A new LC–MS/MS method for the clinical determination of reduced and oxidized glutathione from whole blood

Moore, Tereza; Le, Anthony; Niemi, Anna-Kaisa; Kwan, Tak; Cusmano‐Ozog, Kristina; Enns, Gregory M.; Cowan, Tina M.

doi:10.1016/j.jchromb.2013.04.004

Cited by 107 publications

(88 citation statements)

References 24 publications

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“…Under these acidic conditions (pH =2–2.3) NEM cannot react with thiols. More recently, a new LC-MS/MS method was developed, where compounds were separated by liquid chromatography using a Hypercarb column, particularly suitable for small polar compounds [43]. Also in this work, whole blood was mixed with a precipitating solution containing both NEM and sulfosalicylic acid.…”

Section: Suggested Methods To Measure Gsh and Gssgmentioning

confidence: 99%

“…For instance, Moore et al reported 900±140 μM for GSH, 1.17±0.43 μM for GSSG and 880±370 for the GSH/GSSG ratio in whole blood of 59 healthy adults [43]. In whole blood of 9 subjects, Fahrenholz et al reported 1.46±0.07 μmol/g for GSH and 2.4±0.4 nmol/g for GSSG, yielding a mean GSH/GSSG ratio of 608.…”

Section: Suggested Methods To Measure Gsh and Gssgmentioning

confidence: 99%

“…It should be also reminded that NEM was used at different experimental. In some cases NEM was added to the deproteinizing solution [24,43]. This procedure should be less affective in protecting GSH from its oxidation since NEM alkylation needs a neutral pH.…”

Section: Suggested Methods To Measure Gsh and Gssgmentioning

confidence: 99%

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Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: An elephant in the room

Giustarini

Tsikas

Colombo

et al. 2016

Journal of Chromatography B

120

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Glutathione (GSH) is the most abundant low-molecular-mass thiol within cells and one of the major antioxidant compounds in body fluids. Under pro-oxidant conditions, two GSH molecules donate one electron each and are converted into glutathione disulfide (GSSG). The GSH/GSSG molar ratio is considered a powerful index of oxidative stress and disease risk. Despite high interest in GSH/GSSG titration as measures of thiol redox balance, no broad agreement has yet been reached as to the best pre-analytical and analytical methods for the quantitation of these molecules in biological samples. Consequently, measured concentrations of GSH and GSSG and calculated GSH/GSSG molar ratios vary widely among laboratories. Here, we describe in detail the main analytical and pre-analytical problems related to the artificial oxidation of the sulfhydryl (SH) group of GSH that occur during sample manipulation. We underline how this aspect has been neglected for long time after its first description more than fifty years ago. Finally, selected reliable procedures and methods to measure GSH and GSSG in biological samples are discussed.

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Section: Suggested Methods To Measure Gsh and Gssgmentioning

confidence: 99%

Section: Suggested Methods To Measure Gsh and Gssgmentioning

confidence: 99%

See 1 more Smart Citation

Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: An elephant in the room

Giustarini

Tsikas

Colombo

et al. 2016

Journal of Chromatography B

120

View full text Add to dashboard Cite

show abstract

“…Several methods have been reported for the determination of GSH. These, include chemiluminescence 18 , High Performance Liquid Chromatography (HPLC) [19][20][21] , capillary electrophores [22][23][24][25][26] , derivatization methods 27 , liquid chromatography-tandem mass spectrometry (LC-MS/MS) 28 , liquid chromatography -electrospray mass spectrometric (HPLC-ESI-MS) 29 , liquid chromatography-Electrochemical Method [30][31][32] , Electrochemical methods provide useful alternatives since they allow faster, cheaper and safer analysis [33][34][35][36][37][38][39][40] .…”

Section: Oriental Journal Of Chemistrymentioning

confidence: 99%

A Sensitive Voltammetric Sensor for Determination of Glutathione Based on Multiwall Carbon Nanotubes Paste Electrode Incorporating Pyrogallol Red

Keyvanfard¹,

Alizad²

2014

Orient. J. Chem.

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A new sensitive and selective electrochemical sensor was developed for determination of glutathione (GSH) at the surface of carbon paste electrode (CPE) modified with multi-wall carbon nanotubes (MWCNTs) as a sensor and pyrogallol red (PGR) as a mediator. The mechanism of GSH electrochemical behavior at the modified electrode surface was investigated by various electrochemical techniques including chronoamperometry, cyclic voltammetry (CV) and square wave voltammetry (SWV). A linear calibration curve was obtained in the concentration range of GSH of 0.3-500 µmol L -1 , with a limit of detection of 0.19 µmol L -1 . The method was applied to the determination of GSH in urine samples with satisfactory results.

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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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A new LC–MS/MS method for the clinical determination of reduced and oxidized glutathione from whole blood

Cited by 107 publications

References 24 publications

Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: An elephant in the room

Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: An elephant in the room

A Sensitive Voltammetric Sensor for Determination of Glutathione Based on Multiwall Carbon Nanotubes Paste Electrode Incorporating Pyrogallol Red

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

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