A rapid method for the determination of vitamin D3 in milk and infant formula by liquid chromatography/tandem mass spectrometry

A rapid method for the determination of vitamin D3 applicable to milk and infant formula products is described. Samples are saponified at high temperature, and lipophilic components are extracted into isooctane in a single tube. Vitamin D3 is derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) to form a Diels-Alder adduct, which is re-extracted into a small volume of acetonitrile and analyzed by UHPLC-MS/MS with quantification accomplished by an internal standard technique utilizing deuterium-labeled vitamin D3. The analysis of vitamin D3 as the PTAD adduct offers a significant increase in sensitivity and selectivity, allowing for rapid sample preparation and short chromatographic run times. The method was shown to be accurate, with spike recoveries of 94.7-104.7% and no statistical bias against both a certified reference material (P = 0.37, α = 0.05) and a reference LC-UV analytical method (P = 0.09, α = 0.05). Acceptable precision was confirmed with a repeatability RSD of 1.4-4.5% and corresponding HorRat values of 0.1-0.2. This high-throughput method is ideal for routine compliance testing, with more than 50 samples/day achievable by a single analyst.