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FOR EXPERT REVIEW PANEL USE ONLY
NOT FOR DISTRIBUTION
1
Infant Formula and Adult Nutrition - Review Form
Evaluation of Method VitD-18
#1
Title:
Analysis of Vitamin D2 and Vitamin D3 by LC-MS/MS in Milk Powders, Infant Formulas, and Adult
Nutritionals
Author:
Brendon Gill/Fonterra
Summary of Method:
Method Principle
Liquid samples are prepared directly with powder samples reconstituted per SMPR guidelines prior to
sampling. Pyrogallol is added to protect against oxidation and hexa-deuterated analogues for vitamin D2
and D3 are added as internal standards prior to sample preparation. Samples are prepared by alkaline
saponification at 70C for 1 hr. Lipid-soluble components including vitamins D2 and D3 are extracted into
isooctane. A portion of the isooctane extraction layer is transferred to a second vial and washed.
Following centrifugation, an aliquot of the isooctane layer is transferred to a separate centrifuge tube to
which 75-mcL of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) solution is added. The Diels-Alder reaction is
completed in 5 minutes at ambient temperature producing high molecular mass, easily ionisable Vitamin D-
PTAD adducts. The vitamin D-adducts are re-extracted into a small volume of acetonitrile and analysed by
reversed-phase liquid chromatography. Detection is by MS/MS using MRM.
Instrument:
Chromatographic separation for the SLV study was completed using a Shimadzu UHPLC system equipped
with a 2.1 mm x 50 mm x 2.6micron Phenomenex Kinetex C18 column.
Mass Spectral analysis was completed using ABSciex 6500 QTrap triple quadrupole mass spectrometer with
Electrospray (+ion ) ionization. Two mass transitions, one for quantitation and a second for qualitative
verification, are used for each analyte, vitamin D
2
, D
3
and each IS compound.
Method Scope/Applicability:
The method submitted includes single-laboratory validation data for all SPIFAN materials.
Applicability is consistent with SMPR definition for Infant Formula and Adult Nutritional products; Vitamins
D
2
and D
3
.
General comments about the method:
The method is very well written and thorough.
Hexa-deuterated internal standards are used for both vitamin D2 and D3 which contribute significantly to
the robustness of the method.
VitD-18 Review Forms