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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