A specific assay of vitamin D-3 in human serum

Abstract

A specific mass fragmentographic method for determination of free vitamin D-3 in biological fluids has been developed. The method involves addition of vitamin D-2 to the sample, followed by freeze-drying and extraction with Chloroform/methanol. The amount of vitamin D-2 added should be more than a hundred-fold the amount of vitamin D-3 expected in the sample. The vitamin D and previtamin D present in the extract are heat-equilibrated with each other and then subjected to thin-layer chromatography. Under the Chromatographie conditions employed, there is an only partial separation between vitamin D-2 and vitamin D-3. The material in the appropriate Chromatographie zone is converted into trimethylsilyl ether and analyzed by gas chromatography-mass spectrometry with use of a multiple ion detector. The amount of vitamin D-3 is determined from the ratio between the recordings at m/e 325 and m/e 337. The two ions used correspond to the M — 131 peak in the mass spectrum of trimethylsilyl ether of vitamin D-3 and vitamin D-2, respectively. The method was applied to the determination of vitamin D-3 in human serum and the accuracy of this application was ascertained by different recovery experiments. Considerably lower concentration of vitamin D-3 in serum was found with the present technique than with previous techniques. Thus analysis of serum from 19 healthy men and women gave values ranging from 0 to 6.3 ng/ml (mean about 2 ng/ml). There was little or no increase in concentration after shortterm irradiation of part of body area with UV light. Psoriatic patients subjected to treatment with UV light for 3–5 weeks, however, had considerably higher serum level of vitamin D-3 than normal. Administration of vitamin D-3 per os to one subject gave a transient increase in concentration of vitamin D-3 in serum with a peak value about 4 h after the administration.