柱前衍生化RP-HPLC法对黄杨宁片原料中环维黄杨星D和杂质的定性定量研究

目的：建立黄杨宁片原料中环维黄杨星D准确定量和全面反映杂质色谱信息的分析方法。方法：选择可使主成分和所有杂质衍生化反应的试剂进行柱前衍生化反应，采用RP-HPLC法对衍生产物进行分析，C₈柱分离，乙腈-0.01%甲酸水溶液（71∶29）洗脱，流速1 mL·min^-1，检测波长230 nm，柱温25℃。结果：新建立的方法能使环维黄杨星D与有关生物碱的最低分离度达1.5，检测到的12个杂质峰，其中9个可被定量。环维黄杨星D检测限为0.35 ng（RSD=2.2%，n=5），定量限为1.37ng（RSD=4.6%，n=5）；平均回收率为102.4%，RSD为1.4%（n=6）。来自4个厂家的10批样品中环维黄杨星D含量范围为78.28%~85.48%；9个杂质总含量范围为10.14%~15.57%。结论：该法既可使主成分与杂质完全分离，又可全面反映杂质色谱信息，能同时用于准确测定原料中主成分含量和杂质检查。

Objective: To develop an analytical method for accurate determination of cyclovirobuxine D and impurities profile of the API of Huangyangning tablets.Methods: The suitable reagent was selected for precolumn derivatization of main component and its related alkaloids. RP-HPLC method was adopted to analyze the derivatives. C₈ column was adopted for separation and acetonitrile-0.01% formic acid solution(71:29)was used as the mobile phase at the flow rate of 1.0 mL·min^-1. The detection wavelength was set at 230 nm and the column temperature was 25℃.Results: The new method could separate cyclovirobuxine D from its related alkaloids with resolutions higher than 1.5.12 impurity peaks were detected and 9 were determined. LOD and LOQ of cyclovirobuxine D were 0.35 ng(RSD=2.2%, n=5)and 1.36 ng(RSD=4.1%, n=5)respectively, and the average recovery was 102.41%(RSD=1.4%, n=6). The contents of cyclovirobuxine D in ten samples from four manufacturers were 78.28%-85.48%, and the total contents of 9 impurities were 10.14%-15.57%. Conclusion: The method could separate cyclovirobuxine D from its impurities and reveal comprehensive chromatographic information of the related alkaloids, thus offered an accurate way for assay and impurity examination of the API of Huangyangning tablets.

[1] 胡国强,许启泰,张保国,等.环维黄杨星D分离方法改进[J]. 中国天然药物, 2004, 2(3):155 HU GQ,XU QT,ZHANG BG,et al. An improved separation method of cyclovirobuxine D from the total alkaloids of Buxus[J]. Chin J Nat Med, 2004, 2(3):155
[2] 梁秉文,邓晨安,王学斌,等.黄杨木生物碱Ⅰ、Ⅱ、Ⅲ、Ⅳ的分离与结构研究[J].药学通报,1981,16(4):3 LIANG BW, DENG CA, WANG XB, et al. Isolation and structural elucidationofBuxus alkaloids Ⅰ,Ⅱ,Ⅲ,Ⅳ[J]. Chin Pharm Bull, 1981,16(4):3
[3] 于民权,许立,方泰惠,等.环维黄杨星D的药理及毒理研究概况[J].安徽医药, 2008,12(10):885 YU MQ,XU L,FANG TH, et al. Pharmacology and toxicology over view of cyclovirobuxine D[J]. Anhui Med Pharm J,2008,12(10):885
[4] 周玖瑶.环维黄杨星D药理研究概况[J].广州中医药大学学报, 2005, 22(1):70 ZHOU JY. Pharmacologia over view of cyclovirobuxine D[J]. J Guangzhou UnivTradit Chin Med,2005, 22(1):70
[5] 吴捷,毛幼桦.黄杨宁心血管药理作用[J].心脏杂志, 2003, 15(2):169 WU J, MAO YH. Cardiovascular pharmacological effects of Huangyangning[J]. Chin Heart J, 2003, 15(2):169
[6] HU D, LIU XY, WANG YY, et al. Cyclovirobuxine D amelioates acute myocardial ischemia by K(ATP)channel opening, nitric oxide release and anti-thrombosis[J]. Eur J Pharmacol, 2007, 569(1-2):103
[7] YU B, FANG TH, LV GH, et al. Beneficial effect of cyclovirobuxine D on heart failure rats following myocardial infarction[J]. Fitoterapia, 2011, 82(6):868
[8] NERI M, CERRETANI D, FIASCHI AI, et al. Correlation between cardiac oxidative stress and myocardial pathology due to acute and chronic norepinephrine administration in rats[J]. J Cell Mol Med, 2007,11(1):156
[9] 刘洁,杭太俊,张正行. HPLC法测定黄杨宁中环维黄杨星D和有关生物碱检查[J].药物分析杂志,2006, 26(4):446 LIU J, HANG TJ, ZHANG ZX. HPLC determination of cyclovirobuxine D and its related alkaloids in Huangyangning[J]. Chin J Pharm Anal, 2006, 26(4):446
[10] 梁秉文,张正行,方泰惠,等.黄杨碱与心脑血管疾病[M].北京:化学工业出版社, 2011, 118 LIANG BW, ZHANG ZX, FANG TH, et al. Buxus Alkaloids and Cardiovascular Disease[M]. Beijing:Chemical Industry Press, 2011,118
[11] 邱明华,聂瑞麟.黄杨生物碱及其植物资源[J].天然产物研究与开发. 1992, 4(4):41 QIU MH, NIE RL. Buxus alkaloids and its plant resources[J]. Nat Product Res Dev. 19924(4):41
[12] 中国药典2000年版.一部[S]. 2000:153 ChP 2000. Vol Ⅰ[S]. 2000:153
[13] 中国药典2005年版.一部[S]. 2005:276 ChP2005. Vol Ⅰ[S]. 2005:276
[14] 中国药典2010年版.一部[S]. 2010:575 ChP2010. Vol Ⅰ[S]. 2010:575
[15] 中国药典2015年版.一部[S]. 2015:406 ChP2015. Vol Ⅰ[S]. 2015:406
[16] 谢昀,于治国,徐新盛,等.反相离子对色谱法测定环维黄杨星D含量[J].药物分析杂志,2006, 26(7):888 XIE Y, YU ZG, XU XS, et al. Determination of cyclovirobuxine D by ion pair RP-HPLC[J]. Chin J Pharm Anal, 2006, 26(7):888
[17] 郭青,吕霞,孙姗,等.黄杨宁原料及片中环维黄杨星D含量及含量均匀度测定[J].药物分析杂志,2009, 29(10):1651 GUO Q, LÜX, SUN S, et al. Determination and uniformity examination of cyclovirobuxine D in Huangyangning materials and its tablets[J]. Chin J Pharm Anal, 2009, 29(10):1651
[18] 陈勇,樊雪君,龚青. HPLC梯度洗脱法测定黄杨宁片中环维黄杨星D的含量[J].中国中药杂志,2006, 31(14):1155 CHEN Y, FAN XJ, GONG Q. Determination of cyclovirobuxine-D in Huangyangning tablets by RP-HPLC with gradient elution[J]. China J Chin Mater Med, 2006, 31(14):1155
[19] 吕霞,胡明通,杨春,等. HPLC-ELSD法测定黄杨宁片中环维黄杨星D的含量[J].现代中药研究与实践, 2014, 28(3):52 LÜ X, HU MT, YANG C, et al. Determination of cyclovirobuxine D in Huangyangningtablets by HPLC-ELSD[J]. Chin Med J Res Pract, 2014, 28(3):52
[20] 郭青,吕霞,伍乃英,等.黄杨宁片及原料中环维黄杨星D含量测定的3种HPLC方法评价[J].中国现代应用药学杂志,2009, 26(11):929 GUO Q, LÜ X, WU NY, et al. Evaluation on three HPLC methods on the determination of cyclovirobuxine D in Huangyangning tablets and raw materials[J]. Chin J Mod Appl Pharm,2009,26(11):929
[21] 徐新军,张正行,盛龙生,等.柱前荧光衍生化RP-HPLC测定环维黄杨D含量[J].药学学报, 2002,37(5):359 XU XJ, ZHANG ZX, SHENG LS, et al. Determination of cyclovirobuxine D by RP-HPLC with precolumn fluorescenced dervatization[J]. Acta Pharm Sin, 2002,37(5):359
[22] 黄勤安,黄海燕,鲁静,等.高效液相色谱法测定黄杨宁片中环维黄杨星D的含量和含量均匀度[J].药物分析杂志,2007,27(2):264 HUANG QA, HUANG HY, LU J, et al. HPLC determination of content and uniformity of cyclovirobuxine D in Huangyangning tablets[J]. Chin J Pharm Anal, 2007,27(2):264
[23] 李欣,沈敏雅,杨倩,等.对甲基苯磺酰氯柱前衍生法测定水中哌嗪[J].分析试验室, 2012, 31(12):48 LI X, SHEN MY, YANG Q, et al. Determination of piperazine in water using pre-column derivatization with p-tosyl chloride[J]. Chin J Anal Lab, 2012, 31(12):48
[24] DING L, HU JJ, JIANG M, et al. Sensitive HPLC-APCI-MS method for the determination of cyclovirobuxine D in human plasma[J]. J Chromatogr B, 2006, 843(1):78
[25] YU P, ZOU LM, LIU WY, et al. High performance liquid chromatography-tandem mass spectrometric determination of cyclovirobuxine D in human plasma[J]. J Pharm Biomed Anal, 2006, 42(4):488
[26] MU LL, YU P, HE LH. Determination of cyclovirobuxine D in human plasma by liquid chromatography tandem mass spectrometry and application in a pharmacokinetic study[J]. Acta Pharm Sin B, 2011, 1(3):184