Simultaneous quantitative analysis of eight vitamin D analogues in milk using liquid chromatography–tandem mass spectrometry

Milk is an important source of nutrients for various risk populations, including infants. The accurate measurement of vitamin D in milk is necessary to provide adequate supplementation advice for risk groups and to monitor regulatory compliance. Currently used liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods are capable of measuring only four analogues of vitamin D in unfortified milk. We report here an accurate quantitative analytical method for eight analogues of vitamin D: Vitamin D₂ and D₃ (D₂ and D₃), 25-hydroxy D₂ and D₃, 24,25-dihydroxy D₂ and D₃, and 1,25-dihydroxyD₂ and D₃. In this study, we compared saponification and protein precipitation for the extraction of vitamin D from milk and found the latter to be more effective. We also optimised the pre-column derivatisation using 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD), to achieve the highest sensitivity and accuracy for all major vitamin D forms in milk. Chromatography was optimised to reduce matrix effects such as ion-suppression, and the matrix effects were eliminated using co-eluting stable isotope labelled internal standards for the calibration of each analogue. The analogues, 25-hydroxyD₃ (25(OH)D₃) and its epimer (3-epi-25(OH)D₃) were chromatographically resolved, to prevent over-estimation of 25(OH)D₃. The method was validated and subsequently applied for the measurement of total vitamin D levels in human, cow, mare, goat and sheep milk samples. The detection limits, repeatability standard deviations, and recovery ranges were from 0.2 to 0.4 femtomols, 6.30–13.5%, and 88.2–105%, respectively.