Metabolomics in food analysis: application to the control of forbidden substances Gaud Dervilly-Pinel, a Frédérique Courant, a Sylvain Chéreau, a Anne-Lise Royer, a Fanny Boyard-Kieken, a,c Jean-Philippe Antignac, a,b Fabrice Monteau a and Bruno Le Bizec a * Metabolomics is a science of interest in food analysis to describe and predict properties of food products and processes. It includes the development of analytical methods with the ultimate goal being the identification of so-called ‘quality markers’, (i.e. sets of metabolites that correlate with, for example, quality, safety, taste, or fragrance of foodstuffs). In turn, these metabolites are influenced by factors as genetic differences of the raw food ingredients (such as animal breed or crop species differences), growth conditions (such as climate, irrigation strategy, or feeding) or production conditions (such as temperature, acidity, or pressure). In cases where the routine-based measurement of a food property faces some limitations such as the lack of knowledge regarding the target compounds to monitor, monitoring based on a limited set of crucial biomarkers is a good alternative, which is of great interest for food safety purposes regarding growth promoting practices. Such an approach may be more efficient than using a classic approach based on a limited set of known metabolites of anabolic compounds. In this context, screening strategies allow- ing detection of the physiological response resulting from anabolic compound administration are promising approaches to detect their misuse. The global metabolomics workflow implemented for such studies is presented and illustrated through various exam- ples of biological matrices profiling (tissue, blood, urine) and for different classes of anabolic compounds (steroids, b-agonists and somatotropin). © 2012 John Wiley & Sons, Ltd. Keywords: untargeted profiling; metabolomics; growth promoters; steroids; growth hormone; b-agonists; screening Introduction Growth-promoting practices for animal fattening purposes are still encountered all around the world (e.g. use of clenbuterol in pigs, recombinant growth hormone in fish, natural steroids in cocktails, in bovines or hypothetic but realistic anabolic strategies consisting either on upstream disruption of the hypothalamo- pituitary axis (secretagogues, ...) or even worse on direct genes modification). They are banned in food producing livestock in Europe [1,2] and in other parts of the world. Detection of illegal practices typically relies on residue monitoring in a targeted approach and methods based on gas chromatography (GC) or liquid chromatography (LC) coupled to (tandem) mass spectrometry ((MS/)MS) are today considered as the state-of the-art. [3] These strategies are, however, challenged when facing new xenobiotic growth-promoting agents or new ways of appli- cation, such as the administration of low-dose cocktails. To deal with such anabolic practices, alternative means of detecting the abuse are urgently needed. In this context, strategies based on the detection of physiological actions of anabolic practices are promising approaches to screen for their misuse. Profiling biological matrices to reveal biological effects of a drug can either be performed in a targeted focus on a particular class of compounds, such as the steroidome [4–9] or in an untargeted way using global strategies such as transcriptomics, proteomics, or metabolomics. [10,11] These emerging strategies are promis- ing ways to highlight candidate biomarkers to tackle illegal practices. [12] In particular, metabolomics has recently drawn the attention of the scientific community in the detection of anabolic practices and several studies have indeed demonstrated the efficiency of such mass-spectrometric-based fingerprinting to discriminate animals that have been subjected to anabolic treatment from control animals. Several approaches have shown potential to discriminate through analysis of urine samples of ani- mals treated with a range of anabolic formulations: DHEA and preg- nenolone in bovines, [13] clenbuterol in calves, [14] recombinant equine growth hormone in horses, [15–17] and nandrolone and estradiol in calves. [18] Metabolomics is an emerging field of ‘omics’ research that focuses, in an untargeted mode, on large-scale and high-throughput measurement of small molecules (so-called metabolites) in biological matrices. While metabonomics focuses on understanding systemic change through time in complex multicellular systems, [19] metabolomics refers to an analytical description of biological samples, the metabolome consisting in ‘...the complete set of metabolites/low-molecular-weight inter- mediates, which are context dependent, varying according to the physiology, developmental or pathological state of the cell, tissue, * Correspondence to: Prof. Bruno Le Bizec, Oniris, École nationale vétérinaire, agroalimentaire et de l’alimentation Nantes-Atlantique (Oniris), Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), Atlanpole - La Chantrerie, BP 40706, Nantes, F-44307, France. E-mail: laberca@oniris-nantes.fr a LUNAM Université, Oniris, Laboratoire d’Étude des Résidus et Contaminants dans les Aliments (LABERCA), Nantes, France b INRA, Nantes, France c LCH, Verrières le Buisson, France Drug Test. Analysis 2012, 4 (Suppl. 1), 59–69 © 2012 John Wiley & Sons, Ltd. Policy article Drug Testing and Analysis Published online in Wiley Online Library (www.drugtestinganalysis.com) DOI 10.1002/dta.1349 59