一测多评法测定六神丸中的6个蟾蜍二烯内酯类化合物

目的：建立同时测定六神丸中6个蟾蜍二烯内酯类成分含量的一测多评法（QAMS），并验证其准确性和可行性。方法：采用高效液相色谱法，使用Waters Xbridge C₁₈（250 mm×4.6 mm，5 μm）色谱柱，以乙腈-0.2%磷酸水溶液为流动相，梯度洗脱，流速1 mL·min^-1，柱温30℃，检测波长296 nm。以华蟾酥毒基为内参物，建立其与远华蟾毒精、蟾毒它灵、华蟾毒它灵、蟾毒灵、脂蟾毒配基5个成分的相对校正因子（RCF），并进行含量计算，实现一测多评；同时采用外标法（ESM）测定六神丸中蟾酥的6个成分的含量，比较一测多评法计算值与外标法实测值的差异。结果：在一定线性范围内，华蟾酥毒基与远华蟾毒精、蟾毒它灵、华蟾毒它灵、蟾毒灵、脂蟾毒配基的RCF值分别为0.897、0.965、1.023、0.844、0.964，且在不同实验条件下重现性良好（RSD<5.0%），一测多评法测得15批样品中华蟾酥毒基、远华蟾毒精、蟾毒它灵、华蟾毒它灵、蟾毒灵、脂蟾毒配基的含量范围分别为2.26~9.27 mg·g^-1、0.95~2.06 mg·g^-1、1.12~2.50 mg·g^-1、1.83~3.48 mg·g^-1、1.10~2.81 mg·g^-1、0.90~3.65 mg·g^-1，与外标法实测值无显著性差异。结论：所建立的同时测定六神丸中的6个蟾蜍二烯内酯类化合物的一测多评法准确可靠，可用于六神丸中多个成分的定量分析质量评价。

Objective: To determine six bufadienolides in Liushen pills simultaneously with a quantitative analysis of multi-components by single-marker (QAMS) and accuracy and validate feasibility of the method.Methods: A Waters Xbridge C₁₈ (250 mm×4.6 mm, 5 μm) column was used in the high performance liquid chromatography (HPLC) assay.Acetonitrile-0.2% phosphoric acid aqueous solution with gradient elution was employed as the mobile phase.The flow rate was 1 mL·min^-1, the column temperature was maintained at 30℃ and the detection wavelength was 296 nm.Cinobufagin was used as the internal reference substance to calculate the relative correction factors (RCFs) of elocinobufagin, bufotalin, cinobufotalin, bufalin and resibufogenin and evaluate their contents accordingly.Moreover, the external standard method (ESM) was used to quantify the six components in Liushen pills.The results of the two methods were compared.Results: In certain linear range, RCFs of telocinobufagin, bufotalin, cinobufotalin, bufalin and resibufogenin with reference to cinobufagin were 0.897, 0.965, 1.023, 0.844 and 0.964 respectively.The results were repeatable under different conditions with RSDs less than 5%.The content ranges of cinobufagin, telocinobufagin, bufotalin, cinobufotalin, bufalin and resibufogenin by the QAMS method were 2.26-9.27 mg·g^-1, 0.95~2.06 mg·g^-1, 1.12~2.50 mg·g^-1, 1.83~3.48 mg·g^-1, 1.10~2.81 mg·g^-1, 0.90~3.65 mg·g^-1.Significant difference was not found between the the multi-evaluation method and the external standard method.Conclusion: The established QAMS can quantify six bufadienolides in Liushen pills accurately and reliably.This developed method can be used to determine multiple components in Liushen pills.

[1] 王欣,覃瑶,王德江,等.一测多评法在中药质量控制中的应用进展[J].中成药,2016,38(2):395 WANG X,QIN Y,WANG DJ,et al.Progress in the application of QAMS in quality control of traditional Chinese medicine[J].Chin Tradit Pat Med,2016,38(2):395
[2] 范成杰."一测多评"法在中药质量评价和控制中的应用概况[J].中药与临床,2013,4(2):18 FAN CJ.Application situation of multi-components quantitation by one marker new method for quality evaluation and control of Chinese herbal medicine[J].Pharm Clin Chin Mater Med,2013,4(2):18
[3] 李听弦,李蒙,张志,等.一测多评法测定不同味连饮片中6个生物碱类成分的含量[J].药物分析杂志,2018,38(7):1132 LI TX,LI M,ZHANG Z,et al.Simultaneous determination of 6 alkaloids in prepared slides of Coptis chinensis Franch.by QAMS[J].Chin J Pharm Anal,2018,38(7):1132
[4] 成林,谢巍,朱斌,等.一测多评法测定白背叶中3个黄酮碳苷成分的含量[J].药物分析杂志,2018,38(7):1152 CHENG L,XIE W,ZHU B,et al.Determination of three flavone C-glycosides in the leaves of Mallotus apelta by quantitative analysis of multi-components by single marker[J].Chin J Pharm Anal,2018,38(7):1152
[5] 翟宏宇,单柏宇,王海洋,等.一测多评法测定保心宁片中6个丹参类指标性成分的含量[J].药物分析杂志,2018,38(6):973 ZHAI HY,SHAN BY,WANG HY,et al.Simultaneous determination of six index components in Baoxinning tablets by a single marker[J].Chin J Pharm Anal,2018,38(6):973
[6] 王晓燕,霍甜甜,李振国.一测多评法同时测定杞菊地黄口服液中4种有效成分的含量[J].药物分析杂志,2017,37(2):290 WANG XY,HUO TT,LI ZG.Simultaneous determination of 4 active components in Qiju Dihuang oral liquid by QAMS method[J].Chin J Pharm Anal,2017,37(2):290
[7] 李思阳,孔庆新.一测多评法同时测定复生康胶囊中8个成分的含量[J].药物分析杂志,2019,39(3):518 LI SY,KONG QX.Simultaneous determination of eight components in Fushengkang capsule by QAMS[J].Chin J Pharm Anal,2019,39(3):518
[8] 裴迎霞,郑琦,侯炜,等.六神丸抗肿瘤作用的研究进展[J].中医药导报,2018,24(8):40 PEI YX,ZHENG Q,HOU W,et al.Progress in anti-tumor effects of Liushen pills[J].Guide J Tradit Chin Med Pharm,2018,24(8):40
[9] 中华人民共和国药典2015年版.一部[S].2015:383 ChP 2015.Vol Ⅰ[S].2015:383
[10] 寇冠军,秦姿凡,邓雅芳,等.蟾酥的研究进展[J].中草药,2014,45(21):3185 KOU GJ,QIN ZF,DENG YF,et al.Research progress on Bufonis Venenum[J].Chin Tradit Herb Drugs,2014,45(21):3185
[11] 陈瀛澜,郝艳艳,郭夫江,等.蟾酥化学成分及药理活性研究进展[J].中草药,2017,48(12):2579 CHEN YL,HAO YY,GUO FJ,et al.Research progress on chemical constituents and pharmacological activities of Bufonis Venenum[J].Chin Tradit Herb Drugs,2017,48(12):2579
[12] SHEN S,ZHANG Y,WANG Z,et al.Bufalin induces the interplay between apoptosis and autophagy in glioma cells through endoplasmic reticulum stress[J].Int J Biol Sci,2014,10(2):212
[13] 孙崇峰,范圣此,罗毅,等.蟾酥化学成分及其人工合成的研究进展[J].中草药,2018,49(13):3183 SUN CF,FAN SC,LUO Y,et al.Research progress on chemical constituents and artificial synthesis of Bufonis Venenum[J].Chin Tradit Herb Drugs,2018,49(13):3183
[14] 王智民,钱忠直,张启伟,等.一测多评法建立的技术指南[J].中国中药杂志,2011,36(6):657 WANG ZM,QIAN ZZ,ZHANG QW,et al.Technical manual of QAMS[J].China J Chin Mater Med,2011,36(6):657
[15] 李娟,狄留庆,李俊松,等.HPLC-MS法同时测定六神丸中9种蟾蜍二烯内酯类化合物[J].中草药,2017,48(4):700 LI J,DI LQ,LI JS,et al.Simultaneous determination of nine bufadienolides in Liushen pills by HPLC-MS[J].Chin Tradit Herb Drugs,2017,48(4):700