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2
Applicability generally meets SMPR.
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 and contributes to method selectivity
Internal standardization is used to facilitate analyte quantitation and method robustness.
Instrument operation detail (chromatography conditions and mass spectrometer parameter) are provided
in good detail.
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 for both vitamin D2 and D3 meet SMPR. Over-spike recovery
data for vitamin D2, while within SMPR range, are consistently below 100%. More data is needed.
Method suitability requirements, calculation detail, standard curve detail are generally sparse or absent.
Method written for internal use – more detail and are sparse as are reagent details
Linearity is discussed for both vitamin D2 and D3 with sparse details in how the curve is generated. I would
also like to see additional curve quality criteria including residuals to assess concentration-dependent error.
Precision was evaluated but only in SRM 1849a and in overspike experiments. As such the data are
somewhat limited
Method Clarity:
Method is generally well written but appears to be written as an internal document. Suggest more detail
to be added to address consistent application between labs.
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 is absent in common plant
sterols.
Internal standardization is used to facilitate analyte quantitation and method robustness.
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.
Cons/Weaknesses
Does not include an internal standard for vitamin D2 which could reduce robustness for this analyte
relative to SMPR.
Method detail is a thin. The method would benefit from inclusion of more detail around the sample
preparation procedure (eg, saponification overnight), method suitability requirements, typical method
performance (chromatography), calculation of standard curve metrics, to ensure robustness.
Location of deuterium labelling may not be as robust as others available.
pre-vitamin D, D2 or D3, is not addressed as required by SMPR
Applicability to SPIFAN has been demonstrated but additional data is needed to fully evaluate the method.
Need more data – only four matrices presented in addition to SRM 1849a.
VitD-17 Review Forms