Specific estimation of 24,25-dihydroxyvitamin D in plasma by gas chromatography-mass spectrometry.
This paper describes a specific mass-fragmentographic method, involving a stable-isotope-labeled internal standard, for measurement of 24,25-dihydroxyvitamin D in human plasma. Vitamin D metabolites were rapidly extracted from plasma by using Sep-Pak C18 cartridges and separated into fractions on Sep-Pak SIL cartridges. The polar fraction, containing the dihydroxylated metabolites, was further purified by "high-performance" liquid chromatography on Zorbax SIL. The fraction containing 24,25-dihydroxyvitamin D was collected, evaporated, and converted to the 24:25-cyclic n-butyl boronate-3-trimethylsilyl ether derivative before analysis by gas chromatography-mass spectrometry. The intensity of the mass fragment (m/z 449, m/z 455 for the hexadeuterated internal standard) arising from the loss of one of the angular methyls and the 3-silanol group [( M-90-15]+) was monitored. The minimum limit of detection for this method is about 0.1 microgram/L. Inter- and intra-assay reproducibility was acceptable, and analytical recovery of added 24,25-dihydroxyvitamin D3 over the concentration range 1.0 to 5.0 micrograms/L was quantitative. Concentrations of 24,25-dihydroxyvitamin D3 in plasma of 21 apparently healthy volunteers were between 0.55 and 5.39 micrograms/L, higher values being obtained after prolonged exposure to the sun. No 24,25-dihydroxyvitamin D2 could be detected in any plasma sample examined.
D015650:24,25-Dihydroxyvitamin D 3; D002851:Chromatography, High Pressure Liquid; D003903:Deuterium; D004100:Dihydroxycholecalciferols / Q000097:blood* / Q000302:isolation & purification / Q000592:standards; D004872:Ergocalciferols / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007553:Isotope Labeling; D012015:Reference Standards; D012016:Reference Values; D012621:Seasons; D014297:Trimethylsilyl Compounds / Q000302:isolation & purification
D004100:Dihydroxycholecalciferols; D004872:Ergocalciferols; D014297:Trimethylsilyl Compounds; D015650:24,25-Dihydroxyvitamin D 3; D003903:Deuterium; C026032:25,26-dihydroxycholecalciferol