Analysis of glutathione and glutathione disulfide in human saliva using hydrophilic interaction chromatography with mass spectrometry.
A sensitive method for the determination of glutathione (GSH) and glutathione disulfide (GSSG) in human saliva was developed and validated. GSH was captured and stabilized by the addition of N-ethylmaleimide (NEM). Solid-phase extraction (SPE) using an Oasis MAX extraction cartridge was employed for sample preparation and analysis was performed on a Shimadzu LCMS-2010 A that was operated in the single ion monitoring mode using positive ion electrospray ionization (ESI) as the interface. The monitored ion for GSH-NEM was m/z 433 and that for GSSG was m/z 613. Chromatography was carried out on an Atlantis HILIC silica column (150 mm x 2.1 mm, 5 microm) with acetonitrile and formate buffer as the mobile phase at the flow rate of 0.2 ml/min. The calibration curve was linear over the range of 0.1-100 microM for GSH-NEM. The extraction recoveries of GSH-NEM spiked at concentrations of 25 and 50 microM were 97.1 and 104.4%, respectively. Similar results were obtained for GSSG. The newly developed hydrophilic interaction chromatography with mass spectrometry (HILIC/MS) method showed superior sensitivity for the determination of GSH and GSSG in human saliva samples.
Iwasaki
Yusuke
Y
Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan.
Hoshi
Miyuki
M
Ito
Rie
R
Saito
Koichi
K
Nakazawa
Hiroyuki
H
D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D005033:Ethylmaleimide / Q000737:chemistry; D019803:Glutathione Disulfide / Q000032:analysis* / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D012015:Reference Standards; D015203:Reproducibility of Results; D012463:Saliva / Q000737:chemistry*
D005033:Ethylmaleimide; D019803:Glutathione Disulfide