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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
#3
Title:
Analysis of Vitamin D2 and Vitamin D3 by LC-MS/MS in Milk Powders, Infant Formulas,
and Adult Nutritionals
Author:
Fonterra
Summary of Method:
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 D2 and vitamin D3 internal standards are used for quantitation
to correct for losses in extraction and any variation in derivatisation and ionisation efficiencies.
Method Scope/Applicability:
This method is applicable for vitamin D
2
or vitamin D
3
supplemented milk powders, infant formulas, and adult
nutritional products.
General comments about the method:
This method is a well written method. The procedure is easy to understand. The derivatization procedure is
simple, use stable isotope labled vitamin D2 and D3 as internal standards. The detection is sensitive and
selective. The chromatography is within 5 min.
The saponification was conducted at 70oC for 1 hour
The PTAD dirivatization was conducted for 5 min
SPIFAN samples and NIST SRM 1849a were used in the single lab validation.
Method Clarity:
The method was easy to follow.
Method Safety Concerns:
No particular safety concerns.
Pros/Strengths:
Use of stable isotope labeled Vitamin D2 and D3 as internal standards.
PTAD derivatization improves the method sensitivity and selectivity.
Cons/Weaknesses
Concern about the previtamin determination.
VitD-18 Review Forms