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Method Reference #
Method Title
Method Principle
Parameter
Method Performance
(1)
Weighting
factor for
parameter
Suitability Ranking (1-3-5)
(select from list, 5 = best)
SPIFAN matrices
SPIFAN Kit utlised for SLV study. Sample list includes: NIST
SRM1849a, Infant Formula Powder Partially Hydrolysed Milk-Based,
Infant Formula Powder Partially Hydrolysed Soy-Based, Toddler
Formula Powder Milk-Based, Infant Formula Powder Milk-Based,
Adult Nutritional Powder Low Fat, Child Formula Powder, Infant
Elemental Powder, Infant Formula Powder FOS/GOS-Based, Infant
Formula Powder Milk-Based, Infant Formula Powder Soy-Based,
Infant Formula RTF Milk-Based, Adult Nutritional RTF High Protein,
Adult Nutritional RTF High Fat
1
All analytes defined in the
applicability statement are
measured.
Method applicable to vitamin D
2
(includes pre-vitamin D
2
) and vitamin
D
3
(inlcudes pre-vitamin D
2
).
1
Vitamin D2
0.04–7.3 μg hg
-1
RTF
Vitamin D3
0.06–11.3 μg hg
-1
RTF
Vitamin D2
0.013 μg hg
-1
RTF
Vitamin D3
0.018 μg hg
-1
RTF
Vitamin D2
0.016 μg hg
-1
RTF
Vitamin D3
0.028 μg hg
-1
RTF
Vitamin D2
97–101% (mean = 98%)
Vitamin D3
94–101% (mean = 98%)
Vitamin D2
no CRM value available
Vitamin D3
no bias against NIST 1849a (p = 0.25, α =0.05)
Vitamin D2
≤ 11%
Vitamin D
2
product not available so no repeability data
Vitamin D3
≤ 11%
1.5–5.2% (mean = 3.0%)
Vitamin D2
4.4–7.4% (mean = 5.5%)
Vitamin D3
3.1–7.9% (mean = 5.0%)
Vitamin D2
≤ 15%
Not applicable for SLV
Vitamin D3
≤ 15%
Not applicable for SLV
1
1
1
1
1
1
1
1
≤0.120 μg hg
-1
RTF
90–110%
No criteria in SMPR
No criteria in SMPR
Analysis of Vitamin D
2
and Vitamin D
3
by LC MS/MS in Supplemented Milk Powders, Infant Formulas, and Adult Nutritionals
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. The analyte 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). Deuterium-labelled 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.
Applicable to all forms of infant, adult, and/or pediatric formula (powders, ready-to-
feed liquids, and liquid concentrates).
Any combination of milk, soy, rice, whey, hydrolyzed protein, starch, and amino
acids, with and without intact protein.
Vitamin D2 is defined as ergocalciferol (CAS 8017-28-5) and its previtamin isomer;
and vitamin D3 is defined as cholecalciferol (CAS 67-97-0) and its previtamin
isomer.
Move to Final Action/repeal/remove/expand 2 year term
0
No unique proprietary equipment/accessories are required.
None
Method does not require any special safety precautions.
Overall Score
Decision by ERP
Bias against established method
Analytical equipment
1
Adequate proof of performance via system suitability
Bias evaluated against AOAC 2002.05 and no significant bias was found (p = 0.09, α =0.05)
Analytical equipment is commonly available in most labs. While LC-MS/MS instrument are not as common as LC-UV, many labs
have this instrumentation.
3
1
2
2
3
AOAC SMPR: 2011.04
Limit of detection (LOD)
1
(1)
Concentrations apply to (1) "ready to feed liquids" "as-is"; (2) reconstituted powders (25 g into 200 g water); and (3) liquid concentrates diluted 1:1 by weight.
Did Method Author Consider ERP’s Method Specific
Recommendations
:
Not applicable
Proprietary equipment
Notes:
Recommendation of ERP
2 years after First Action Status
Laboratory safety
Other Considerations
Accuracy as Recovery
Accuracy as Bias vs CRM
0.12–5.1 μg hg
-1
RTF
≤0.020 μg hg
-1
RTF
Evaluation of Method Performance vs. SMPR requirements
Feedback from Users of the Method since being awarded
First Action
Official Methods
Status
Repeatability (RSD
r
)
Reproducibility (RSD
R
)
Highly selective LC-MS/MS used for analysis, single peak only for analyte typically seen in sample SRM chromatogram.
ADDITIONAL EVALUATION PARAMETERS
Not applicable
Intermediate Reproducibility
(RSD
iR
)
Limit of quantification (LOQ)
SMPR Requirement
Analytical Range
1
SPIFAN ERP Checklist v 1.6
27.06.2013
VitD-18 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE