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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