Journal
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume 410, Issue 27, Pages 7153-7161Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-018-1318-x
Keywords
Differential mobility spectrometry; Short LC; Quantification; Blood; Glutathione
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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 N-2, 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 mu M for GSSG and 3.2 mu M for GS-N-ethylmaleimide (NEM) using a blood volume of 60 mu L. The method is linear over a wide dynamic concentration range of 0.4 to 400 mu M for GSSG and from 3.2 to 3200 mu M 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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