Facile synthesis of [16,16,17-2H3]-testosterone, -epitestosterone and their glucuronides and sulfates.

Abstract

The analysis of trace components in complex biological matrices requires the use of reliable internal standards. For the gas chromatography/mass spectrometry (GC/MS) and high performance liquid chromatography/mass spectrometry (HPLC/MS) analyses, the stable isotope-labelled analogues of the analyte molecules are the most appropriate internal standards. In this work high-yield synthetic procedures for stably labelled and isotopically pure [16,16,17-2H3]-testosterone and- epitestosterone are reported. Synthetic methodologies for the glucuronidation and sulfation were established with the commercially available epitestosterone. Structure characterization of 4-androsten-17 alpha-ol-3-one methyl-2',3',4'-tri-O-acetyl-beta-D-glucuronate was made by two-dimensional nuclear magnetic resonance (COSY). Subsequently glucuronidation of [16,16,17-2H3]-testosterone and sulfation of [16,16,17-2H3]-epitestosterone were carried out at greater than 60% yield. However, the yield from the glucuronidation of epitestosterone was not as high. Electrospray mass spectrometry of four conjugates: testosterone sulfate, epitestosterone sulfate, testosterone glucuronide and epitestosterone glucuronide was carried out in the positive ion mode at a number of orifice voltages (50-95 V). Studies of the collisionally induced dissociation at both the interface and in the collision cell (MSMS) confirmed that the glycosidic bond of epitestosterone glucuronide was more labile than that of testosterone glucuronide. Use of the deuterated internal standards is reported to demonstrate the direct analysis of the steroid conjugates by HPLC/MS.