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Introduction
The purpose of this report is to document the single laboratory validation (SLV) validation of an LC-MS/MS
method against SPIFAN matrices for the purposes of selection of the method as AOAC First Action and
recommendation to continue to Multi-Laboratory Validation (MLT).
Method
The LC-MS/MS method validated in this study is as published previously [3] for the analysis vitamin D
3
(cholecalciferol) in milk powder and infant formulas with additional method development to include
vitamin D
2
(ergocalciferol).
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 form the vitamin D adducts. The analytes are then re-extracted into a small volume of acetonitrile
and analysed by reverse-phase chromatography. Detection is by triple quadrupole mass spectrometer
using a selective transition: 572.2
Ѝ
298.0
m/z
for vitamin D
2
and 560.2
Ѝ
298.0
m/z
for vitamin D
3
. Internal
standards, deuterium-labelled vitamin D
2
(26,26,26,27,27,27-d6 ergocalciferol) or vitamin D
3
(26,26,26,27,27,27-d6 cholecalciferol) are used to correct for losses during extraction and variation in
derivatisation and ionisation efficiencies.
Validation
The method was initially developed and validated for the quantitation of vitamin D
3
in infant formulas and
fortified whole milk powders [3]. This study was undertaken to validate modifications to the original
published method to include vitamin D
2
and adult nutritional products within the method scope. The
validation protocol for inclusion in this report is based upon the SPIFAN SLV guidelines described
recently [1].
Samples
A range of infant formulas provided in the SPIFAN kit and in-house vitamin D
3
reference sample were used
in the evaluation of this method (Table 1). This SPIFAN kit was found during method validation to contain
no samples fortified with vitamin D
2
.
VitD-18 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE