title: Analysis of Vitamin D2 and Vitamin D3 in Fortified Milk Powders and Infant and Nutritional Formulas by Liquid Chromatography-Tandem Mass Spectrometry: Single-Laboratory Validation, First Action 2016.05.
abstract: A method for the determination of vitamin D2 and vitamin D3 in fortified milk powders and infant and adult nutritional formulas is described. Samples are saponified at high temperature and lipid-soluble components are extracted into isooctane. A portion of the isooctane layer is transferred and washed, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione is added to derivatize the vitamin D to form a high-molecular-mass, easily ionizable adduct. The vitamin D adduct is then re-extracted into a small volume of acetonitrile and analyzed by RPLC. Detection is by tandem MS, using multiple reaction monitoring. Stable isotope-labeled vitamin D2 and vitamin D3 internal standards are used for quantitation to correct for losses in extraction and any variation in derivatization and ionization efficiencies. A single-laboratory validation of the method using AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) kit samples was performed and compared with parameters defined according to the vitamin D Standard Method Performance Requirements (SMPR(®)). Linearity was demonstrated over the range specified in the SMPR, with the LOD being estimated at below that required. Method spike recovery (vitamin D2, 97.0-99.2%; and vitamin D3, 96.0-101.0%) and RSDr (vitamin D3, 1.5-5.2%) were evaluated and compared favorably with limits in the vitamin D SMPR. Acceptable bias for vitamin D3 was demonstrated against both the certified value for National Institute of Standards and Technology 1849a Standard Reference material (P(α = 0.05) = 0.25) and AOAC INTERNATIONAL reference method 2002.05 (P(α = 0.05) = 0.09). The method was demonstrated to meet the requirements of the vitamin D SMPR as defined by SPIFAN, and was recently approved for Official First Action status by the AOAC Expert Review Panel on SPIFAN Nutrient Methods.
authors.affiliation: Fonterra Co-operative Group Ltd, PO Box 7, Waitoa 3341, New Zealand.
authors.forename: Brendon D
authors.initials: BD
authors.lastname: Gill
authors.affiliation: Fonterra Research and Development Centre, Dairy Farm Rd, Palmerston North 4442, New Zealand.
authors.forename: Grant A
authors.initials: GA
authors.lastname: Abernethy
authors.affiliation: Fonterra Co-operative Group Ltd, PO Box 7, Waitoa 3341, New Zealand.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Green
authors.affiliation: Fonterra Co-operative Group Ltd, PO Box 7, Waitoa 3341, New Zealand.
authors.forename: Harvey E
authors.initials: HE
authors.lastname: Indyk
mesh_terms: D000818:Animals; D002762:Cholecalciferol / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D004872:Ergocalciferols / Q000032:analysis*; D005527:Food, Fortified / Q000032:analysis*; D006801:Humans; D007223:Infant; D041943:Infant Formula / Q000737:chemistry*; D007753:Laboratories / Q000592:standards; D008892:Milk / Q000737:chemistry*; D011208:Powders; D053719:Tandem Mass Spectrometry
chemical_list: D004872:Ergocalciferols; D011208:Powders; D002762:Cholecalciferol