The estimation of vitamin D and some metabolites in human plasma by mass fragmentography

A mass fragmentographic assay for vitamin D3, 25-hydroxyvitamin D3, 25-hydroxyvitamin D2 and 24,25-dihydroxyvitamin D3 is described. After extraction of plasma with methanol and dichloromethane, Lipidex 5000 chromatography was used to separate the plasma extract into three fractions (a vitamin D fraction, a 25-hydroxyvitamin D fraction and a 24,25-dihydroxyvitamin D fraction). Cholesterol was removed from the vitamin D fractio by thin-layer chromatography. :After addition of vitamin D2 to each fraction, isotachysterol trimethylsilyl ethers were formed. Recoveries prior to gas chromatography-mass spectrometry were assessed by the use of tritiated standards added to the plasma before extraction. Using this procedure, normal ranges were established (vitamin D3: 3--17.7, 25-hydroxyvitamin D3: 5.9--35.2, 25-hydroxyvitamin D2: 0.6---1.0 and 24,25-dihydroxyvitamin D3: 0.6--2.9 micrograms/l).