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FOR EXPERT REVIEW PANEL USE ONLY
NOT FOR DISTRIBUTION
1
Expert Review Panel for Infant Formula and Adult Nutrition
Evaluation of Method VITD-18; 2016.05
Title: Analysis of Vitamin D2 and Vitamin D3 by LC-MS/MS in Infant Formulas and Nutritional
Products
Author:
Brendon Gill (Fonterra)
Summary of Method:
The method presented, AOAC 206.05 (VitD-10) details the analysis of vitamins D
2
and D
3
by Liquid
Chromatography and tandem mass spectrometry.
Samples are prepared by alkaline saponification at high temperature. Pyrogallol is added to
samples prior to saponification protect against oxidative loss of vitamin D during sample
preparation and extraction. Hexa-deuterated analogs for both vitamin D
2
and D
3
are added also
prior to saponification as internal standards. Following saponification, 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 (PTAD) is added to derivatize vitamin D to form a
high molecular mass, easily ionizable adduct. The vitamin D-adducts are tune extracted into a
small volume of acetonitrile. Analysis is by reversed-phase liquid chromatography and detection is
by tandem mass spectrometry using multiple-reaction monitoring (MRM). Stable isotope labelled
d
6
-vitamin D
2
and
d
6
-vitamin D
3
internal standards are used for quantitation to correct for losses in
extraction and any variation in derivatization and ionization efficiencies.
Method Scope/Applicability:
Applicability of the method is consistent with the SPIFAN SMPR for vitamin D.
General comments about the method / Method Clarity:
The MLT report and associated data submitted by the Study Director is well written with data
compared consistently to SMPR parameters. The report provided by the study director includes
both comments received from participating laboratories as well as responses back to the
laboratory. One comment of particular note referenced a request for higher centrifuge speeds to
improve phase separation.