使用LC-MS/MS方法进行生物样本分析时对溶血样本的处理策略
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摘要
在进行生物样本分析时,临床生物样本发生溶血是较常见现象。临床血浆/血清样本在发生溶血后会不同程度地改变其物理、化学性质,使得其基质与正常血浆/血清样本有所不同,对溶血样本中的目标化合物进行测定时,可能会造成浓度测定的不准确。一般而言,溶血的发生会影响待测物的色谱分离,溶血基质中可能会有一些特有的、与待测物质的共流出的内源性干扰;也可能影响待测物在质谱检测中的基质效应,甚至红细胞中释放出的酶可能影响待测物的稳定性。评价溶血基质对于生物分析的影响具有重要意义,虽然关于溶血基质效应的考察已在我国的生物分析实验室中引起了重视,但关于溶血基质效应的研究尚未达成共识。本文从生物分析行业研究的现状调查入手,探讨溶血对生物分析结果的影响以及溶血基质效应考察的规定等,结合多方面的考虑,给出了使用液相色谱-质谱联用方法进行生物样本分析时对溶血样本处理可行性的策略。
Hemolysis,which results in additional endogenous components being released from the lysed red blood cells(RBCs),occurs frequently during the bioanalysis of clinical samples. The physical and chemical propertie of a hemolyzed clinical sample would change,which might cause additional matrix interferences. The effects of the degree of hemolysis on the method accuracy and precision should be evaluated when hemolyzed study samples were analyzed. Generally,the impact of hemolysis on bioanalysis includes:1)Chromatography Separation—additional endogenous interferences might co-elute with the target analyte in hemolyzed samples;2)Matrix Effects—the endogenous components in hemolyzed samples may cause ion suppression or enhancement in LC-MS/MS analysis. 3)Analytes Stability—analytes might not be stable in hemolyzed plasma/serum due to the enzymes released from RBCs. Therefore,it is important to evaluate the impact of hemolysis on sample analysis in LC-MS. Although the investigations of hemolysis matrix effect have drawn increasing attention in recent years,especially in cases where the sample concentrations are critical for pharmacokinetic calculation,there is no established procedure to objectively assess the risk of reporting potentially inaccurate bioanalytical results from hemolyzed samples. In this article,we investigated the proportion of hemolyzed samples in clinical samples,discussed the effects of hemolysis on determination of the concentrations of analytes. In addition,we proposed the strategy for dealing with the hemolyzed sample during LC-MS/MS bioanalysis.
Hemolysis,which results in additional endogenous components being released from the lysed red blood cells(RBCs),occurs frequently during the bioanalysis of clinical samples. The physical and chemical propertie of a hemolyzed clinical sample would change,which might cause additional matrix interferences. The effects of the degree of hemolysis on the method accuracy and precision should be evaluated when hemolyzed study samples were analyzed. Generally,the impact of hemolysis on bioanalysis includes:1)Chromatography Separation—additional endogenous interferences might co-elute with the target analyte in hemolyzed samples;2)Matrix Effects—the endogenous components in hemolyzed samples may cause ion suppression or enhancement in LC-MS/MS analysis. 3)Analytes Stability—analytes might not be stable in hemolyzed plasma/serum due to the enzymes released from RBCs. Therefore,it is important to evaluate the impact of hemolysis on sample analysis in LC-MS. Although the investigations of hemolysis matrix effect have drawn increasing attention in recent years,especially in cases where the sample concentrations are critical for pharmacokinetic calculation,there is no established procedure to objectively assess the risk of reporting potentially inaccurate bioanalytical results from hemolyzed samples. In this article,we investigated the proportion of hemolyzed samples in clinical samples,discussed the effects of hemolysis on determination of the concentrations of analytes. In addition,we proposed the strategy for dealing with the hemolyzed sample during LC-MS/MS bioanalysis.
引文
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[8]GAROFOLO F,ROCCI ML,DUMONT I,et al.2011 White Paper on recent issues in bioanalysis and regulatory findings from audits and inspections[J].Bioanalysis,2011,3(18):2081
[9]DESILVA B,GAROFOLO F,ROCCI M,et al.2012 White Paper on recent issues in bioanalysis and alignment of multiple guidelines[J].Bioanalysis,2012,4(18):2213
[10]AMARAVADI L,SONG A,MYLER H,et al.2015 White Paper on recent issues in bioanalysis:focus on new technologies and biomarkers(Part 3-LBA,biomarkers and immunogenicity)[J].Bioanalysis,2015,7(24):3107
[11]TANG D,THOMAS E.Strategies for dealing with hemolyzed samples in regulated LC-MS/MS bioanalysis[J].Bioanalysis,2012,4(22):2715
[12]LIU G,JI QC,ARNOLD ME,et al.Identifying,evaluating,and controlling bioanalytical risks resulting from nonuniform matrix ion suppression/enhancement and nonlinear liquid chromatography-mass spectrometry assay response[J].Anal Chem,2010,82(82):9671
[13]龙跃兵,朱伟斌,章美燕.溶血对生化检验结果准确性的影响及校正方法的探讨[J].广东医学,2011,32(12):1562LONG YB,ZHU WB,ZHANG MY.Investigation of influence by sample hemolysis on biochemical detection outcomes and its calibration methods[J].Guangdong Med J,2011,32(12):1562
[14]BéRUBéER,TAILLON MP,FURTADO M,et al.Impact of sample hemolysis on drug stability in regulated bioanalysis[J].Bioanalysis,2011,3(18):2097
[15]BéRUBéER,LACASSE MC,FURTADO M,et al.Severe impact of hemolysis on stability of phenolic compounds[J].Bioanalysis,2013,5(12):1491
[16]European Medicines Agency(EMA).Guideline of Bioanalytical Method Validation[S/OL].(2012)[2012-02-01].http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf
[17]Food and Drug Administration(FDA).Guidance for Industry:BioanalyticalMethodValidation[S/OL].(2013)[2013-09].Availableat:http://www.fda.gov/downloads/Drugs/Guidance Compliance Regulatory Information/Guidances/UCM368107.pdf
[18]INGELSE B,BARROSO B,GRAY N,et al.European bioanalysis forum:recommendationondealingwithhemolyzedand hyperlipidemic matrices[J].Bioanalysis,2014,6(23):3113
[19]HUGHES NC,BAJAJ N,FAN J,et al.Assessing the matrix effects of hemolyzed samples in bioanalysis[J].Bioanalysis,2009,1(6):1057
[20]FUNG EN,AUBRY AF,ALLENTOFF A,et al.Utilizing internal standard responses to assess risks on reporting bioanalytical results from hemolyzed samples[J].AAPS J,2015,17(5):1168
[21]CHAMBERS E,WAGROWSKIDIEHL DM,LU Z,et al.Systematic and comprehensive strategy for reducing matrix effects on LC/MS/MS analysis[J].J Chromatogr B Anal Technol Biomed Life Sci,2007,852(1-2):22
[22]刘小娟,杜晓琳,黄娟,等.生物分析中溶血对检测的影响及其对策[J].中国新药与临床杂志,2017,36(4):181LIU XJ,DU XL,HUANG J,et al.Impact of hemolysis on detection in bioanalysis and its countermeasures[J].Chin J New Drugs Clin Rem,2017,36(4):181
[23]杨永怀.标本溶血对生化检验结果的影响及处理对策探析[J].中国现代药物应用,2017,11(13):42YANG YH.Investigation of influence by sample hemolysis on biochemical detection outcomes and its treatment countermeasures[J].Chin J Mod Drug Appl,2017,11(13):42.
[24]刘婧,冯立民,龚人杰.标本溶血的患者因素分析及对策探讨[J].实验与检验医学,2017,35(1):117LIU J,FENG LM,GONG RJ.Investigation of patients factors of hemolysis and its countermeasures[J].Exp Mol Lab Med,2017,35(1):117
[25]钟大放,李高,刘昌孝.生物样品定量分析方法指导原则:草案[J].药物评价研究,2011,34(6):409ZHONG DF,LI G,LIU CX,et al.Guidance on bioanalysis:method validation and analysis of study samples(draft)[J].Drug Eval Res,2011,34(6):409
[26]冯晓杰,杜丽英,冯章英,等.LC-MS/MS法测定人血药浓度的基质效应研究进展[J].中国新药杂志,2015,24(13):1488FENG XJ,DU LY,FENG ZY,et al.Research progress of matrix effect on determining blood concentration by LC-MS/MS[J].Chin J New Drugs,2015,24(13):1488
[27]姚梦侃,马秉亮,马越鸣.生物样品液质联用分析中的基质效应研究概况[J].药物分析杂志,2010,30(12):2436YAO MK,MA BL,MA YM.Overview of the matrix effect on quantitative liquid chrimatography/mass spectrometry analyses of biological samples[J].Chin J Pharm Anal,2010,30(12):2436
[28]张人杰,柴逸峰,朱臻宇,等.LC-MS/MS生物分析中试验样品再分析失败后的调查和解决方案[J].药物分析杂志,2016,36(4):559ZHANG RJ,CHAI YF,ZHU ZY,et al.Investigation and resolution of incurred sample reanalysis failures in LC-MS/MS bioanalysis[J].Chin J Pharm Anal,2016,36(4):559
[2]LIPPI G,PLEBANI M,SOMMA SD,et al.Hemolyzed specimens:a major challenge for emergency departments and clinical laboratories[J].Crit Rev Clin Lab Sci,2011,48(3):143
[3]陈姚,马继红,曾蓉君.真空静脉采血标本溶血原因分析及对策[J].护理管理杂志,2010,10(9):677CHEN Y,MA JH,ZENG RJ.Cause analysis and countermeasures of hemolysis of vacuum venous blood specimens[J].J Nurs Adm,2010,10(9):677
[4]刘海燕.溶血现象对临床生化检验项目影响的观察及预防对策[J].当代医学,2011,17(27):33LIU HY.Strategies for observation and precaution of influence of sample hemolysis on biochemical detection outcomes and its treatment countermeasures[J].Contemp Med,2011,17(27):33
[5]张勇军,郭越文.标本溶血对常规生化检验结果的影响[J].检验医学与临床,2013,10(10):1239ZHANG YJ,GUO YW.Influence of hemolysis on biochemical detection[J].Lab Med Clin,2013,10(10):1239
[6]中华人民共和国药典2015年版.四部[S].2015:363《9012生物样品定量分析方法验证指导原则》ChP 2015.VolⅣ[S].2015:363《Guidance on Bioanalytical Method Validation》
[7]SAVOIE N,GAROFOLO F,Van AMSTERDAM P,et al.2010white paper on recent issues in regulated bioanalysis&global harmonization of bioanalytical guidance[J].Bioanalysis,2010,2(12):1945
[8]GAROFOLO F,ROCCI ML,DUMONT I,et al.2011 White Paper on recent issues in bioanalysis and regulatory findings from audits and inspections[J].Bioanalysis,2011,3(18):2081
[9]DESILVA B,GAROFOLO F,ROCCI M,et al.2012 White Paper on recent issues in bioanalysis and alignment of multiple guidelines[J].Bioanalysis,2012,4(18):2213
[10]AMARAVADI L,SONG A,MYLER H,et al.2015 White Paper on recent issues in bioanalysis:focus on new technologies and biomarkers(Part 3-LBA,biomarkers and immunogenicity)[J].Bioanalysis,2015,7(24):3107
[11]TANG D,THOMAS E.Strategies for dealing with hemolyzed samples in regulated LC-MS/MS bioanalysis[J].Bioanalysis,2012,4(22):2715
[12]LIU G,JI QC,ARNOLD ME,et al.Identifying,evaluating,and controlling bioanalytical risks resulting from nonuniform matrix ion suppression/enhancement and nonlinear liquid chromatography-mass spectrometry assay response[J].Anal Chem,2010,82(82):9671
[13]龙跃兵,朱伟斌,章美燕.溶血对生化检验结果准确性的影响及校正方法的探讨[J].广东医学,2011,32(12):1562LONG YB,ZHU WB,ZHANG MY.Investigation of influence by sample hemolysis on biochemical detection outcomes and its calibration methods[J].Guangdong Med J,2011,32(12):1562
[14]BéRUBéER,TAILLON MP,FURTADO M,et al.Impact of sample hemolysis on drug stability in regulated bioanalysis[J].Bioanalysis,2011,3(18):2097
[15]BéRUBéER,LACASSE MC,FURTADO M,et al.Severe impact of hemolysis on stability of phenolic compounds[J].Bioanalysis,2013,5(12):1491
[16]European Medicines Agency(EMA).Guideline of Bioanalytical Method Validation[S/OL].(2012)[2012-02-01].http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf
[17]Food and Drug Administration(FDA).Guidance for Industry:BioanalyticalMethodValidation[S/OL].(2013)[2013-09].Availableat:http://www.fda.gov/downloads/Drugs/Guidance Compliance Regulatory Information/Guidances/UCM368107.pdf
[18]INGELSE B,BARROSO B,GRAY N,et al.European bioanalysis forum:recommendationondealingwithhemolyzedand hyperlipidemic matrices[J].Bioanalysis,2014,6(23):3113
[19]HUGHES NC,BAJAJ N,FAN J,et al.Assessing the matrix effects of hemolyzed samples in bioanalysis[J].Bioanalysis,2009,1(6):1057
[20]FUNG EN,AUBRY AF,ALLENTOFF A,et al.Utilizing internal standard responses to assess risks on reporting bioanalytical results from hemolyzed samples[J].AAPS J,2015,17(5):1168
[21]CHAMBERS E,WAGROWSKIDIEHL DM,LU Z,et al.Systematic and comprehensive strategy for reducing matrix effects on LC/MS/MS analysis[J].J Chromatogr B Anal Technol Biomed Life Sci,2007,852(1-2):22
[22]刘小娟,杜晓琳,黄娟,等.生物分析中溶血对检测的影响及其对策[J].中国新药与临床杂志,2017,36(4):181LIU XJ,DU XL,HUANG J,et al.Impact of hemolysis on detection in bioanalysis and its countermeasures[J].Chin J New Drugs Clin Rem,2017,36(4):181
[23]杨永怀.标本溶血对生化检验结果的影响及处理对策探析[J].中国现代药物应用,2017,11(13):42YANG YH.Investigation of influence by sample hemolysis on biochemical detection outcomes and its treatment countermeasures[J].Chin J Mod Drug Appl,2017,11(13):42.
[24]刘婧,冯立民,龚人杰.标本溶血的患者因素分析及对策探讨[J].实验与检验医学,2017,35(1):117LIU J,FENG LM,GONG RJ.Investigation of patients factors of hemolysis and its countermeasures[J].Exp Mol Lab Med,2017,35(1):117
[25]钟大放,李高,刘昌孝.生物样品定量分析方法指导原则:草案[J].药物评价研究,2011,34(6):409ZHONG DF,LI G,LIU CX,et al.Guidance on bioanalysis:method validation and analysis of study samples(draft)[J].Drug Eval Res,2011,34(6):409
[26]冯晓杰,杜丽英,冯章英,等.LC-MS/MS法测定人血药浓度的基质效应研究进展[J].中国新药杂志,2015,24(13):1488FENG XJ,DU LY,FENG ZY,et al.Research progress of matrix effect on determining blood concentration by LC-MS/MS[J].Chin J New Drugs,2015,24(13):1488
[27]姚梦侃,马秉亮,马越鸣.生物样品液质联用分析中的基质效应研究概况[J].药物分析杂志,2010,30(12):2436YAO MK,MA BL,MA YM.Overview of the matrix effect on quantitative liquid chrimatography/mass spectrometry analyses of biological samples[J].Chin J Pharm Anal,2010,30(12):2436
[28]张人杰,柴逸峰,朱臻宇,等.LC-MS/MS生物分析中试验样品再分析失败后的调查和解决方案[J].药物分析杂志,2016,36(4):559ZHANG RJ,CHAI YF,ZHU ZY,et al.Investigation and resolution of incurred sample reanalysis failures in LC-MS/MS bioanalysis[J].Chin J Pharm Anal,2016,36(4):559