Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography-electrospray mass spectrometry.
We developed a sensitive and specific liquid chromatography-electrospray mass spectrometric (HPLC-ESI-MS) assay for the simultaneous determination of reduced and oxidized glutathione (GSH and GSSG) in peripheral blood mononuclear cells (PBMC). Following derivatization with N-ethylmaleimide to prevent GSH auto-oxidation, addition of thiosalicylic acid as internal standard, and protein precipitation with cold acetonitrile, the samples were injected into a diol column, eluted with acetonitrile-1% aqueous acetic acid (25:75) and detected by the ESI-MS system. The optimized method exhibited a good detection limit for both analytes (0.01 and 0.05 microM for GSH and GSSG, respectively). Good linearity was reached in the 0.01-20 microM range for GSH and 0.05-20 microM for GSSG. The mean recoveries of GSH and GSSG were 98.5-100.6% and 105.8-111.5%, respectively. The run-to-run repeatability for retention time and peak area was RSD% 0.06 and 1.75 for GSH and 0.18 and 2.50 for GSSG. The optimized method was applied to GSH and GSSG assay in PBMC analyzing 20 healthy individuals.
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Istituto Dermatologico San Gallicano (IRCCS), Rome, Italy. picardo@ifo.it
Rinaldi
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Briganti
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Picardo
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Fanali
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D002853:Chromatography, Liquid / Q000379:methods*; D005033:Ethylmaleimide / Q000737:chemistry; D005978:Glutathione / Q000097:blood* / Q000737:chemistry; D006801:Humans; D009000:Monocytes / Q000378:metabolism*; D010084:Oxidation-Reduction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D005978:Glutathione; D005033:Ethylmaleimide