SPECIFIC MASS FRAGMENTOGRAPHIC ASSAY FOR 25,26-DIHYDROXYVITAMIN-D IN HUMAN-PLASMA USING A DEUTERATED INTERNAL STANDARD

被引:17
作者
COLDWELL, RD
TRAFFORD, DJH
MAKIN, HLJ
VARLEY, MJ
KIRK, DN
机构
[1] UNIV LONDON LONDON HOSP, COLL MED, DEPT CHEM PATHOL, STEROID LAB, TURNER ST, LONDON E1 2AD, ENGLAND
[2] UNIV LONDON WESTFIELD COLL, DEPT CHEM, LONDON NW3 7ST, ENGLAND
来源
JOURNAL OF CHROMATOGRAPHY | 1985年 / 338卷 / 02期
关键词
D O I
10.1016/0378-4347(85)80100-6
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
A specific mass fragmentographic assay for the measurement of 25,26-dihydroxyvitamin D3 [25,26(OH)2D3] in human plasma, using a stable isotope labeled internal standard {[26,27-2H5]25,26(OH)2D3} was described. Plasma samples (5 ml) were extracted with acetonitrile and applied to a C18 Sep-Pak cartridge, from which the vitamin D metabolites were eluted with methanol. The metabolites were then applied to a Sep-Pak SIL cartridge and 3 fractions were collected. The most polar fraction, containing the polyhydroxylated metabolites, was further purified by high-performance liquid chromatography on Zorbax SIL. The eluent containing 25,26(OH)2D3 was collected and the 25,26-n-butylboronate cyclic ester 3-trimethylsilyl ether derivative was formed. Gas chromatography-mass spectrometry was carried out, monitoring the intensities of the ions at m/z 449 and m/z 454 (for the internal standard). These ions represent the loss of a methyl group and the 3-silanol group, (M-90-15)+. The minimum limit of detection of the assay was estimated to be .apprx. 0.05 .mu.g/l. Inter-assay (3.7%) and intra-assay (8.0%) precision was acceptable and added 25,26(OH)2D3, over the concentration range 0.5-1.5 .mu.g/l, was recovered quantitatively. The plasma 25,26(OH)2D3 level was estimated in 26 healthy volunteers and ranged from 0.05-1.30 .mu.g/l, with a mean value of 0.54 .mu.g/m.
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页码:289 / 302
页数:14
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