Work performed on the MET-A-FOR project has assessed the ability of metabolism studies using in vitro incubation methodologies to rapidly predict patterns of in vivo metabolism of SARM compounds. The aim of in vitro approaches is to accurately reflect the metabolite profile of illegally administered drugs in the absence of extensive elimination and pharmacokinetic studies and thereby facilitate rapid development of mass spectrometry based methods to more quickly detect new and emerging designer drug abuse as evidence arises that they are being used. Work on the project has prioritised key classes/types of SARM compounds (both from reputable sources but also unverifiable (e.g. web-based) vendors) based on their current availability and on the likely level of risk of compounds to actually be used illegally in sport and food animals – work has also examined new SARM compounds emerging from clinical focused pharmaceutical drug development. Liver/microsomal preparations/fractions from different species have been used in conjunction with LC-MS analysis to profile and identify phase 1 and 2 metabolites of SARMs to help predict and compare to actual in vivo drug metabolism. Various methods (LC-MS and 2D HR-NMR) were applied to the profiling of metabolomic responses to SARMs in urine/plasma (and tissues) and to identify key signatures (metabolites) of SARM compound administration. Proteomic analyses of target tissue (muscle/liver) responses to SARMs has additionally revealed new information on the physiological affects of these compounds in vivo.
The project has focused on the extensive development and optimisation of sample extraction, liquid chromatography and mass spectrometry analysis that can be applied to the analytical detection of SARMs of interest in various test matrices across a range of relevant target species (e.g. equine, bovine, canine, murine, human), with a particular emphasis on urine/blood of bovine/equine origin. Fit-for-purpose sample extraction and UHPLC-MS/MS-based analytical methods have thereby been developed within the project for the screening of 15 SARM compounds under commercially offered or regulatory control testing environments. The developed methods have been validated in accordance with relevant legislation such as the EU Commission Decision 2002/657/EC criteria and Community Reference Laboratories Residues (CRLs) guidelines – with an analytical LC-MS run-time of between 12-14 minutes, a total of 50 samples can be analysed in a single day using these high-throughput assays.