title: Simultaneous quantitative analysis of eight vitamin D analogues in milk using liquid chromatography-tandem mass spectrometry.
abstract: 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.
authors.affiliation: School of Pharmacy, The University of Queensland, Queensland 4072, Australia.
authors.forename: Fabio P
authors.initials: FP
authors.lastname: Gomes
authors.affiliation: School of Pharmacy, The University of Queensland, Queensland 4072, Australia.
authors.forename: P Nicholas
authors.initials: PN
authors.lastname: Shaw
authors.affiliation: School of Pharmacy, The University of Queensland, Queensland 4072, Australia.
authors.forename: Karen
authors.initials: K
authors.lastname: Whitfield
authors.affiliation: School of Pharmacy, The University of Queensland, Queensland 4072, Australia. Electronic address: a.hewavitharana@pharmacy.uq.edu.au.
authors.forename: Amitha K
authors.initials: AK
authors.lastname: Hewavitharana
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D006041:Goats; D006801:Humans; D057230:Limit of Detection; D008892:Milk / Q000737:chemistry*; D012756:Sheep; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000032:analysis*
chemical_list: D014807:Vitamin D
keywords: Derivatisation; Liquid chromatography–mass spectrometry; Milk; Protein precipitation; Saponification; Vitamin D analogues; Vitamin D metabolites