142 / 159
FOR EXPERT REVIEW PANEL USE ONLY
NOT FOR DISTRIBUTION
1
Expert Review Panel for Infant Formula and Adult Nutrition
Evaluation of Method:
VitD-17
#3
Title:
Determination of Vitamin D in Milk Products by UPLC-MS-MS with 4-Phenyl-1,2,4-triazoline-3,5-dione
(PTAD) Derivatization
Author:
CDC-Shanghai
Summary of Method:
Powder samples were reconstituted and diluted in pyrogallic acid/ethanol, and KOH solutions. After
saponification, liquid-liquid extraction was performed, followed by derivatization using PTAD. The
derivatized sample solution was analyzed by UPLC-MS-MS analysis.
Method Scope/Applicability:
Infant Formula and Adult/Pediatric Formulas
General comments about the method:
The method describes the analysis of vitamin D2 and vitamin D3 using a PTAD derivatisation technique
followed by UPLC-MS/MS. The use of PTAD derivatisation adds an extra degree of specificity to the analysis
and provides clean chromatograms free from isobaric interferences.
Meets SMPR applicability statement by measuring both vitamin D and pre-vitamin D as single result.
Method Clarity:
Clear directions for operation of the method, instruments. Clear equations describing how the standards
and samples are quantitated is required.
Method Safety Concerns:
None, beyond standard laboratory safety
Pros/Strengths:
VitD-PTAD analysis provides high molecular weight analyte which is free from matrix interferences that can
cause problems in typical vitamin D methods. Additionally, the PTAD-adduct ionizes readily allowing for
increase sensitivity.
Cons/Weaknesses
The choice of internal standard is not ideal given the site of deuteration coincides with the site of PTAD
derivatisation. This has been shown in a recent publication to cause problems with accurate analysis.
No SIL vitamin D2 std is used and so questionable about whether accurate quantitation for this analyte.
Supporting Data
The SPIFAN kit was not used for validation.
VitD-17 Review Forms