title: Development of a reliable method based on ultra-performance liquid chromatography coupled to tandem mass spectrometry to measure thiol-associated oxidative stress in whole blood samples.
abstract: The aminothiols are biological compounds with numerous vital functions. One of the most relevant is their role as antioxidant systems. The reduced to oxidized ratios are extremely useful indicators of oxidative stress and cellular redox status. We have validated an ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method to determine the levels of glutathione, cysteine, homocysteine, and their respective oxidized compounds in whole blood samples. Results showed excellent linearity for all the analytes with correlation coefficients between 0.990 and 0.997, suitable precision with intra-day coefficient of variation ≤20%, and satisfactory accuracy with recoveries between 75 and 130%. The limits of detection in whole blood samples were 1.16 nmol L(-1) for glutathione, 115.8 nmol L(-1) for oxidized glutathione, 9.3 nmol L(-1) for homocystine, 92.6 nmol L(-1) for homocysteine, 347 nmol L(-1) for cystine and 0.23 nmol L(-1) for cysteine. The suitability of the method was ascertained in whole blood samples (n=80) from a consolidated experimental model of hypoxia-reoxygenation in newborn piglets.
authors.affiliation: Neonatal Research Group, Health Research Institute La Fe, Valencia, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Escobar
authors.affiliation: Neonatal Research Group, Health Research Institute La Fe, Valencia, Spain.
authors.forename: Ángel
authors.initials: Á
authors.lastname: Sánchez-Illana
authors.affiliation: Neonatal Research Group, Health Research Institute La Fe, Valencia, Spain.
authors.forename: Julia
authors.initials: J
authors.lastname: Kuligowski
authors.affiliation: Neonatal Research Group, Health Research Institute La Fe, Valencia, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Torres-Cuevas
authors.affiliation: Department of Pediatric Research, University of Oslo, Oslo University Hospital, Rikshospitalet, Norway.
authors.forename: Rønnaug
authors.initials: R
authors.lastname: Solberg
authors.affiliation: Department of Pediatric Research, University of Oslo, Oslo University Hospital, Rikshospitalet, Norway.
authors.forename: Håvard T
authors.initials: HT
authors.lastname: Garberg
authors.affiliation: Department of Pediatric Research, University of Oslo, Oslo University Hospital, Rikshospitalet, Norway.
authors.forename: Marianne U
authors.initials: MU
authors.lastname: Huun
authors.affiliation: Department of Pediatric Research, University of Oslo, Oslo University Hospital, Rikshospitalet, Norway.
authors.forename: Ola D
authors.initials: OD
authors.lastname: Saugstad
authors.affiliation: Neonatal Research Group, Health Research Institute La Fe, Valencia, Spain; Division of Neonatology, University & Polytechnic Hospital La Fe, Valencia, Spain.
authors.forename: Máximo
authors.initials: M
authors.lastname: Vento
authors.affiliation: Neonatal Research Group, Health Research Institute La Fe, Valencia, Spain. Electronic address: m.consuelo.chafer@uv.es.
authors.forename: Consuelo
authors.initials: C
authors.lastname: Cháfer-Pericás
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000975:Antioxidants / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003545:Cysteine / Q000097:blood / Q000737:chemistry; D005978:Glutathione / Q000097:blood / Q000737:chemistry; D019803:Glutathione Disulfide / Q000097:blood / Q000138:chemical synthesis; D006710:Homocysteine / Q000097:blood / Q000737:chemistry; D057230:Limit of Detection; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000502:physiology*; D013438:Sulfhydryl Compounds / Q000097:blood* / Q000737:chemistry*; D013552:Swine; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000975:Antioxidants; D013438:Sulfhydryl Compounds; D006710:Homocysteine; D005978:Glutathione; D003545:Cysteine; D019803:Glutathione Disulfide
keywords: Aminothiols; Blood; Liquid chromatography; Mass spectrometry; Oxidative stress