title: Combined Measurement of 6 Fat-Soluble Vitamins and 26 Water-Soluble Functional Vitamin Markers and Amino Acids in 50 μL of Serum or Plasma by High-Throughput Mass Spectrometry.
abstract: Targeted metabolic profiling characterized by complementary platforms, multiplexing and low volume consumption are increasingly used for studies using biobank material. Using liquid-liquid extraction, we developed a sample workup suitable for quantification of 6 fat- and 26 water-soluble biomarkers. 50 μL of serum/plasma was mixed with dithioerythritol, ethanol, and isooctane/chloroform. The organic layer was used for analysis of the fat-soluble vitamins all-trans retinol (A), 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, α-tocopherol (E), γ-tocopherol (E), and phylloquinone (K1) by LC-MS/MS. The remaining aqueous fraction was mixed with ethanol, water, pyridine, and methylchloroformate (in toluene) to derivatize the water-soluble biomarkers. The resulting toluene layer was used for GC-MS/MS analysis of alanine, α-ketoglutarate, asparagine, aspartic acid, cystathionine, total cysteine, glutamic acid, glutamine, glycine, histidine, total homocysteine, isoleucine, kynurenine, leucine, lysine, methionine, methylmalonic acid, ornithine, phenylalanine, proline, sarcosine, serine, threonine, tryptophan, tyrosine, and valine. Isotope-labeled internal standards were used for all analytes. Chromatographic run times for the LC-MS/MS and GC-MS/MS were 4.5 and 11 min, respectively. The limits of detection (LOD) for the low-concentration analytes (25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and phylloquinone) were 25, 17, and 0.33 nM, respectively, while all other analytes demonstrated sensitivity significantly lower than endogenous concentrations. Recoveries ranged from 85.5-109.9% and within- and between-day coefficients of variance (CVs) were 0.7-9.4% and 1.1-17.5%, respectively. This low-volume, high-throughput multianalyte assay is currently in use in our laboratory for quantification of 32 serum/plasma biomarkers in epidemiological studies.
authors.affiliation: Bevital AS , Laboratory Building, Ninth Floor, Jonas Lies veg 87, 5021 Bergen, Norway.
authors.forename: Øivind
authors.initials: Ø
authors.lastname: Midttun
authors.affiliation: Bevital AS , Laboratory Building, Ninth Floor, Jonas Lies veg 87, 5021 Bergen, Norway.
authors.forename: Adrian
authors.initials: A
authors.lastname: McCann
authors.affiliation: Bevital AS , Laboratory Building, Ninth Floor, Jonas Lies veg 87, 5021 Bergen, Norway.
authors.forename: Ove
authors.initials: O
authors.lastname: Aarseth
authors.affiliation: Bevital AS , Laboratory Building, Ninth Floor, Jonas Lies veg 87, 5021 Bergen, Norway.
authors.forename: Marit
authors.initials: M
authors.lastname: Krokeide
authors.affiliation: Bevital AS , Laboratory Building, Ninth Floor, Jonas Lies veg 87, 5021 Bergen, Norway.|Department of Clinical Science, University of Bergen , 5021 Bergen, Norway.
authors.forename: Gry
authors.initials: G
authors.lastname: Kvalheim
authors.affiliation: Bevital AS , Laboratory Building, Ninth Floor, Jonas Lies veg 87, 5021 Bergen, Norway.
authors.forename: Klaus
authors.initials: K
authors.lastname: Meyer
authors.affiliation: Department of Clinical Science, University of Bergen , 5021 Bergen, Norway.|Laboratory of Clinical Biochemistry, Haukeland University Hospital , 5021 Bergen, Norway.
authors.forename: Per M
authors.initials: PM
authors.lastname: Ueland
mesh_terms: D000596:Amino Acids / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D006801:Humans; D057230:Limit of Detection; D018401:Sample Size; D012995:Solubility; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D014801:Vitamin A / Q000097:blood; D014807:Vitamin D / Q000097:blood; D014810:Vitamin E / Q000097:blood; D014815:Vitamins / Q000097:blood*
chemical_list: D000596:Amino Acids; D015415:Biomarkers; D014815:Vitamins; D014801:Vitamin A; D014807:Vitamin D; D014810:Vitamin E