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Simultaneous determination of reduced and oxidized glutathione in tissues by a novel liquid chromatography-mass spectrometry method: application in an inhalation study of Cd nanoparticles

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Abstract

The paper presents the development of an advanced extraction and fast analytical LC MS/MS method for simultaneous analyses of reduced and oxidized glutathione (GSH and GSSG, respectively) in different animal tissues. The simultaneous determination of GSH and GSSG is crucial because the amount and ratio of both GSH and GSSG may be altered in response to oxidative stress, an important mechanism of toxicity. The method uses the derivatization of free thiol groups in GSH. Its performance was demonstrated for less explored tissues (lung, brain, and liver) in mouse. The combined extraction and analytical method has very low variability and good reproducibility, maximum coefficients of variance for within-run and between-run analyses under 8 %, and low limits of quantification; for GSH and GSSG, these were 0.2 nM (0.06 ng/mL) and 10 nM (6 ng/mL), respectively. The performance of the method was further demonstrated in a model experiment addressing changes in GSH and GSSG concentrations in lung of mice exposed to CdO nanoparticles during acute 72 h and chronic 13-week exposures. Inhalation exposure led to increased GSH concentrations in lung. GSSG levels were in general not affected; nonsignificant suppression occurred only after the longer 13-week period of exposure. The developed method for the sensitive detection of both GSH and GSSG in very low tissue mass enables these parameters to be studied in cases where only a little sample is available, i.e. in small organisms or in small amounts of tissue.

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Acknowledgments

We thank Ing. Filip Mika, PhD, from the Institute of Scientific Instruments, CAS v.v.i., Brno, Czech Republic for performing the STEM analysis. The work was supported by the Czech Science Foundation grant No. P503/11/2315, project LO1214 (Research Centre for Toxic Compounds in the Environment) funded by the National Sustainability Programme of the Czech Republic, European Social Fund and the state budget of the Czech Republic (OPVK—operational programme education for competitiveness).

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Authors and Affiliations

  1. Masaryk University, Faculty of Science, RECETOX, Kamenice 753/5, Building A29, 62500, Brno, Czech Republic

    L. Bláhová, J. Kohoutek, J. Lebedová, L. Bláha & K. Hilscherová

  2. Academy of Sciences of the Czech Republic, Institute of Analytical Chemistry, v.v.i., 61200, Brno, Czech Republic

    Z. Večeřa

  3. Academy of Sciences of the Czech Republic, Institute of Animal Physiology and Genetics, v.v.i., 60200, Brno, Czech Republic

    M. Buchtová & I. Míšek

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  1. L. Bláhová
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  2. J. Kohoutek
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  3. J. Lebedová
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  4. L. Bláha
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  5. Z. Večeřa
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  6. M. Buchtová
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  7. I. Míšek
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  8. K. Hilscherová
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Correspondence to L. Bláhová.

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Bláhová, L., Kohoutek, J., Lebedová, J. et al. Simultaneous determination of reduced and oxidized glutathione in tissues by a novel liquid chromatography-mass spectrometry method: application in an inhalation study of Cd nanoparticles. Anal Bioanal Chem 406, 5867–5876 (2014). https://doi.org/10.1007/s00216-014-8033-z

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  • DOI: https://doi.org/10.1007/s00216-014-8033-z

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