动态显色法检测4种清热解毒类中药注射液中细菌内毒素含量
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摘要
目的:建立定量检测4种清热解毒类中药注射液中细菌内毒素的试验方法。方法:采用动态显色法,测定温度为(37±0.5)℃,检测波长为405 nm,预设吸光度(A)值为0.1,反应时间为3 600 s。依据2015年版《中国药典》(四部)通则细菌内毒素检查法中的光度测定法及其指导原则,进行标准曲线可靠性试验和干扰试验,确定4个品种(清开灵注射液、复方半边莲注射液、茵栀黄注射液、双黄连注射液)的最大不干扰浓度并进行细菌内毒素定量检测。结果:可靠性试验中标准曲线的相关系数r为0.999;清开灵注射液、复方半边莲注射液、茵栀黄注射液、双黄连注射液分别在原液200、125、75、12.5倍及以下稀释倍数时对鲎试剂检测无干扰作用;干扰试验中回收率均在50%~200%;样品中细菌内毒素含量分别小于1.66、1.04、0.62、0.104 EU/m L,均低于各样品规定的限值。结论:采用动态显色法可检测这4种中药注射液的细菌内毒素,且方法简单快捷,结果稳定可靠。
OBJECTIVE:To set up the method for quantitative determination of bacterial endotoxin in 4 kinds of heat-clearing and detoxifying TCM injections. METHODS: The kinetic chromogenic assay(KCA) method was used. The determination conditions included detection temperature(37 ± 0.5)℃,detection wavelength 405 nm,preset absorbance(A) 0.1,and reaction time 3 600 s. According to photometry method and guidance principle general rule of bacterial endotoxin test in 2015 edition of Chinese Pharmacopoeia(part Ⅳ),reliability test and interference test of standard curve were conducted. The non-interference concentration of 4 type(Qingkailing injection, Compound Banbianlian injection,Yinzhihuang injection and Shuanghuanglian injection) was determined and quantitative determination of bacterial endotoxin was also conducted. RESULTS:The correlation coefficient r of standard curve was 0.999. Qingkailing injection,Compound Banbianlian injection,Yinzhihuang injection and Shuanghuanglian injection didn't interfere with the detection of tachypleus amebocyte lysate in 200,125,75 and 12.5 times and below dilution times respectively. The recoveries were 50%-200%,and the contents of bacterial endotoxin in samples were 1.66,1.04,0.62,0.104 EU/m L,which were all lower than the limits of samples. CONCLUSIONS:KCA is suitable for the detection of bacterial endotoxin in 4 kinds of TCM injections. The method is simple,convenient,stable and reliable.
OBJECTIVE:To set up the method for quantitative determination of bacterial endotoxin in 4 kinds of heat-clearing and detoxifying TCM injections. METHODS: The kinetic chromogenic assay(KCA) method was used. The determination conditions included detection temperature(37 ± 0.5)℃,detection wavelength 405 nm,preset absorbance(A) 0.1,and reaction time 3 600 s. According to photometry method and guidance principle general rule of bacterial endotoxin test in 2015 edition of Chinese Pharmacopoeia(part Ⅳ),reliability test and interference test of standard curve were conducted. The non-interference concentration of 4 type(Qingkailing injection, Compound Banbianlian injection,Yinzhihuang injection and Shuanghuanglian injection) was determined and quantitative determination of bacterial endotoxin was also conducted. RESULTS:The correlation coefficient r of standard curve was 0.999. Qingkailing injection,Compound Banbianlian injection,Yinzhihuang injection and Shuanghuanglian injection didn't interfere with the detection of tachypleus amebocyte lysate in 200,125,75 and 12.5 times and below dilution times respectively. The recoveries were 50%-200%,and the contents of bacterial endotoxin in samples were 1.66,1.04,0.62,0.104 EU/m L,which were all lower than the limits of samples. CONCLUSIONS:KCA is suitable for the detection of bacterial endotoxin in 4 kinds of TCM injections. The method is simple,convenient,stable and reliable.
引文
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[12]党京丹.细菌内毒素的研究及临床应用[J].山西医药杂志,2014,43(7):771-773.
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[21]曹春雨,刘婷.显色基质法检测6种中药注射液内毒素含量[J].中国实验方剂学杂志,2010,16(9):88-91.
[2]吴剑波,何志民.清开灵注射液联合糖皮质激素对大叶性肺炎患者血清IL-10及TNF-α水平的影响[J].中国生化药物杂志,2016,36(4):152-154.
[3]杨聪琴.复方半边莲治疗小儿急性支气管炎疗效观察[J].实用中医药杂志,2005,21(3):136-137.
[4]陈军,陈辉扬,刘志承,等.茵栀黄注射液对黄疸型病毒性肝炎的退黄效果与安全性的系统评价[J].中国循证医学杂志,2005,5(6):461-465.
[5]赵振寰,荆伟丽,姜艳,等.双黄连注射液治疗慢性阻塞性肺疾病急性加重期患者的临床观察[J].中国药房,2016,27(29):4096-4098.
[6]徐媛媛,熊俊,张俊华,等.双黄连注射剂与抗生素比较治疗小儿肺炎疗效的系统评价[J].时珍国医国药,2011,22(7):1790-1793.
[7]桂曼青.双黄连注射剂与抗生素比较治疗小儿肺炎疗效的系统评价[J].航空航天医学杂志,2014,25(5):692-693.
[8]张海英,任晓蕾,李玉珍.双黄连注射液治疗小儿肺炎有效性的Meta分析[J].中国药房,2010,21(44):4205-4207.
[9]常学文,赵桂清.茵栀黄注射液致不良反应23例临床分析[J].中国社区医师,2008,10(10):79-80.
[10]陈文贵.浅谈中药及其制剂的过敏反应[J].中国药房,1995,6(5):47-48.
[11]刘文生.静脉用中药注射剂集中配置稳定性探讨[J].中国药房,2008,19(24):1919-1920.
[12]党京丹.细菌内毒素的研究及临床应用[J].山西医药杂志,2014,43(7):771-773.
[13]孙哲.细菌内毒素检验[J].世界最新医学信息文摘,2012,12(9):128-130.
[14]HIRATA N,YANAQAWA Y,SATOH M,et al.Dendritic cell-derived TNF-alpha is responsible for development of IL-10-producing CD4+T cell[J].Cell Immunol,2010,261(1):37-41.
[15]NEWBY AC.Metalloproteinase expression in monocytes and macrophages and its relationship to atheroselerotic plaque instability[J].Arterioscler Thromb Vasc Biol,2008,28(12):2108-2114.
[16]邵英光,李京华,魏桂林,等.内毒素的去除策略[J].广州化学,2003,28(2):38-40.
[17]国家药典委员会.中华人民共和国药典:四部[S].2015年版.北京:中国医药科技出版社,2015:156-157、401.
[18]黄继全.大青叶抗内毒素的实验研究[J].江西中医学院学报,2007,19(2):70-71.
[19]谢恬,魏杭英.大黄对家兔内毒素性发热及血浆c AMP和c GMP含量的影响[J].上海中医药杂志,1991(2):46-48.
[20]郝然,刘婷,易艳,等.动态浊度法检测5种中药注射液的内毒素含量[J].中国实验方剂学杂志,2011,17(24):120-123.
[21]曹春雨,刘婷.显色基质法检测6种中药注射液内毒素含量[J].中国实验方剂学杂志,2010,16(9):88-91.