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Analysis of Vitamin D
2
and Vitamin D
3
by LC-MS/MS in Milk
Powders, Infant Formulas, and Adult Nutritionals
Page 1 of 10
Introduction
Vitamin D
3
(cholecalciferol) is generated in the skin of animals when a precursor molecule, 7-dehydrocholesterol,
absorbs light energy. Vitamin D is thus not a true vitamin, because individuals with adequate exposure to sunlight
do not require dietary supplementation. The major biological function of vitamin D is to maintain normal blood
levels of calcium and phosphorus. Vitamin D aids in the absorption of calcium, helping to form and maintain strong
bones preventing rickets in children. There is a high health risk associated with consuming too much vitamin D.
Vitamin D toxicity occurs at relatively low levels causing nausea, poor appetite, constipation, and weakness with
further long-term complications.
Scope
This method is applicable for vitamin D
2
or vitamin D
3
supplemented milk powders, infant formulas, and adult
nutritional products.
Principle
Samples are saponified at high temperature then lipid soluble components are extracted into isooctane. A portion
of the isooctane layer is transferred, washed, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione is added to
derivatise vitamin D to form a high molecular mass, easily ionisable adduct. The vitamin D-adduct is then
re-extracted into a small volume of acetonitrile and analysed by reverse-phase liquid chromatography. Detection is
by triple quadrupole mass spectrometer using multiple reaction monitoring (MRM). Stable isotope labelled (SIL)
vitamin D
2
and vitamin D
3
internal standards are used for quantitation to correct for losses in extraction and any
variation in derivatisation and ionisation efficiencies.
Reference
Gill, B.D.; Zhu, X.; Indyk, H.E. (2015) A rapid method for the determination of vitamin D
3
in milk and infant formula
by liquid chromatography-mass spectrometry.
Journal of AOAC International
, 98, 431
–
435.
Apparatus
x
UHPLC system, Nexera (Shimadzu) or equivalent
x
Triple quadrupole mass spectrometer, 6500 QTrap (ABSciex) or equivalent
x
C
18
2.6
P
m, 2.1 x 50 mm column, Kinetex C
18
core-shell (Phenomenex) or equivalent
x
UV-Spectrophotometer
x
Balance, 4 decimal places
x
Schott bottles: 500 mL, 1 L
x
Boiling tubes: 60 mL
x
Measuring cylinder: 500 mL, 1 L
x
Volumetric flasks: 5 mL, 10 mL, 25 mL, 50 mL, 100 mL
x
Adjustable volume pipettes, 20 μL, 200 μL, mL, 10 mL
x
Disposable centrifuge tubes with screw top lids: 15 mL
x
Disposable plastic containers with screw top lids: 150 mL
x
Centrifuge
x
Glass Pasteur pipettes
x
Water baths: cold 20 °C, hot 70 °C
x
Horizontal shaker
x
Parafilm
x
Eppendorf vials: 2 mL
x
Syringe: 1 mL
x
Syringe filter: PTFE, 0.45 μm, 13 mm
x
HPLC vials, septa, and caps
x
Cryogenic vials, 2 mL
VitD-18 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE