Abstract
A multilaboratory testing study was conducted on AOAC First Action Method 2016.05 "Analysis of Vitamin D2 and Vitamin D3 in Fortified Milk Powders, Infant Formulas, and Adult/Pediatric Nutritional Formulas by Liquid Chromatography-Tandem Mass Spectrometry." Nine laboratories participated in the analysis of duplicate samples of 20 nutritional products. The samples were saponified at high temperature with lipid-soluble components extracted into isooctane; an aliquot was washed and vitamin D derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione to form a high-molecular mass, easily ionizable adduct, extracted into acetonitrile and analyzed by reversed-phase LC-tandem MS. Stable isotope-labeled internal standards were used for quantitation to correct for losses in extraction and variation in derivatization and ionization efficiencies. Acceptable precision as RSD was demonstrated; repeatability ranged from 1.9 to 5.8% RSDr and reproducibility values ranged from 6.4 to 12.7% RSDR, with samples meeting the precision limits specified in the vitamin D Standard Method Performance Requirements and the guidelines recommended for the Horwitz ratio. Method accuracy was assessed using NIST 1849a Standard Reference Material, with a P-value of 0.32, indicating an absence of bias against the certified value. As expected, placebo samples not fortified with vitamin D returned negligible results.
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