title: Determination of vitamins A, D and E in a small volume of human plasma by a high-throughput method based on liquid chromatography/tandem mass spectrometry.
abstract: We have developed an automated high-throughput assay for the determination of vitamin A (retinol), ergocalciferol (25-OH D2), cholecalciferol (25-OH D3) and vitamin E (α-tocopherol) in a small volume of human plasma. Sample preparation involved mixing 50 μL of plasma with 100 μL of ethanol containing isotope-labelled internal standards, followed by mixing with isooctane/chloroform (3:1, 300 μL). The organic phase was evaporated, and the sample reconstituted in 50 μL methanol. The analysis was performed using reversed-phase liquid chromatography with a gradient mobile phase containing water, methanol and ammonium formate. Chromatographic run-time was 5 min, and positive mode electrospray tandem mass spectrometry (MS/MS) was used for detection. The limits of detection were 0.10 μM for all-trans retinol and 3.3 nM for 25-OH D2 and 25-OH D3. Recoveries were 91.9-105.0%, and within- and between-day coefficients of variance (CVs) 2.4-5.3 and 3.1-8.2, respectively. The assay is presently being used in large-scale studies.
authors.affiliation: Bevital A/S, Laboratory Building 9th Floor, N-5021 Bergen, Norway. nkjbm@uib.no
authors.forename: Øivind
authors.initials: Ø
authors.lastname: Midttun
authors.forename: Per M
authors.initials: PM
authors.lastname: Ueland
mesh_terms: D000328:Adult; D000368:Aged; D000704:Analysis of Variance; D002762:Cholecalciferol / Q000097:blood*; D056148:Chromatography, Reverse-Phase / Q000379:methods; D004872:Ergocalciferols / Q000097:blood*; D005260:Female; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D016014:Linear Models; D008297:Male; D008875:Middle Aged; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods; D014801:Vitamin A / Q000097:blood*; D024502:alpha-Tocopherol / Q000097:blood*
chemical_list: D004872:Ergocalciferols; D014801:Vitamin A; D002762:Cholecalciferol; D024502:alpha-Tocopherol