氨甲环酸有关物质及其含量分析方法的研究
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摘要
目的:设计2010年版《中国药典》中氨甲环酸含量测定及有关物质的检查方法,建立反相高效液相色谱法色谱条件。对氨甲环酸中的杂质进行分析,通过精密度试验、线性试验等评价方法的可靠性及耐用性。确定方法之后,检验厂家样品,验证所建立方法的适用性。
方法:在对己有文献分析的基础上,制订氨甲环酸反相高效液相色谱的分析方法,并对方法进行优化,包括对色谱柱、流动相进行筛选,考察柱温对分析的影响,确定检测波长,考察溶剂对测定的影响,从而确定色谱条件。通过对所制定的高效液相色谱法的可行性进行方法学验证,从而确定本方法的可行性与可靠性。
结果:
1、确定色谱条件为:Diamonsil TM C18色谱柱,5μm,4.6mm×250mm;流动相:pH2.5磷酸盐缓冲液(取11.0g无水磷酸二氢钠,溶于500ml水中,加5ml三乙胺与1.4g十二烷基硫酸钠,用稀磷酸调节pH值至2.5,以水稀释至600ml,即得)-甲醇(60:40);检测波长:220nm;流速:1.0ml/min;进样量:20μl;柱温:30℃。
2、在该色谱条件下,氨甲环酸与有关物质可完全分离,空白辅料无干扰,有关物质环烯汀、氨甲苯酸、z-异构体的最低检出限分别为5.2ng、10.4ng和200ng。检测了三批样品的有关物质均符合规定(应≤0.5%)。
3、氨甲环酸注射液的含量测定采用的是高效液相色谱法,对测定方法进行了方法学的验证,测定了湖南洞庭药业股份有限公司提供的共九批氨甲环酸注射液的含量,均符合规定(应为标示量的93.0%～107.0%),线性范围为0.515mg/ml～4.12mg/ml,相关系数r=0.9999,平均回收率分别为99.91%、99.98%、100.01%,RSD=0.05%(n=9)。
结论:本课题建立的方法可以用来测定氨甲环酸的含量,也可用来控制氨甲环酸中z-异构体、氨甲苯酸、环烯烃等杂质的量,该法简便、快速、准确,能满足制剂质量标准的要求。
Objective To design the controlling method of related substance of 2010 edition CH.P in Tranexamic Acid, to establish the RP-HPLC chromatographic condition. To separate the impurities from Tranexamic Acid,and analyze the separated impurities and confirm reliability and robustness of the method by precision test and linearity test . After establishing the method, analyze the sample from manufacturer to validate the applicability of the method.
Method Based on the analysis of reported references, a RP-HPLC analysis method was designed and the RP-HPLC condition was optimized, including chromatographic column and mobile phase. Methodology validation was performed to confirm the feasibility and reliability of the established HPLC method.
Results
1. Diamonsil TM C18 column(5μm, 4.6mm×250mm) was used. The mobile phase was pH2.5 phosphate buffer solution (takes the 11.0g anhydrous sodium dihydrogen phosphate, dissolves in the 500ml water, adds the 5ml triethylamine and 1.4g lauryl sodium sulfate, adjusts the pH value with thin phosphoric acid to 2.5, dilutes by water to 600ml, namely) - methyl alcohol (60:40). The detection wave length was set at 220nm. The flow rate was 1.0ml/min. Specimen handling quantity: 20μl; The column temperature was 30℃.
2. Related substances and tranexamic acid could be separated well. Additives did not interfere with the quantitation of related substances in tranexamic acid injection, The LOQ was 5.2 ng ,10.4 ng , and 200ng.
3. Assaying of tranexamic acid was carried out with high performance liquid chromatography which was validated. and the contents of nine batches samles were all qualified (93.0%～107.0% of signal quantity ). Its linear range was0.515mg/ml～4.12mg/ml , and the average recovery was 99.91%、99.98%、100.01 % , RSD=0.05%(n=9).
Conclusion The established method could not only be used to analyze the content of major component in tranexamic acid, but to control the contents of impurities in the products. This method is simple, rapid and accurate, and can fulfill the requirement of quality control.
方法:在对己有文献分析的基础上,制订氨甲环酸反相高效液相色谱的分析方法,并对方法进行优化,包括对色谱柱、流动相进行筛选,考察柱温对分析的影响,确定检测波长,考察溶剂对测定的影响,从而确定色谱条件。通过对所制定的高效液相色谱法的可行性进行方法学验证,从而确定本方法的可行性与可靠性。
结果:
1、确定色谱条件为:Diamonsil TM C18色谱柱,5μm,4.6mm×250mm;流动相:pH2.5磷酸盐缓冲液(取11.0g无水磷酸二氢钠,溶于500ml水中,加5ml三乙胺与1.4g十二烷基硫酸钠,用稀磷酸调节pH值至2.5,以水稀释至600ml,即得)-甲醇(60:40);检测波长:220nm;流速:1.0ml/min;进样量:20μl;柱温:30℃。
2、在该色谱条件下,氨甲环酸与有关物质可完全分离,空白辅料无干扰,有关物质环烯汀、氨甲苯酸、z-异构体的最低检出限分别为5.2ng、10.4ng和200ng。检测了三批样品的有关物质均符合规定(应≤0.5%)。
3、氨甲环酸注射液的含量测定采用的是高效液相色谱法,对测定方法进行了方法学的验证,测定了湖南洞庭药业股份有限公司提供的共九批氨甲环酸注射液的含量,均符合规定(应为标示量的93.0%～107.0%),线性范围为0.515mg/ml～4.12mg/ml,相关系数r=0.9999,平均回收率分别为99.91%、99.98%、100.01%,RSD=0.05%(n=9)。
结论:本课题建立的方法可以用来测定氨甲环酸的含量,也可用来控制氨甲环酸中z-异构体、氨甲苯酸、环烯烃等杂质的量,该法简便、快速、准确,能满足制剂质量标准的要求。
Objective To design the controlling method of related substance of 2010 edition CH.P in Tranexamic Acid, to establish the RP-HPLC chromatographic condition. To separate the impurities from Tranexamic Acid,and analyze the separated impurities and confirm reliability and robustness of the method by precision test and linearity test . After establishing the method, analyze the sample from manufacturer to validate the applicability of the method.
Method Based on the analysis of reported references, a RP-HPLC analysis method was designed and the RP-HPLC condition was optimized, including chromatographic column and mobile phase. Methodology validation was performed to confirm the feasibility and reliability of the established HPLC method.
Results
1. Diamonsil TM C18 column(5μm, 4.6mm×250mm) was used. The mobile phase was pH2.5 phosphate buffer solution (takes the 11.0g anhydrous sodium dihydrogen phosphate, dissolves in the 500ml water, adds the 5ml triethylamine and 1.4g lauryl sodium sulfate, adjusts the pH value with thin phosphoric acid to 2.5, dilutes by water to 600ml, namely) - methyl alcohol (60:40). The detection wave length was set at 220nm. The flow rate was 1.0ml/min. Specimen handling quantity: 20μl; The column temperature was 30℃.
2. Related substances and tranexamic acid could be separated well. Additives did not interfere with the quantitation of related substances in tranexamic acid injection, The LOQ was 5.2 ng ,10.4 ng , and 200ng.
3. Assaying of tranexamic acid was carried out with high performance liquid chromatography which was validated. and the contents of nine batches samles were all qualified (93.0%～107.0% of signal quantity ). Its linear range was0.515mg/ml～4.12mg/ml , and the average recovery was 99.91%、99.98%、100.01 % , RSD=0.05%(n=9).
Conclusion The established method could not only be used to analyze the content of major component in tranexamic acid, but to control the contents of impurities in the products. This method is simple, rapid and accurate, and can fulfill the requirement of quality control.
引文
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[2]欧洲药典2001版[S].2001:1270.
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[4]英国药典2007版.二部[S].2007:2919～2920.
[5]刘成广.反相高效液相色谱法测定氨甲环酸葡萄糖注射液中氨甲环酸的含量.医药导报,2007,26(l0);1214-1215.
[6]中华人民共和国药典2005版.二部[S].2005:611～612.
[7]范爱玲,李省云,李钠.荷移分光光度法测定氨甲环酸注射液的含量[ J ].太原师范学院学报(自然科学版) , 2002, 1 (1) : 37 - 39.
[8]中华人民共和国药典2000版.二部[S].2005:611～612.
[9] CHANG Q, YIN OQ, CHOW MS. Liquid chromatography-tandem mass spectrometry method for the determination of tranexamic acid in human plasma [J]. Chromatogr B Analyt Technol Biomed Life Sci, 2004, 805(2): 275-280.
[10] VávrováK, Hrabálek A, Dolezal P, Holas T, KlimentováJ. Biodegradable derivatives of tranexamic acid as transdermal permeation enhancers. [J]. J Control Release,2005, 104(1): 41-49.
[11]胡亮.反相高效液相色谱( HPLC )技术及其发展.中国科技信息.2006,(9):298-304
[12]凌大奎.有关物质及其高效液相色谱测定法[J].中国药学杂志,2000,35 (8):567-570.
[13]谢沐风,如何建立高效液相色谱法测定有关物质的方法[J].中国医药工业杂志,2007 38(1):45~48.
[14]王平.药品质量控制与质量标准[J].中国执业药师,2008,5(7):37-39.
[1]新药审批办法[S].国家药监局.1999.
[2] Avallone HL.Drug substance purity[J].Pharm Tech,1992,16(12):48～54.
[3]化学药物杂质研究的技术指导原则[S].国家药监局,指导原则编号:【H】GPH3-1.
[4]窦爱兰,钟化人,郝丽晓.氨苄青霉素钠中有机溶媒残留量的测定与考察[J].药物分析杂志.1996,6:382～384.
[5] YIN Yan,ZHANG Qi et.Enantioseparation of Zolmitriptan by HPLC.Journal of Chinese Pharmaceutical Sciences.2006,15(1):55～58.
[6]朱康玲,康月菊,尹翠英.关于阿莫西林中聚合物杂质的研究[J]..石家庄职业技术学院学报.2006,18(2):9～10.
[7]华丹宇,杨良衍,周英兰.盐酸班布特罗多晶型的鉴别[J].中国医药工业杂志.1999,30(7):315～316.
[8]周红华,高松梅,王尔华等.加替沙星主要相关物质的分离和签定[J].药学学报.2002.37(6):462～464.
[9] Horvath P,Balogh G,Brlik J,et al.Estimation of impurity profiles of drugs and related materials part 16:identification of side products of the ethinylation step in the synthesis of contraceptive gestogens.J Pharma Biomed A nal,1997 ,15:1343～1349.
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