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FOR EXPERT REVIEW PANEL USE ONLY
NOT FOR DISTRIBUTION
2
The resulting chromatography is very clean and addresses a primary chromatography concerns observed in
some LC-MS/MS methods previously evaluated. S/N is also significantly improved vs direct measurement
of vitamins D2 and D3.
Method Clarity:
Method is very well written.
Pros/Strengths:
Method is an LC-MS/MS method offering selectivity as a strength.
Diels-Alder reaction is specific to vitamin D2 and D3 (conjugated diene) which are absent in common plant
sterols.
Internal standardization is used to facilitate analyte quantitation and method robustness.
Location of deuterium labelling is robust
Multiple mass transitions are used, one for quantitation and qualitative confirmation.
The PTAD-D adducts produce simplified mass spectra contributing to good S/N.
Method repeatability, accuracy, recovery and LOQ for both vitamin D2 and D3 meet SMPR.
Method performance was evaluated against AOAC 2002.05 for bias. While not an SMPR requirement, the
method compares quite favorably to this commonly used method.
Cons/Weaknesses
There are very few weaknesses in the method as presented.
Vitamin D and pre-D are measured as an aggregate, that is pre-D is not directly measured. Vitamins D2 and
D3, along with their pre-vitamin D isomers, contain the necessary conjugated diene structure to facilitate
the Diels-Alder reaction to form PTAD-D adduct.
Supporting Data
General Comment:
A full set of SLV data are provided and completed per Appendix L; AOAC Recommended Guidelines for
Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Single-Laboratory Validation.
Performance Characteristics:
Analytical Range:
Vitamin D2: 0.04 – 7.3 mcg/100g RTF
Vitamin D3: 0.06 – 11.3 mcg/100g RTF
SMPR: 0.12 – 5.1 mcg/100g RTF
LOQ:
Vitamin D2: 0.016 mcg/100g RTF (≤0.120 mcg/100g RTF)
Vitamin D3: 0.028 mcg/100g RTF (≤0.120 mcg/100g RTF)
VitD-18 Review Forms