Vitamin D3 in plasma: quantitation by mass fragmentography

Combined gas chromatography-mass spectrometry with multiple-ion detection is used to quantitate vitamin D3 in plasma samples. Extensive cleanup of the plasma extract prior to analysis is required before mass fragmentographic analysis is possible, i.e., solvent extraction, digitonin precipitation, Lipidex column chromatography, and derivatization. Low resolution mass fragmentography is performed by monitoring simultaneously the molecular ions of the heptafluorobutyric esters of the all-trans isomer of vitamin D3 and dihydrotachysterol2, which is used as an internal standard. This new method uses 5 ml of plasma and it is specific and sensitive to 600 pg of vitamin D3. This assay shows that in plasma of normal adult subjects there is a vitamin D3 concentration of 5 to 11 ng/ml, lower than those values reported for biological and competitive protein binding assays. Specifically the method described offers potential as an eventual reference method.