Gas chromatography—mass spectrometry in the investigation of on-column dehydration of steroid hormones during gas—liquid chromatography

Abstract Some underivatized steroids when injected onto conventional packed columns for gas—liquid chromatography underwent varying degrees of dehydration. This problem was traced to the presence of small pieces of broken glass on the top of the column at the point of injection. This observation provoked an examination of the effect of pre-column dehydration on a number of different types of steroids. Powdered aluminium was placed in the injection liner of a Hewlett—Packard gas chromatograph fitted with an HP1 capillary column connected to a mass selective detector, and injections were made using a new high temperature septumless injection system at temperatures between 200 and 400°C. 5α-androstan-3α-ol, a simple monofunctional C19 steroid chosen as a model to establish optimum conditions, underwent dehydration at injection temperatures greater than 250°C and the product reached a maximum at 400°C when no unchanged steroid was present. Monohydroxylated androgens and oestrogens underwent dehydration at 400°C producing products whose mass spectra indicated they were monenes, although the position of the double bond could not be assigned. Polyfunctional androgens and oestrogens and corticosteroids underwent complex changes producing a number of products some of whose structures could not be determined. The dehydration products had the advantage that they had relatively intense high mass ions and for suitable steroids this might provide enhanced sensitivity of detection during mass fragmentography. In such cases dehydration was reproducible and straight line standard curves were obtained. C27 and C28 secosteroids (vitamins D 2 and D 3 ) and some of their metabolises (e.g. 25-hydroxyvitamin D) underwent efficient dehydration, again producing products with intense molecular ions. In the case of 24,25-dihydroxyvitamin D 3 and 25,26- dihydroxyvitamin D 3 , dehydration produced different products which were easily resolved in the chromatographic system used. Dehydration of vitamin D metabolites eliminates the need for derivatization and gives enhanced sensitivity of measurement by gas chromatography—mass spectrometry.