Review Team (February 2016)

VitD-18 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Evaluation of Method Performance vs. SMPR requirements

AOAC SMPR: 2011.04

Method Reference # Method Title

Analysis of Vitamin D 2 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. and Vitamin D 3

Method Principle

Weighting factor for parameter

Suitability Ranking (1-3-5) (select from list, 5 = best)

SMPR Requirement

Method Performance (1)

Parameter

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.

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

SPIFAN matrices

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.

Method applicable to vitamin D 2

(includes pre-vitamin D 2

) and vitamin

All analytes defined in the applicability statement are measured.

(inlcudes pre-vitamin D 2 ).

D 3

1

0.04–7.3 μg hg -1 RTF 0.06–11.3 μg hg -1 RTF

Vitamin D2 Vitamin D3 Vitamin D2 Vitamin D3 Vitamin D2 Vitamin D3 Vitamin D2 Vitamin D3 Vitamin D2 Vitamin D3 Vitamin D2 Vitamin D3 Vitamin D2 Vitamin D3 Vitamin D2 Vitamin D3

0.12–5.1 μg hg -1 RTF

1

Analytical Range

0.013 μg hg -1 RTF 0.018 μg hg -1 RTF 0.016 μg hg -1 RTF 0.028 μg hg -1 RTF

≤0.020 μg hg -1 RTF

Limit of detection (LOD)

1

≤0.120 μg hg -1 RTF

Limit of quantification (LOQ)

2

97–101% (mean = 98%) 94–101% (mean = 98%) no CRM value available

Accuracy as Recovery

2

90–110%

Accuracy as Bias vs CRM

3

No criteria in SMPR

no bias against NIST 1849a (p = 0.25, α =0.05)

≤ 11% ≤ 11%

Vitamin D 2

product not available so no repeability data

Repeatability (RSD r )

3

1.5–5.2% (mean = 3.0%) 4.4–7.4% (mean = 5.5%) 3.1–7.9% (mean = 5.0%) Not applicable for SLV Not applicable for SLV

Intermediate Reproducibility (RSD iR )

1

No criteria in SMPR

≤ 15% ≤ 15%

Reproducibility (RSD R )

1

ADDITIONAL EVALUATION PARAMETERS

Adequate proof of performance via system suitability

Highly selective LC-MS/MS used for analysis, single peak only for analyte typically seen in sample SRM chromatogram.

1

Did Method Author Consider ERP’s Method Specific Recommendations : Feedback from Users of the Method since being awarded First Action Official Methods Status

Not applicable

1

Not applicable

1

Bias against established method

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.

1

Analytical equipment

1

No unique proprietary equipment/accessories are required. None Method does not require any special safety precautions.

Proprietary equipment Laboratory safety Other Considerations

1 1 1

0

Overall Score

Decision by ERP

Recommendation of ERP 2 years after First Action Status

Move to Final Action/repeal/remove/expand 2 year term

Notes:

(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.

SPIFAN ERP Checklist v 1.6

27.06.2013