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High-throughput liquid chromatography differential mobility spectrometry mass spectrometry for bioanalysis: determination of reduced and oxidized form of glutathione in human blood

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Abstract

Currently, the measure of the oxidative stress, from oxidized and reduced glutathione (GSSG and GSH respectively), for large cohorts of samples, is generally limited to spectrometric methods. In this study, a high-throughput assay for GSH after derivatization with N-ethylmaleimide and GSSG in blood sample was developed with an analysis time of 1.5 min. The method combines protein precipitation and a short LC (10-mm length) column where compounds were trapped in front-flush mode and eluted in back-flush mode. This setup is combined with modifier-assisted differential ion mobility spectrometry (DMS, SelexIon) and detection is performed in the selected reaction monitoring mode using positive electrospray ionization. In DMS, various modifiers were investigated including N2, methanol, toluene, ethanol, acetonitrile, and isopropanol to improve assay selectivity. Using EtOH as modifier, the limit of quantification (LOQ) was found to be 0.4 μM for GSSG and 3.2 μM for GS-N-ethylmaleimide (NEM) using a blood volume of 60 μL. The method is linear over a wide dynamic concentration range of 0.4 to 400 μM for GSSG and from 3.2 to 3200 μM for GS-NEM. The inter-assay precision of QC samples were ≤ 6.7%, with accuracy values between 98.3 and 103%. The method was further cross-validated with a LC Hypercarb-DMS-MS/MS method by the analysis of human blood samples. The bias between both assays ranged from − 0.3 to 0.2%.

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Acknowledgments

The authors are grateful to Dr. Yves LeBlanc and Dr. Brad Schneider (Sciex) for fruitful discussions.

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Authors and Affiliations

  1. Life Sciences Mass Spectrometry, Department of Inorganic and Analytical Chemistry, University of Geneva, 24 Quai Ernest Ansermet, 1211, Geneva 4, Switzerland

    Sophie Bravo-Veyrat & Gérard Hopfgartner

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  1. Sophie Bravo-Veyrat
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  2. Gérard Hopfgartner
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Corresponding author

Correspondence to Gérard Hopfgartner.

Ethics declarations

The blood samples were provided by the Centre de Transfusion Sanguine, University Hospital Geneva, Geneva, Switzerland. The Human Research Act (HRA) does not apply for the anonymized blood samples analyzed in the present work (Art. 2 para. 2 let. b and c).

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The authors declare that they have no conflict of interest.

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Bravo-Veyrat, S., Hopfgartner, G. High-throughput liquid chromatography differential mobility spectrometry mass spectrometry for bioanalysis: determination of reduced and oxidized form of glutathione in human blood. Anal Bioanal Chem 410, 7153–7161 (2018). https://doi.org/10.1007/s00216-018-1318-x

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  • DOI: https://doi.org/10.1007/s00216-018-1318-x

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