The development and evaluation of methods for the screening of steroids using molecular imprinted polymers coupled with comprehensive gas chromatography/mass spectrometry and aptamers coupled with liquid chromatography/mass spectrometry

Abstract

The profiling of hormones is an important requirement in sports dope testing and it is increasingly being used for clinical applications. The screening of samples for the detection of steroids is usually undertaken in urine and these analyses present many challenges. Issues related to the complexity of the matrix, the separation of exogenous from endogenous species and the very low detection limits have placed increasing demands on clinical and International Olympic Commission accredited laboratories. Whilst liquid chromatography (LC) systems provide a highly sensitive detection method for targeted species, gas chromatography, especially when coupled to mass spectrometry, is considered preferable for the screening of a wider range of target and unknown species. Existing chromatography methods are not without their limitations, not least the difficulty in reproducibly derivatising the wide range of steroids that can be exogenously administered, in particular the so called ‘designer steroids’ which deliberately hinder this process. Initially, a pilot study was conducted to investigate whether aptamer-coated magnetic beads could provide a simple, low cost method for the extraction of estradiol from urine, prior to its detection and quantification with mass spectrometry. In reality, the process necessitated the use of additional solvent extraction steps to the standard method applied previously. These additional steps added complexity and were not without issue and the limit of detection was not found to be adequate for the proposed clinical purpose. The project explored the application of comprehensive gas chromatography/mass spectrometry (GC x GC/MS) coupled to bespoke molecular imprinted polymers (MIPS) as an alternative solution to the problem of the screening of multiple steroids in urine. Twelve structurally similar and commercially relevant steroids were used to develop the chromatographic method. Once optimised, these compounds were then extracted from synthetic urine using a MIP templated with testosterone and developed during the project. Significant improvements compared to commercially available C-18 materials were found. All the steroids were identified and detected when spiked at the 10 ng/ml level, as required by World Anti-Doping Agency (WADA) at the time. The method was further validated and then successfully applied to the extraction of designer steroids from synthetic urine, in the presence of the original steroids. Epistane, which has a different structure to testosterone, was not detected indicating the efficacy of the templating process.