Specific mass fragmentographic assay for 25,26-dihydroxyvitamin D in human plasma using a deuterated internal standard

A specific mass fragmentographic assay for the measurement of 25,26-dihydroxyvitamin D 3 [25,26(OH) 2D 3] in human plasma, using a stable isotope labelled internal standard {[26,27- 2H 5]25,26(OH) 2D 3}, is described. Plasma samples (5 ml) were extracted with acetonitrile and applied to a C 18 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 three 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) 2D 3 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 approximately 0.05 μg/l. Inter-assay (3.7%) and intra-assay (8.0%) precision was acceptable and added 25,26(OH) 2D 3, over the concentration range 0.5–1.5 μg/l, was recovered quantitatively. The plasma 25,26(OH) 2D 3 level was estimated in 26 healthy volunteers and ranged from 0.05 to 1.30 μg/l, with a mean value of 0.54 μg/l.