title: A simultaneous quantitative method for vitamins A, D and E in human serum using liquid chromatography-tandem mass spectrometry.
abstract: Non-classical roles of fat-soluble vitamins (FSVs) in many pathologies including cancer have been identified. There is also evidence of hormonal interactions between two of these vitamins, A and D. As a result of this enhanced clinical association with disease, translational clinical research and laboratory requests for FSV measurement has significantly increased. However there are still gaps in the analytical methods available for the measurement of these vitamins. This study aimed to develop a method for simultaneous quantification of 25-hydroxyvitamin-D2 (25-OHD2), 25-hydroxyvitamin-D3 (25-OHD3) and its 3-epimer (epi-25-OHD3), retinol and α-tocopherol in human serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The procedure was developed and validated across two LC-MS/MS platforms, using commercial calibrators referenced to certified reference materials, controls, and deuterated internal standards. The samples were prepared by liquid-liquid extraction prior to injection and LC separation (using a Pursuit-PFP column) on two Agilent MS/MS systems (6410 and 6490) in electrospray ionisation positive mode with multiple reaction monitoring. Identification and quantification of 25-OHD3 from its 3-epimer as well as 25-OHD2, retinol and α-tocopherol were achieved. The dynamic ranges were 4-160 nmol/L for 25-OHD2 and epi-25-OHD3, 4-200 nmol/L for 25-OHD3, 0.1-4.0μmol/L for retinol and 4-70μmol/L for α-tocopherol with correlation (r(2)) of 0.997-0.998. Based on participation in an external quality assurance program, the overall performance of the simultaneous methods were: imprecision (CV%) and inaccuracy (average bias) 3.0% and 3.2 nmol/L, respectively, for 25-OHD3; 5.0% and 0.04μmol/L, respectively, for retinol; and 4.7% and 0.2μmol/L, respectively, for α-tocopherol. In summary, two simple LC-MS/MS methods were successfully developed and validated for the simultaneous quantification of the three vitamin D metabolites (25-OHD2, 25-OHD3 and 3-epimer of 25-OHD3), vitamin A (retinol) and vitamin E (α-tocopherol) in serum.
authors.affiliation: School of Health and Biomedical Sciences, RMIT University, Victoria, Australia; Security Forces Hospital, Dammam, Saudi Arabia.
authors.forename: Ali A
authors.initials: AA
authors.lastname: Albahrani
authors.affiliation: School of Health and Biomedical Sciences, RMIT University, Victoria, Australia; Invasion and Metastasis Unit, University of Melbourne Department of Surgery, St. Vincent's Hospital, Melbourne, Australia.
authors.forename: Victor
authors.initials: V
authors.lastname: Rotarou
authors.affiliation: School of Health and Biomedical Sciences, RMIT University, Victoria, Australia.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Roche
authors.affiliation: School of Health and Biomedical Sciences, RMIT University, Victoria, Australia; Centre for Hormone Research, Murdoch Children's Research Institute, Victoria, Australia; Australasian Association of Clinical Biochemists, Vitamins Working Party, Australia. Electronic address: ronda.greaves@rmit.edu.au.
authors.forename: Ronda F
authors.initials: RF
authors.lastname: Greaves
mesh_terms: D000328:Adult; D001774:Blood Chemical Analysis / Q000379:methods / Q000592:standards; D002112:Calcifediol / Q000097:blood* / Q000302:isolation & purification; D002853:Chromatography, Liquid; D006801:Humans; D012015:Reference Standards; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry; D024505:Tocopherols / Q000097:blood* / Q000302:isolation & purification; D014801:Vitamin A / Q000097:blood* / Q000302:isolation & purification
chemical_list: D014801:Vitamin A; D002112:Calcifediol; D024505:Tocopherols
keywords: Cholecalciferol; Fat-soluble vitamins; Mass spectrometry; Retinol; Tocopherol