浙江大学学报（医学版）
浙江大學學報（醫學版）
절강대학학보（의학판）
JOURNAL OF ZHEJIANG UNIVERSITY MEDICAL SCIENCES
2014年
2期
129-134
,共6页
孔思思%涂美娟%杨希%赵垒%周慧%曾苏%蒋惠娣
孔思思%塗美娟%楊希%趙壘%週慧%曾囌%蔣惠娣
공사사%도미연%양희%조루%주혜%증소%장혜제
酮类%铁螯合剂%质谱分析法%色谱法, 液相%有机阳离子转运子%有机阴离子转运子1
酮類%鐵螯閤劑%質譜分析法%色譜法, 液相%有機暘離子轉運子%有機陰離子轉運子1
동류%철오합제%질보분석법%색보법, 액상%유궤양리자전운자%유궤음리자전운자1
Ketones%Iron chelating agents%Mass spectrometry%Chromatography,liquid%Organic cation transporters%Organic anion transporter 1
目的：建立细胞裂解液中去铁酮测定的液相色谱-串联质谱（ LC-MS/MS）方法，体外考察去铁酮与人有机阳离子转运体（ hOCTs）及有机阴离子转运体1（hOAT1）的相互作用。方法：以Agilent Eclipse Plus C18柱（2．1 mm ×50 mm，3．5μm）为分析柱；0．1％甲酸-水（ v/v）和0．1％甲酸-乙腈（ v/v）为流动相，梯度洗脱；应用电喷雾离子源（ ESI源）、多反应监测（ MRM）模式进行检测；应用稳定表达hOCTs及hOAT1的细胞模型（ MDCK-hOCTs和MDCK-hOAT1）考察去铁酮对经典底物在细胞内积聚的影响；比较去铁酮在转空载体的mock细胞及MDCK-hOCTs中积聚的差异，以及经典抑制剂对其积聚的影响。结果：去铁酮在5～300 nmol/L的浓度范围内线性关系良好；准确度大于94％，日内相对标准偏差小于2％。300μmol/L去铁酮使hOCTs经典底物MPP＋在MDCK-hOCTs的积聚降低至阴性对照的70．4％～87．1％；去铁酮在MDCK-hOCTs细胞和mock细胞中的积聚无明显差别，且经典抑制剂对去铁酮的积聚无明显抑制作用；100μmol/L去铁酮对hOAT1经典底物6-CF在MDCK-hOAT1细胞中的积聚无显著影响。结论：本实验建立的方法适用于细胞裂解液中去铁酮的定量分析；去铁酮对hOCT1和hOCT3有一定的抑制作用，对hOCT2和hOAT1则无明显抑制作用；hOCTs在去铁酮跨膜转运中不起主导作用。
目的：建立細胞裂解液中去鐵酮測定的液相色譜-串聯質譜（ LC-MS/MS）方法，體外攷察去鐵酮與人有機暘離子轉運體（ hOCTs）及有機陰離子轉運體1（hOAT1）的相互作用。方法：以Agilent Eclipse Plus C18柱（2．1 mm ×50 mm，3．5μm）為分析柱；0．1％甲痠-水（ v/v）和0．1％甲痠-乙腈（ v/v）為流動相，梯度洗脫；應用電噴霧離子源（ ESI源）、多反應鑑測（ MRM）模式進行檢測；應用穩定錶達hOCTs及hOAT1的細胞模型（ MDCK-hOCTs和MDCK-hOAT1）攷察去鐵酮對經典底物在細胞內積聚的影響；比較去鐵酮在轉空載體的mock細胞及MDCK-hOCTs中積聚的差異，以及經典抑製劑對其積聚的影響。結果：去鐵酮在5～300 nmol/L的濃度範圍內線性關繫良好；準確度大于94％，日內相對標準偏差小于2％。300μmol/L去鐵酮使hOCTs經典底物MPP＋在MDCK-hOCTs的積聚降低至陰性對照的70．4％～87．1％；去鐵酮在MDCK-hOCTs細胞和mock細胞中的積聚無明顯差彆，且經典抑製劑對去鐵酮的積聚無明顯抑製作用；100μmol/L去鐵酮對hOAT1經典底物6-CF在MDCK-hOAT1細胞中的積聚無顯著影響。結論：本實驗建立的方法適用于細胞裂解液中去鐵酮的定量分析；去鐵酮對hOCT1和hOCT3有一定的抑製作用，對hOCT2和hOAT1則無明顯抑製作用；hOCTs在去鐵酮跨膜轉運中不起主導作用。
목적：건립세포렬해액중거철동측정적액상색보-천련질보（ LC-MS/MS）방법，체외고찰거철동여인유궤양리자전운체（ hOCTs）급유궤음리자전운체1（hOAT1）적상호작용。방법：이Agilent Eclipse Plus C18주（2．1 mm ×50 mm，3．5μm）위분석주；0．1％갑산-수（ v/v）화0．1％갑산-을정（ v/v）위류동상，제도세탈；응용전분무리자원（ ESI원）、다반응감측（ MRM）모식진행검측；응용은정표체hOCTs급hOAT1적세포모형（ MDCK-hOCTs화MDCK-hOAT1）고찰거철동대경전저물재세포내적취적영향；비교거철동재전공재체적mock세포급MDCK-hOCTs중적취적차이，이급경전억제제대기적취적영향。결과：거철동재5～300 nmol/L적농도범위내선성관계량호；준학도대우94％，일내상대표준편차소우2％。300μmol/L거철동사hOCTs경전저물MPP＋재MDCK-hOCTs적적취강저지음성대조적70．4％～87．1％；거철동재MDCK-hOCTs세포화mock세포중적적취무명현차별，차경전억제제대거철동적적취무명현억제작용；100μmol/L거철동대hOAT1경전저물6-CF재MDCK-hOAT1세포중적적취무현저영향。결론：본실험건립적방법괄용우세포렬해액중거철동적정량분석；거철동대hOCT1화hOCT3유일정적억제작용，대hOCT2화hOAT1칙무명현억제작용；hOCTs재거철동과막전운중불기주도작용。
Objective: To develop a LC-MS/MS method for determination of deferiprone in cell lysate and to study the potential interaction between deferiprone and hOCTs or hOAT1 transporters in vitro.Methods: The determination was performed on an Agilent Eclipse Plus C18 column(3.5 μm, 2.1 mm ×50 mm).The gradient mobile phase was composed of solvent A:0.1% formic acid in water, and B:0.1% formic acid in acetonitrile .The mass spectrometer with an electrospray interface was operated in positive ion mode with multiple reaction monitoring ( MRM) scan mode monitored the ion pair of deferiprone at m/z 140→96 , or phenacetin at m/z 180→110 .The effects of deferiprone on the accumulation of typical substrates of hOCTs and hOAT 1 were evaluated by MDCK-hOCTs and MDCK-hOAT1 cells respectively .The accumulation of deferiprone was also investigated in MDCK-hOCTs cells and mock cells with or without typical inhibitors .Results: The standard curve was linear over the range of 5-300 nmol/L.The assay recovery of deferiprone was above 94%, and the intra-day precision (RSD) was less than 2.0%.The accumulation of MPP +in MDCK-hOCTs cells with 300 μmol/L deferiprone were 73 .5%, 87 .1% and 70 .4%, respectively .The uptake of deferiprone in MDCK-hOCTs and mock cells did not show significant difference . Deferiprone of 100 μmol/L did not significantly affect the accumulation of 6-CF in MDCK-hOAT1 cell. Conclusion: The method is sensitivity and suitable for the determination of deferiprone in cell lysate .Deferiprone can significantly inhibit hOCT 1 and hOCT3, but has no effects on hOCT2 and hOAT1.hOCTs may not play a major role in the transport of deferiprone .
