From targeted methods to metabolomics based strategies to screen for growth promoters misuse in horseracing and livestock: A review

Despite banned for public health, societal and economic reasons, and therefore strictly regulated, growth promoters can still be used in farm animals to increase production yields or in racing animals to enhance their performance. The introduction of specific regulations has led to the establishment of control systems, in particular through the development of analytical methods to detect doping practices. Although high-performance analytical strategies, most often based on targeted mass spectrometry approaches, are implemented in testing laboratories, some challenges still remain, particularly those specifically implemented to circumvent controls (e.g. the use of natural hormones, low-dose cocktails, or the use of new synthetic drugs). To overcome some limitations of these targeted methods and to deal with some difficult cases, especially at the critical screening stage, a new strategy emerged in the early 20s: the investigation of effect biomarkers, based on non-targeted profiling involving genomics, transcriptomics, proteomics, and metabolomics approaches for a global analysis of genes, RNAs, proteins and endogenous metabolites, respectively, as potential biomarkers of interest. With hindsight now, metabolomics is recognized the most promising area to highlight biomarkers related to animal doping and their subsequent monitoring in a control perspective. Several studies have already demonstrated the proof of concept of the metabolomics-based workflow and its suitability for alternative screening for a range of growth promoters (e.g., β-agonists, steroids) but also various species (e.g., bovine, horse), it is now time to move on to a robust implementation for monitoring purpose. In particular, the next research question in metabolomics is how to validate resulting non-targeted methods and transfer to routine frameworks, complying standards and regulatory expectations. This state of the art aims at providing an overview of the evolution of screening methods, from direct strategies targeting exposure biomarkers to indirect methods looking for new effect biomarkers based on semi-targeted or non-targeted methods. With the official implementation of these innovative effect biomarkers-based screening strategies in mind, this review article also aims to detail, the metabolomics workflow and best practices from sample preparation to the evaluation of performances of such approaches and ways to validate the corresponding methods. •Review on the evolution of anaboli