The development of 19F NMR as a tool for analysing steroid degradation in active urine samples

Abstract

Abstract
The detection of prohibited performance-enhancing drugs in sports is often carried out using urine samples. The reason for this is that urine samples can be collected under non-sterile conditions and do not require the presence of a sanctioned medical officer. Improper storage of the urine samples from athletes can lead to microbial contamination, which can cause changes in the steroids profile, leading to false positive or false negative results for a particular athlete. To address this problem, a new analytical method was proposed that employs a fluorinated steroid as an internal standard and fluorine-19 nuclear magnetic resonance spectroscopy (19F-NMR) spectroscopy to identify both microbial and thermally-induced changes in the urine samples.
In Chapter 2, synthesis of fluorinated steroids was carried out using method that involve the reaction of Selectfluor® with enolates/enols of steroids. A range of fluorinated steroids was prepared (2 novel F-steroids) in moderate yields and varying diastereoselectivities. Several synthesised steroids were recrystallized and crystals suitable for X-ray were obtained.
In Chapter 3, selected fluorinated steroids were incubated with microorganisms such as Escherichia coli, Bacillus subtilis, Bacillus megaterium and Streptomyces griseus. Fluorinated steroids were transformed to various oxidised metabolites upon incubation with Streptomyces griseus. Escherichia coli did not produce any metabolites due to lack of cytochrome P450 enzymes. Incubation of Bacillus subtilis and Bacillus megaterium was not successful and therefore metabolites were not detected.
In Chapter 4, hydroxy steroids were reacted with pentafluoropyridine (PFP) to form perfluoropyridine ethers in good yield. Several novel steroids were synthesised and the structures of 4 perfluoropyridine ethers were confirmed for the first time by X-ray structure. It was found that the hydroxy steroid PFP adducts have very similar 19F NMR spectra however they can be distinguished using this technique. This novel derivatisation technique could be potentially used for identification of hydroxy steroids in biological material by 19F NMR.