posted on 2022-12-15, 16:06authored byLaura
S. Bailey, Dilip V. Prajapati, Kari B. Basso
Liquid chromatography (LC)–mass
spectrometry (MS)/MS
lipidomic
normalization is generally performed by equalizing pre-extraction
sample materials or via DNA or protein pre-quantitation methods, which
have known measurement inaccuracies. We propose the use of the sulfo-phospho-vanillin
assay (SPVA), a total lipid colorimetric analysis, as a pre-quantitation
method to normalize lipids in lipidomic LC–MS/MS applications.
The assay has been applied to a 300 μL well volume in a 96-well
plate and tested using Avanti total lipid standards of porcine brain
and E. coli. Assay parameters for lipid
sample volume, sulfuric acid, vanillin/phosphoric acid, post-reaction
incubation time, and wavelength are optimized for robust application
to biologically sourced lipid samples. Standard test samples were
prepared using three concentrations covering approximately 100 μg/mL
range. The optimized assay yielded test sample errors less than 10%,
indicating a precise and accurate assay performance. The test samples
were then analyzed by LC–MS/MS and normalized using SPVA pre-quantitation
and pseudo-mass normalization. The detected lipids showed smaller
standard deviations and greater relative concentration differences
compared to the pseudo-mass normalized lipids, showing promise as
a normalization method.