Brendon Gill PhD MSc(hons) GradDipT FAOACI


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.

Brendon D. Gill, Grant A. Abernethy, Rebecca J . Green, Harvey E. Indyk

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.


Gill et al. (2016) J. AOAC. Int., 99, In Press.Journal Link
Gill et al. (2016) J. AOAC Int., 99, In Press.Article PDF
Reference Style

Journal: Journal of AOAC International
Publisher: AOAC International

Gill, B.D., Abernethy, G.A., Green, R.J., & Indyk, H.E. (2016) J. AOAC Int. 99, 1321‑1330
doi: 10.5740/jaoacint.16-0160


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