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 D-3 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 D-3 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 D-3. The analysis of vitamin D-3 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, alpha = 0.05) and a reference LC-UV analytical method (P = 0.09, alpha = 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.