AOAC Official Method 2016.05; Analysis of Vitamin D2 and Vitamin D3 in Fortified Milk Powders, Infant Formulas, and Adult/Pediatric Nutritional Formulas


The method for the analysis of vitamin D2 and vitamin D3 performed successfully in ten global laboratories during its MLT study, and was endorsed as AOAC Official Final Action method 2016.05 and ISO Standard 20636:2018 with subsequent adoption by Codex Alimentarius as a Type II Reference Method. In recognition of the application of innovative technology, the critical need for a new reference method internationally, and the difficultly of the analysis, this method was awarded 2017 AOAC Method of the Year.

Method Principle: Samples are saponified at high temperature; then 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 (PTAD) is added to derivatize 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 MS using multiple reaction monitoring (MRM). Stable isotope-labeled (SIL) d6‑vitamin D2 and d6‑vitamin D3 internal standards are used for quantitation to correct for losses in extraction and any variation in derivatization and ionization efficiencies.


Method Link
Reference Style

Journal: Journal of AOAC International
Publisher: AOAC International

AOAC International (2016) Analysis of Vitamin D2 and Vitamin D3 in Fortified Milk Powders, Infant Formulas, and Adult/Pediatric Nutritional Formulas: Gaithersburg MD, United States of America. Official Method 2016.05


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