Direct glutathione quantification in human blood by LC–MS/MS: comparison with HPLC with electrochemical detection

Squellerio, Isabella; Caruso, Donatella; Porro, Benedetta; Veglia, Fabrizio; Tremoli, Elena; Cavalca, Viviana

doi:10.1016/j.jpba.2012.08.013

Cited by 85 publications

(53 citation statements)

References 50 publications

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“…The baseline for GSSG was noisier in both standards and blanks; however, with a response factor more than 5 times greater than GSH, the LOD and LOQ were 0.5 and 1.6 nM, respectively, or 39 and 120 fmol of analyte on-column. Among the assays in which GSH is not derivatized [11,27,28,33,[35][36][37], the LOD and LOQ for the assay presented here are second lowest, bettered only by Robin and coworkers, who achieved LOD values equivalent to 8 and 7 fmol on-column for GSSG and GSH, respectively, using postcolumn reaction with silver ions [35]. The linear ranges for GSH and GSSG cover three orders of magnitude, making this assay comparable to other assays using underivatized glutathione.…”

Section: Selection and Identification Of Multiple Reaction Monitoringmentioning

confidence: 65%

“…A study of GSH and GSSG ratios in whole blood and red blood cell samples by Rossi and coworkers determined that the use of strong acids, such as trichloroacetic acid and sulfosalicylic acid, to precipitate protein acidified the supernatant and minimized GSH oxidation [26]. Other authors have confirmed the limited stability in whole blood [11,27] and mouse liver tissue [28]. Supernatant samples collected in triplicate over the course of 1 day were reproducible, with relative standard deviations under 6% for GSH and under 9% for GSSG, showing no sign of oxidative degradation over the course of the data collection.…”

Section: Chromatographic Proceduresmentioning

confidence: 99%

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Quantitation of protein S-glutathionylation by liquid chromatography–tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide

Bukowski

Bucklin

Picklo

2015

Analytical Biochemistry

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Section: Selection and Identification Of Multiple Reaction Monitoringmentioning

confidence: 65%

Section: Chromatographic Proceduresmentioning

confidence: 99%

Quantitation of protein S-glutathionylation by liquid chromatography–tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide

Bukowski

Bucklin

Picklo

2015

Analytical Biochemistry

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“…GSH and GSSG were measured by LC-MS/MS method on whole blood, after proteins precipitation with trichloroacetic acid [42]. Levels of GSH and GSSG were expressed as μ mol/g Hb.…”

Section: Methodsmentioning

confidence: 99%

Nitric Oxide Synthetic Pathway in Patients with Microvascular Angina and Its Relations with Oxidative Stress

Porro

Eligini

Veglia

et al. 2014

Oxidative Medicine and Cellular Longevity

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A decreased nitric oxide (NO) bioavailability and an increased oxidative stress play a pivotal role in different cardiovascular pathologies. As red blood cells (RBCs) participate in NO formation in the bloodstream, the aim of this study was to outline the metabolic profile of L-arginine (Arg)/NO pathway and of oxidative stress status in RBCs and in plasma of patients with microvascular angina (MVA), investigating similarities and differences with respect to coronary artery disease (CAD) patients or healthy controls (Ctrl). Analytes involved in Arg/NO pathway and the ratio of oxidized and reduced forms of glutathione were measured by LC-MS/MS. The arginase and the NO synthase (NOS) expression were evaluated by immunofluorescence staining. RBCs from MVA patients show increased levels of NO synthesis inhibitors, parallel to that found in plasma, and a reduction of NO synthase expression. When summary scores were computed, both patient groups were associated with a positive oxidative score and a negative NO score, with the CAD group located in a more extreme position with respect to Ctrl. This finding points out to an impairment of the capacity of RBCs to produce NO in a pathological condition characterized mostly by alterations at the microvascular bed with no significant coronary stenosis.

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“…A resulting overproduction of GSSG (GSH oxidation), which often occurs during alkalization and derivatization phase, is the major disadvantage of such sample preparation protocol [9,10]. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the technique of choice for the simultaneous determination of GSH and GSSG (taking into consideration specificity and low limits of detection and quantification), it is inaccessible to many laboratories [11][12][13]. Other methods, such as micellar electrokinetic capillary electrophoresis (MEKC) [14], nuclear magnetic resonance (NMR) [15], fluorescence [16], and chemiluminescence-based methods, have also been proposed [17].…”

Section: Introductionmentioning

confidence: 99%

The simple isocratic HPLC—UV method for the simultaneous determination of reduced and oxidized glutathione in animal tissue

Begić¹,

Djurić²,

Gobeljić³

et al. 2017

Acta Chromatographica

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Summary. The aim of our work was to optimize and apply simple high-performance liquid chromatography method with ultraviolet detection (HPLC-UV) for simultaneous determination of reduced (GSH) and oxidized (GSSG) glutathione in biological matrix (specifically, the rat liver tissue was used herein), since the ratio between oxidized and reduced glutathione forms (GSSG-GSH) has been recognized as an important biological marker of oxidatively depleted GSH in oxidative stress (OS)-associated diseases and poisonings. An isocratic chromatographic separation of GSH and GSSG (2.8 min and 6.3 min, respectively) was performed with the mobile phase consisted of sodium perchlorate solution (pH adjusted to 2.8) at flow rate of 1 mL min −1 , detection set at 215 nm, and column temperature of 40 °C. The method offers short run time, linearity in the range of 0.01-200 µM concentration for both compounds (R 2 = 1), low limits of detection and quantification (GSH: 0.18 µM and 0.56 µM, GSSG: 0.52 µM and 1.58 µM, respectively), precision, accuracy (bias < 2%), and high reproducibility.Through suitable sample handling, an overestimation of GSSG was prevented. High recovery (>99%) was achieved. The method was successfully applied for the analysis of GSH and GSSG in liver homogenates of Wistar rats intraperitoneally exposed to cadmium (Cd) (1 mg kg −1 CdCl2/21 days). Regardless of other Cd-mediated hepatotoxicity mechanisms, herein, we have exclusively interpreted/emphasized oxidative GSH depletion.The presented method is acceptable for a routine analysis of GSH and GSSG in biological matrix, while the calculated ratio GSSG-GSH is considered as a valuable OS marker.

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Direct glutathione quantification in human blood by LC–MS/MS: comparison with HPLC with electrochemical detection

Cited by 85 publications

References 50 publications

Quantitation of protein S-glutathionylation by liquid chromatography–tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide

Quantitation of protein S-glutathionylation by liquid chromatography–tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide

Nitric Oxide Synthetic Pathway in Patients with Microvascular Angina and Its Relations with Oxidative Stress

The simple isocratic HPLC—UV method for the simultaneous determination of reduced and oxidized glutathione in animal tissue

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