动态浊度法定量检测乳糖一水合物中细菌内毒素的含量

目的：应用美洲鲎试剂（Limulus Amebocyte Lysate，LAL）的动态浊度法，定量检测乳糖一水合物中细菌内毒素。方法：依据2015年版《中华人民共和国药典》<1143细菌内毒素检查法>的要求，应用Pyros Kinetix^? Flex（PKF）细菌内毒素定量检测系统，使用葡聚糖抑制缓冲液稀释溶解LAL，建立细菌内毒素的标准曲线并确定稀释倍数，通过测定供试品溶液中外加内毒素的回收率进行干扰试验，并定量测定样品中的细菌内毒素含量。Pyros^? EQS软件自动绘制并出具标准曲线，计算溶液中内毒素的绝对含量。结果：内毒素检测标准曲线浓度线性范围为0.001～0.032 EU·mL^-1（r=-0.985 4），标准曲线回归方程为logT=-0.200logC+2.943；供试品在稀释100、300、500倍时均对试验无干扰作用，细菌内毒素回收率在50%～200%之间；4批样品的内毒素定量测定结果符合规定。结论：在Pyros Kinetix^? Flex（PKF）细菌内毒素定量检测系统平台，使用LAL建立的动态浊度法，可用于乳糖一水合物的细菌内毒素定量检测。LAL有更高的灵敏度，葡聚糖抑制缓冲液可有效阻止由（1→3）-β-D-葡聚糖引起的干扰，提高试验的成功率，符合数据完整性的要求。

Objective: To develope a kinetic turbidimetric method with Limulus Amebocyte Lysate(LAL)to quantify bacterial endotoxin in lactose monohydrate. Methods: According to the requirements in Chapter 1143 Bacterial Endotoxin Test in Chinese Pharmacopoeia 2015 edition,by using Pyros Kinetix^? Flex(PKF)bacterial endotoxin quantitative detection system and LAL dissolved in glucan inhibitory buffer,a standard curve for bacterial endotoxin was established and the dilution factor was determined.The interference test was performed by measuring recovery rate of endotoxin added in the test solution,and bacterial endotoxin in the sample was quantitatively determined.Pyros^? EQS software automatically produced a calibration curve to calculate the absolute content of endotoxin in each solution. Results: Concentration linear ranges of endotoxin detection calibration curve was 0.001-0.032 EU·mL^-1(r=-0.985 4)with recovery rate of 50%-200%,and no interference was observed for test samples diluted 100-500 times.Results of quantitative determination of endotoxin in four batches of samples met the requirements.Conclusion: On the platform of PKF bacterial endotoxin quantitative detection system,the kinetic turbidimetric endotoxin method using LAL can be used for bacterial endotoxin detection of lactose monohydrate.Glucan inhibitory buffer effectively prevented the interference caused by(1→3)-β-D-glucan,improved the test success rate,sensitivity and accuracy of detection.the experiment met data integrity requirements.

[1] 中华人民共和国药典2015年版.四部[S].2015:154 ChP 2015.Vol Ⅳ[S].2015:154
[2] 李薇,刘肃,何华红,等.三磷酸腺苷二钠原料药的细菌内毒素检查法可行性研究[J].中国药房,2010,21(17):1592 LI W,LIU S,HE HH,et al.Study on the practicability of bacterial endotoxins test for adenosine disodium triphosphate raw material[J].China Pharm,2010,21(17):1592
[3] 章亚兵,许雷鸣.注射用福沙匹坦二甲葡胺中细菌内毒素检查法的建立[J].安徽医药,2013,17(8):1298 ZHANG YB,XU LM.Determination of bacterial endotoxin of fosaprepitant dimeglumine for injection[J].Anhui Med Pharm J,2013,17(8):1298
[4] 张广琴,沈剑,黄幸芳,等.动态浊度法定量检测复方氯化钠注射液中细菌内毒素[J].中国药业,2006,15(16):25 ZHANG GQ,SHEN J,HUANG XF,et al.Quantitative detection of bacterial endotoxin in compound sodium chloride injection by dynamic turbidity method[J].China Pharm,2006,15(16):25
[5] 祝清芬,魏霞.动态浊度法测定多西他赛注射液中细菌内毒素的含量[J].中国药房,2011,22(1):54 ZHU QF,WEI X.Content determination of bacterial endotoxin in docetaxel injection by kinetic turbidimetric method[J].China Pharm,2011,22(1):54
[6] 肖鲲,李建华,吴文静,等.动态浊度法检测细菌内毒素试验假阳性结果原因分析[J].中国输血杂志,2012,25(3):274 XIAO K,LI JH,WU WJ,et al.Analysis of the causes of false positive results of bacterial endotoxin test by dynamic turbidimetry[J].Chin J Blood Transfus,2012,25(3):274
[7] 肖贵南.顺铂注射液细菌内毒素的动态浊度法定量研究[J].中国药理学通报,2003,19(4):435 XIAO GN.Quantitative study of bacterial endotoxin in cisplatin injection by dynamic turbidimetry[J].Chin Pharmacol Bull,2003,19(4):435
[8] 苑庆华,芮菁,华晓东,等.动态浊度法定量检测替硝唑注射液中内毒素研究[J].中国药学杂志,2003,38(12):952 YUAN QH,RUI J,HUA XD,et al.Measurement of endotoxin in tinidazole injection using the kinetic-turbidimetric technique[J].Chin Pharm J,2003,38(12):952
[9] 李荣,吴玲.动态浊度法检测乳酸左氧氟沙星氯化钠注射液中的细菌内毒素[J].中国药房,2012,23(9):837 LI R,WU L.Determination of bacterial endotoxin for levofloxacin lactate/sodium chloride injection by kinetic turbidimetric limulus test[J].China Pharm,2012,23(9):837
[10] 张秀萍,赵海山,苑庆华,等.动态浊度法定量检测血塞通注射液中细菌内毒素[J].药物分析杂志,2000,20(4):272 ZHANG XP,ZHAO HS,YUAN QH,et al.Quantitative detection of bacterial endotoxin in Xuesaitong injection by dynamic turbidity method[J].Chin J Pharm Anal,2000,20(4):272
[11] 祝清芬,国明,魏霞,等.米力农注射液动态浊度法细菌内毒素检查方法研究[J].中国新药杂志,2013,22(3):355 ZHU QF,GUO M,WEI X,et al.Detection of bacterial endotoxin in milrinone injection by the method of kinetic turbidimetric limulus test[J].Chin J New Drugs,2013,22(3):355
[12] 李树馀.注射用顺苯磺酸阿曲库铵细菌内毒素检测动态浊度法的研究[J].药物分析杂志,2014,34(2):340 LI SY.Study on bacterial endotoxin test of cisatracurium besilate for injection using the dynamic turbidity method[J].Chin J Pharm Anal,2014,34(2):340
[13] 何进,高军,陈金旺.凝胶法细菌内毒素检查的干扰因素及消除[J].中国药师,2010,13(2):284 HE J,GAO J,CHEN JW.Interference factors and elimination of bacterial endotoxin test by gel method[J].China Pharm,2010,13(2):284