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Quantification of glutathione and cysteine by electrospray ionization mass spectrometry, high performance liquid chromatography and ultra performance liquid chromatography.

机译：通过电喷雾电离质谱，高效液相色谱和超高效液相色谱对谷胱甘肽和半胱氨酸进行定量。

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摘要

Three analytical methods, liquid chromatography mass spectrometry (LC/MS), high performance liquid chromatography (HPLC), and ultra performance liquid chromatography (UPLC) were developed for the simultaneous detection and quantitation of glutathione (GSH) and cysteine (CYS) in urine samples. The aminothiols were derivatized with Ellman's reagent. The samples were then analyzed by electrospray ionization negative mode (ESI-) conditions for the LC/MS system and by reversed-phase chromatography with ultra violet (UV) detection for both the HPLC and UPLC systems. Thiols were derivatized with excess Ellman's reagent to ensure an instantaneous and successful derivatization. Ellman's reagent is a thiol specific reagent that blocks the oxidation of thiols to disulfides. Of the three analytical methods studied, the UPLC method showed the best overall performance. A sensitive, reproducible and robust UPLC method has been validated for the simultaneous determination of CYS and GSH in artificial urine. Aliquots (20muL) of artificial urine were injected into a Waters' UPLC instrument equipped with a Waters' AccQ-Tag(TM) Ultra 2.1 X 100 mm column at a flow rate of 0.7 mL/min. The mobile phase consisted of Waters' AccQ-Tag(TM) Ultra Eluent A concentrate: water (1:10), and Waters' AccQ-Tag(TM) Ultra Eluent B. Detection was performed by UV absorbance at 340 nm. No significant interfering peaks were detected at the retention times of CYS and GSH in artificial urine blanks. The calibration curves were linear in the range of 50.0-1000.0 and 0.50-10.0 muM for CYS and GSH, respectively. Both the limit of detection (LOD) and the limit of quantification (LOQ) values for CYS and GSH were established at 50 muM and 0.5 muM respectively. The intra-day and inter-day precision values for CYS were 0.008% and 0.005%, respectively; for GSH they were 0.019% and 0.006% respectively, showing a high degree of reproducibility. The intra-day and inter-day accuracy for CYS were 111%, and107% respectively while for GSH they were 102%, and 107% respectively. Thus, this method will be useful in metabolomics investigations of conditions of oxidative stress by determination of prominent redox-sensitive thiols found in human urine.
机译：开发了三种分析方法，液相色谱质谱法（LC / MS），高效液相色谱法（HPLC）和超高效液相色谱法（UPLC）用于同时检测和定量尿液中的谷胱甘肽（GSH）和半胱氨酸（CYS）样品。用Ellman试剂将氨基硫醇衍生化。然后通过LC / MS系统的电喷雾负离子模式（ESI-）条件分析样品，并通过HPLC和UPLC系统的反相色谱结合紫外（UV）检测技术进行分析。硫醇用过量的Ellman试剂衍生化，以确保瞬时成功地衍生化。埃尔曼试剂是一种硫醇特异性试剂，可阻止硫醇氧化为二硫化物。在研究的三种分析方法中，UPLC方法显示出最佳的整体性能。已验证了灵敏，可重现且可靠的UPLC方法，可同时测定人造尿中的CYS和GSH。将等分试样（20μL）的人造尿液以0.7mL / min的流速注入配备有Waters的AccQ-Tag TM Ultra 2.1×100mm柱的Waters的UPLC仪器中。流动相由Waters的AccQ-Tag™Ultra Eluent A浓缩液：水（1:10）和Waters的AccQ-Tag™Ultra Eluent B组成。检测是通过340 nm的UV吸光度进行的。在人工尿液空白中，在CYS和GSH的保留时间未检测到明显的干扰峰。 CYS和GSH的校准曲线分别在50.0-1000.0和0.50-10.0μM范围内呈线性关系。 CYS和GSH的检出限（LOD）和定量限（LOQ）值分别设置为50μM和0.5μM。 CYS的日内和日间精度值分别为0.008％和0.005％。对于GSH，它们分别为0.019％和0.006％，显示出高度的可重复性。 CYS的日内准确性和日间准确性分别为111％和107％，而GSH的日间准确性分别为102％和107％。因此，该方法通过测定人尿液中主要的氧化还原敏感硫醇，可用于氧化应激条件的代谢组学研究。

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  • 作者

    Keil, Kim.;

  • 作者单位

    State University of New York at Buffalo.;

  • 授予单位 State University of New York at Buffalo.;
  • 学科 Chemistry Analytical.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2011
  • 页码 239 p.
  • 总页数 239
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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