Measurement of Vitamin D and its Metabolites by Gas Chromatography-Mass Spectrometry

Abstract

Gas-liquid chromatography (GC) has been very successfully applied to the analysis of a number of different groups of biologically important compounds. In particular, the analysis of the mUltiplicity of metabolites of secreted steroid hormones found in human urine has been greatly simpli-fied by the introduction of GC (1). Using conventional packed columns (2) and wall-coated capillary columns (3), 'profiles' of varying degrees of complexity have been produced. Plasma steroids have also been analysed by GC using electron capture detection (4) and more recently using gas chromatography-mass spectrometry (GC-MS, 5). Vitamin D and its hydroxylated metabolites are secosteroids and as such might be expected to behave like steroid hormones. They should therefore be susceptible to analysis by GC using similar methods to those already used for steroids. Vitamin D and its metabolites, like the steroids, are relatively non-volatile compounds and require GC oven temperatures of the order of 200-350 0 for separat ion. There are many parallels between the problems associated with the GC analysis of steroid hormones and vitamin D, which is perhaps not surprising when one considers their common chemical origin. Both groups of steroids can be analysed by GC without formation of derivatives although considerable peak broadening occurs, probably indicat ing adsorpt ion into the 'inert' support of the column. The choice of suitable derivatives can enhance separation and improve peak shape. C2l-corticosteroids containing a l7-hydroxyl group are thermally labile and unless suitable derivatives are formed prior to GC, the high oven temperatures give rise to side chain cleavage, producing C19 steroids. In a similar fashion, vitamin D and its metabolites undergo thermal changes during GC. Injection of both derivatised and underivatised vitamin D onto a GC column gives rise to closure of the B ring, producing two isomers, pyro-(9acH3' lOaH) and isopyro-(9SCH3, 10SH) calciferols. Kumar, R. red.J, VITAMIN D. © 1984. Martinus Nijhoff Publishing, Boston/The HaguelDordrechtlLancaster. All rights reseJved.