改良酶放大免疫法提高甲氨蝶呤检测灵敏度
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摘要
目的:通过对现有检测方法进行改良,将西门子VIVA-E生化分析仪对甲氨喋呤的检测灵敏度从0. 30μmol/L提升至0. 05μmol/L。方法:设置参数,降低试剂用量从180μL降至110μL,同时更改定标点浓度为0、0. 05、0. 10、0. 25、0. 50和1. 00μmol/L,建立新方法。分别使用改良法、商业化方法及LC-MS/MS法测量相同的样本进行对照检验。结果:改良后新方法在0. 30～1. 00μmol/L范围内与商业化的方法有良好的相关性,在0. 05～1. 00μmol/L范围内改良法与LC-MS/MS法有良好的相关性。结论:改良法在提高灵敏度的前提下将单个试剂盒的测量次数从140次提升至210次,显著降低了检测成本,且检测结果准确可靠,可在临床推广应用。
Objective: Through improving the existing detection method,to improve the sensitivity of the Siemens VIVA-E biochemical analyzer to methotrexate from 0. 30 μmol/L to 0. 05 μmol/L. Methods: The new method was established by setting parameters,reducing the reagent dosage from 180 μL to 110 μL,and changing the fixed punctuation concentration to 0,0. 05,0. 10,0. 25,0. 50 and 1. 00μmol/L.The same samples were respectively measured by using the modified method,commercial method and LC-MS/MS method for the control test.Results: The improved method had good correlation with the commercial method in the range of 0. 30-1. 00 μmol/L. The improved method had good correlation with LC-MS/MS in the range of 0. 05-1. 00 μmol/L.Conclusion: Under the premise of improving sensitivity,the improved method increases the number of measurements of a single kit from 140 to 210,which greatly reduces the detection cost; and the detection results are accurate and reliable,and can be applied and promoted in clinical practice.
Objective: Through improving the existing detection method,to improve the sensitivity of the Siemens VIVA-E biochemical analyzer to methotrexate from 0. 30 μmol/L to 0. 05 μmol/L. Methods: The new method was established by setting parameters,reducing the reagent dosage from 180 μL to 110 μL,and changing the fixed punctuation concentration to 0,0. 05,0. 10,0. 25,0. 50 and 1. 00μmol/L.The same samples were respectively measured by using the modified method,commercial method and LC-MS/MS method for the control test.Results: The improved method had good correlation with the commercial method in the range of 0. 30-1. 00 μmol/L. The improved method had good correlation with LC-MS/MS in the range of 0. 05-1. 00 μmol/L.Conclusion: Under the premise of improving sensitivity,the improved method increases the number of measurements of a single kit from 140 to 210,which greatly reduces the detection cost; and the detection results are accurate and reliable,and can be applied and promoted in clinical practice.
引文
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[5]AL-GHOBASHY M A,HASSAN S A,ABDELAZIZ D H,et al.Development and validation of LC-MS/MS assay for the simultaneous determination of methotrexate,6-mercaptopurine and its active metabolite 6-thioguanine in plasma of children with acute lymphoblastic leukemia:Correlation with genetic polymorphism[J].J Chromatogr B Analyt Technol Biomed Life Sci,2016,1038:88-94.
[6]GUO P,WANG X,LIU L,et al.Determination of methotrexate and its major metabolite 7-hydroxymethotrexate in mouse plasma and brain tissue by liquid chromatography-tandem mass spectrometry[J].J Pharm Biomed Anal,2007,43(5):1789-1795.
[7]BOUQUIE R,DESLANDES G,BERNALDEZ B N,et al.A fast LC-MS/MS assay for methotrexate monitoring in plasma:Validation,comparison to FPIA and application in the setting of carboxypeptidase therapy[J].Anal Methods,2013,6(1):178-186.
[8]FOTOOHI K,SKARBY T,SODERHALL S,et al.Interference of7-hydroxymethotrexate with the determination of methotrexate in plasma samples from children with acute lymphoblastic leukemia employing routine clinical assays[J].J Chromatogr B Analyt Technol Biomed Life Sci,2005,817(2):139-144.
[9]BOUQUIR,GRGOIRE M,HERNANDO H,et al.Evaluation of a methotrexate chemiluminescent microparticle immunoassay comparison to fluorescence polarization immunoassay and liquid chromatography tandem mass spectrometry[J].Am J Clin Pathol,2016,146(1):119-124.
[10]FDA Guidelines.Bioanalytical method validation[S].2013-15-18.
[11]GUERRIERO E,SIMON N,NELKEN B,et al.Unexpected overestimation of methotrexate plasma concentrations:Analysis of a single center pediatric population[J].Ther Drug Monit,2014,36(4):499-504.
[2]BORGMAN M P,HIEMER M F,MOLINELLI A R,et al.Improved sensitivity for methotrexate analysis using enzyme multiplied immunoassay technique on the Siemens Viva-Einstrument[J].Ther Drug Monit,2012,34(2):193-197.
[3]MENDU D R,CHOU P P,SOLDIN S J.An improved application for the enzyme multipled immunoassay technique for caffeine,amikacin,and methotrexate assays on the dade-behring dimension rxl max clinical chemistry system[J].Ther Drug Monit,2007,29(5):632-637.
[4]GODEFROID M J,VON MEYER A,PARSCH H,et al.Multicenter method evaluation of the ARKTM Methotrexate immunoassay[J].Clin Chem Lab Med,2014,52(2):13-16.
[5]AL-GHOBASHY M A,HASSAN S A,ABDELAZIZ D H,et al.Development and validation of LC-MS/MS assay for the simultaneous determination of methotrexate,6-mercaptopurine and its active metabolite 6-thioguanine in plasma of children with acute lymphoblastic leukemia:Correlation with genetic polymorphism[J].J Chromatogr B Analyt Technol Biomed Life Sci,2016,1038:88-94.
[6]GUO P,WANG X,LIU L,et al.Determination of methotrexate and its major metabolite 7-hydroxymethotrexate in mouse plasma and brain tissue by liquid chromatography-tandem mass spectrometry[J].J Pharm Biomed Anal,2007,43(5):1789-1795.
[7]BOUQUIE R,DESLANDES G,BERNALDEZ B N,et al.A fast LC-MS/MS assay for methotrexate monitoring in plasma:Validation,comparison to FPIA and application in the setting of carboxypeptidase therapy[J].Anal Methods,2013,6(1):178-186.
[8]FOTOOHI K,SKARBY T,SODERHALL S,et al.Interference of7-hydroxymethotrexate with the determination of methotrexate in plasma samples from children with acute lymphoblastic leukemia employing routine clinical assays[J].J Chromatogr B Analyt Technol Biomed Life Sci,2005,817(2):139-144.
[9]BOUQUIR,GRGOIRE M,HERNANDO H,et al.Evaluation of a methotrexate chemiluminescent microparticle immunoassay comparison to fluorescence polarization immunoassay and liquid chromatography tandem mass spectrometry[J].Am J Clin Pathol,2016,146(1):119-124.
[10]FDA Guidelines.Bioanalytical method validation[S].2013-15-18.
[11]GUERRIERO E,SIMON N,NELKEN B,et al.Unexpected overestimation of methotrexate plasma concentrations:Analysis of a single center pediatric population[J].Ther Drug Monit,2014,36(4):499-504.