traditional herbal medicine[J]. Sci Rep,2016, 6:18997
[20] 孙大赢,赵文法,李广华,等. LC-MS/MS法同时测定参芍胶囊中人参皂苷和芍药苷的含量[J].药物分析杂志, 2018, 38(1):186 SUN DY, ZHAO WF, LI GH, et al. LC-MS/MS method for simultaneous determination of ginsenoside and paeoniflorin in Shenshao capsules[J]. Chin J Pharm Anal,2018, 38(1):186
[21] 洪宗超,吴和珍,杨艳芳,等.基于液相质谱联用测定复方苦参汤中三种生物碱的含量[J].湖北中医药大学学报, 2018, 20(2):39 HONG ZC, WU HZ, YANG YF, et al. Determination of three alkaloids in compound Sophora flavescens decoction by liquid chromatography mass spectrometry[J]. J Hubei Univ Tradit Chin Med, 2018, 20(2):39
[22] 薛刚强,曹宝帅,高淑丽,等. LC-MS/MS法同时测定回春育子颗粒中9种成分的含量[J].中国药房, 2018, 29(9):1213 XUE GQ, CAO BS, GAO SL, et al. LC-MS/MS were used to simultaneously determine the content of 9 components in Huichun Yuzi granules[J]. China Pharm, 2018, 29(9):1213