Detection of gonadotropin-releasing hormone and its analogues in equine and canine urine by high-resolution data-independent acquisition.

Gonadotropin-releasing hormone (GnRH) and its synthetic analogues are considered banned substances by the racing industry. GnRH is used as a pharmaceutical to regulate the female oestrous cycle, but the hormone is also thought to increase the production of testosterone in male animals. Using liquid chromatography in conjunction with high-resolution mass spectrometry (LC-HRMS) and data-independent acquisition (DIA), a method is presented for the detection of intact and truncated peptides of GnRH and its analogues down to the low picogram level in equine urine. The study of the catabolism of GnRH and analogues in plasma, combined with the analysis of urine from administration studies, reveals a common pattern of peptide catabolites that can be used to guide the design of MS-based screens for this class of drugs. This culminated in the successful detection of the peptide in two out-of-competition canine urine samples.

Metabolic study of selective androgen receptor modulator LY2452473 in thoroughbred horses for doping control.

RATIONALE: Since 2010, there has been an increasing number of adverse analytical findings related to selective androgen receptor modulators (SARMs) in competitive sports. It emphasizes the importance of comprehensive doping control analytical procedures that are capable of detecting SARM misuse. METHODS: In this study, it is described how LY2452473, a SARM, was metabolized in thoroughbred horses after a single-dose oral administration and in vitro with equine liver microsome preparations. An investigation of the metabolism of LY2452473 in horses' urine, plasma, and hair matrices was carried out during the study. The plausible structures of the detected metabolites were postulated using high-performance liquid chromatography-high resolution mass spectrometry. RESULTS: Under the experimental conditions 15 metabolites (12 phase I and three conjugates of phase I) were detected (M1-M15). The major phase I metabolites identified were formed by hydroxylation. Side-chain dissociated and methylated metabolites were also detected. In phase II, the glucuronic acid and sulfonic acid conjugates of hydroxy LY2452473 were detected as the major metabolites. In vitro analysis has confirmed the presence of all metabolites found in vivo except for the methylated analogs M11 and M12. A peak concentration of LY2452473 (0.5 pg/mg) in proximal hair segments was achieved 4 weeks after administration, according to hair analysis. CONCLUSIONS: Data obtained will aid in identifying LY2452473 and related substances faster. Furthermore, the results will assist in checking for the illegal use of these substances in competitive sports.

Characterization of growth hormone secretagogue small molecule ibutamoren (MK-0677) and its possible metabolites in thoroughbred horses for doping control.

RATIONALE: It is important to remember that performance-enhancing agents such as non-peptide growth hormone secretagogues present a significant risk of abuse. Ibutamoren (MK-0677) is a potent, long-acting, selective non-peptide growth hormone secretagogue that can be taken orally. METHODS: This study examines ibutamoren and its metabolites in thoroughbred horses after oral administration. Liquid chromatography/high-resolution mass spectrometry was used to determine the probable structures of the detected metabolites. RESULTS: In this study, 22 metabolites of ibutamoren were identified (17 phase I and 5 phase II). Oxidation of ibutamoren leads to hydroxylated metabolites (mono and di). The study also detected dissociated side chains (benzyl group and 2-amino-2-methylpropanamide) and hydrogenated metabolites. The glucuronic acid conjugated analogs of ibutamoren were detected during phase II of the study, but no sulfonic acid conjugated analogs were observed. The major metabolites can be detected up to 96 hours after a single dose, and ibutamoren can persist for up to 72 hours. CONCLUSIONS: These findings will aid in the detection of ibutamoren and the detection of its illegal use in competitive sports.

Detection and confirmation of zilpaterol in equine hair using liquid chromatography-mass spectrometry.

Zilpaterol is a ß2 -adrenergic agonist and a repartitioning agent that has a high potential for abuse in equine performance athletes. Analysis of zilpaterol in hair is an alternative sampling matrix that extends detection time periods beyond those found in urine or blood samples. Our laboratory has been screening for zilpaterol in hair for many years and recently detected and confirmed its presence in official samples. Accordingly, a liquid chromatography-mass spectrometry method was developed and validated to detect and confirm zilpaterol in equine hair. Briefly, equine hair was decontaminated, cut, and pulverized prior to disruption and liquid-liquid extraction in basic conditions. Following extraction, the sample was introduced to an Agilent 1260 HPLC and zilpaterol was separated using a reverse phase gradient with a total run time of 12.5 min. Following chromatographic separation, zilpaterol and its corresponding stable isotope labeled internal standard were introduced via positive mode electrospray ionization to a Thermo Q-Exactive Plus mass spectrometer and spectra collected using parallel reaction monitoring. The methodology was validated using in-house criteria including characterization of accuracy, precision, recovery, linear range, matrix effects, limit of detection, and limit of quantitation, and the method was found to be fit-for-purpose to confirm the presence of zilpaterol in equine hair. This methodology has been used to detect and confirm the presence of zilpaterol from out-of-competition hair samples submitted by regional racing authorities.

In vitro metabolism of the REV-ERB agonist SR-9009 and subsequent detection of metabolites in associated routine equine plasma and urine doping control samples.

SR-9009 is a synthetic compound widely available to purchase online as 'supplement' products due to its potential performance-enhancing effects, presenting a significant threat with regard to doping control in sport. In vitro metabolism with equine liver microsomes was performed to identify potential targets for detection of SR-9009. Six metabolites were identified, with the most abundant consisting of N-dealkylated metabolites (M1-M3). The addition of the identified metabolites to high-resolution accurate mass databases resulted in a positive finding for the N-dealkylated metabolite M1 of SR-9009 in an associated plasma and urine doping sample. Liquid chromatography-high-resolution mass spectrometry was used to verify the presence of the N-dealkylated metabolite (M1) in both matrices, with a low concentration of the parent compound and additional N-desalkyl metabolites (M2 and M3) detected in the plasma sample as supporting evidence of administration. To the best of the authors' knowledge, this is the first report of an adverse analytical finding in an equine sample for SR-9009 or its metabolites in equine doping control.

In vitro and in vivo metabolism of the anabolic-androgenic steroid oxandrolone in the horse.

Oxandrolone is an anabolic-androgenic steroid with favourable anabolic to androgenic ratio, making it an effective anabolic agent with less androgenic side effects. Although its metabolism has been studied in humans, its phase I and II metabolism has not been previously reported in the horse. The purpose of this study was to investigate the in vitro metabolism of oxandrolone (using both equine liver microsomes and S9) and in vivo metabolism following oral administration (three daily doses of 50 mg of oxandrolone to a single Thoroughbred horse), using both gas and liquid chromatography-mass spectrometry techniques. The in vitro phase I transformations observed included 16-hydroxylated (two epimers), 17-methyl-hydroxylated and 16-keto metabolites. In addition to parent oxandrolone and these hydroxylated metabolites, the 17-epimer and a 17,17-dimethyl-18-norandrost-13-ene analogue were detected in biological samples following the administration. 16-keto-oxandrolone was only observed in urine. The 16- and 17-methyl-hydroxylated oxandrolone metabolites were predominantly excreted as sulfate conjugates in urine, whereas parent oxandrolone, its epimer and 17,17-dimethyl-18-norandrost-13-ene derivative were found predominantly in the unconjugated urine fraction. The most abundant analyte detected in both plasma and urine was parent oxandrolone. However, the longest detection period using the developed analytical method was provided by 17-hydroxymethyl-oxandrolone in both matrices. The results of this study provided knowledge of how best to detect the use of oxandrolone in regulatory samples.

Identification of equine in vitro metabolites of seven non-steroidal selective androgen receptor modulators for doping control purposes.

Selective androgen receptor modulators, SARMs, are a large class of compounds developed to provide therapeutic anabolic effects with minimal androgenic side effects. A wide range of these compounds are available to purchase online and thus provide the potential for abuse in sports. Knowledge of the metabolism of these compounds is essential to aid their detection in doping control samples. In vitro models allow a quick, cost-effective response where administration studies are yet to be carried out. In this study, the equine phase I metabolism of the non-steroidal SARMs GSK2881078, LGD-2226, LGD-3303, PF-06260414, ACP-105, RAD-140 and S-23 was investigated using equine liver microsomes. Liquid chromatography coupled to a QExactive Orbitrap mass spectrometer allowed identification of metabolites with high resolution and mass accuracy. Three metabolites were identified for both GSK2881078 and LGD-2226, four for LGD-3303 and RAD-140, five for PF-06260414, twelve for ACP-105 and ten for S-23. The equine metabolism of GSK-2881078, LGD-2226, LGD-3303 and PF-06260414 is reported for the first time. Although the equine metabolism of ACP-105, RAD-140 and S-23 has previously been reported, the results obtained in this study have been compared with published data.

Detection of Methylphenidate in Equine Hair Using Liquid Chromatography-High-Resolution Mass Spectrometry.

Methylphenidate is a powerful central nervous system stimulant with a high potential for abuse in horse racing. The detection of methylphenidate use is of interest to horse racing authorities for both prior to and during competition. The use of hair as an alternative sampling matrix for equine anti-doping has increased as the number of detectable compounds has expanded. Our laboratory developed a liquid chromatography-high-resolution mass spectrometry method to detect the presence of methylphenidate in submitted samples. Briefly, hair was decontaminated, cut, and pulverized prior to liquid-liquid extraction in basic conditions before introduction to the LC-MS system. Instrumental analysis was conducted using a Thermo Q Exactive mass spectrometer using parallel reaction monitoring using a stepped collision energy to obtain sufficient product ions for qualitative identification. The method was validated and limits of quantitation, linearity, matrix effects, recovery, accuracy, and precision were determined. The method has been applied to confirm the presence of methylphenidate in official samples submitted by racing authorities.

Detecting and quantifying marijuana metabolites in serum and urine of 19 dogs affected by marijuana toxicity.

Veterinarians diagnose marijuana toxicity based on clinical signs and history, or in conjunction with an over-the-counter (OTC) human urine drug screen. With the legalization of recreational marijuana use becoming more prevalent in the United States, a more accurate test to aid in the diagnosis of canine marijuana toxicity is needed. We collected urine and serum samples from 19 dogs with confirmed or suspected marijuana toxicosis from multiple veterinary hospitals and analyzed them with a novel UPLC-MS/MS method. Calibrations from 0.1 to 100 ng/mL and QC materials were prepared. Samples were extracted, purified, and eluted with solid-phase extraction. Urine samples were tested with an OTC human urine drug screen. The limit of detection (LOD) and lower limit of quantification (LLOQ) ranges for marijuana metabolites in serum were 0.05-0.25 ng/mL and 0.1-0.5 ng/mL, respectively. In urine, the LOD and LLOQ ranges for the metabolites were 0.05-0.1 ng/mL and 0.1-0.5 ng/mL, respectively. In serum, median and range of metabolite concentrations (ng/mL) detected included: THC, 65.0 (0.14-160); 11-OH-Δ9-THC, 4.78 (1.15-17.8); 11-nor-9-carboxy-Δ9-THC, 2.18 (0.71-7.79); CBD, 0.28 (0.11-82.5); and THC-glucuronide, 2.05 (0.72-18.3). In the 19 urine samples, metabolite: creatinine (ng: mg) values detected included: THC, 0.22 (0.05-0.74); 11-OH-Δ9-THC, 0; 11-nor-9-carboxy-Δ9-THC, 1.32 (0.16-11.2); CBD, 0.19 (0.12-0.26); THC-COOH-glucuronide, 0.08 (0.04-0.11); and THC-glucuronide, 0.98 (0.25-10.7). Twenty of 21 urine samples tested negative for THC on the urine drug screen. All 19 serum samples contained quantifiable concentrations of THC using our novel UPLC-MS/MS method. Utilizing a UPLC-MS/MS method can be a useful aid in the diagnosis of marijuana toxicosis in dogs, whereas using an OTC human urine drug test is not a useful test for confirming marijuana exposure in dogs because of the low concentration of THC-COOH in urine.

The in vivo metabolism of Furazadrol in greyhounds.

Samples of the 'dietary supplement' Furazadrol sourced through the internet have been reported to contain the designer anabolic androgenic steroids [1',2']isoxazolo[4',5':2,3]-5α-androstan-17ß-ol (furazadrol F) and [1',2']isoxazolo[4',3':2,3]-5α-androstan-17ß-ol (isofurazadrol IF). These steroids contain an isoxazole fused to the A-ring and were designed to offer anabolic activity while evading detection, raising concerns over the potential for abuse of this preparation in sports. The metabolism of Furazadrol (F:IF, 10:1) was studied by in vivo methods in greyhounds. Urinary phase II Furazadrol metabolites were detected as glucuronides after a controlled administration. These phase II metabolites were subjected to enzymatic hydrolysis by Escherichia coli ß-glucuronidase to afford the corresponding phase I metabolites. Using a library of synthetically derived reference materials, the identities of seven urinary Furazadrol metabolites were confirmed. Major confirmed metabolites were isofurazadrol IF, 4α-hydroxyfurazadrol 4α-HF and 16α-hydroxy oxidised furazadrol 16α-HOF, whereas the minor confirmed metabolites were furazadrol F, 4ß-hydroxyfurazadrol 4ß-HF, 16ß-hydroxyfurazadrol 16ß-HF and 16ß-hydroxy oxidised furazadrol 16ß-HOF. One major hydroxyfurazadrol and two dihydroxyfurazadrol metabolites remained unidentified. Qualitative excretion profiles, limits of detection and extraction recoveries were established for furazadrol F and major confirmed metabolites. These investigations identify the key urinary metabolites of Furazadrol following oral administration, which can be incorporated into routine screening by anti-doping laboratories to aid the regulation of greyhound racing.

Towards an untargeted mass spectrometric approach for improved screening in equine antidoping.

The emergence of novel doping agents is a continuous issue for analysts who aim to maintain the integrity of horseracing together with the well-being and safety of the animals and riders involved. Untargeted mass spectrometric analysis presents a potential improvement for antidoping as it enables the detection of compounds being indirectly affected by an administered drug. In this study, liquid chromatography-high-resolution mass spectrometry was used to investigate a 12-horse administration study of the synthetic opioid, butorphanol. A mass spectrometric workflow capable of detecting metabolic differences for an extended period of time was successfully developed. This proof-of-concept study demonstrates the potential of untargeted workflows to provide a list of biomarkers of exposure and effect that are indicative of drug administration which may be implemented into routine testing for improved doping control.

Metabolic study of GW1516 in equine urine using liquid chromatography/electrospray ionization Q-Exactive high-resolution mass spectrometry for doping control.

RATIONALE: The use of GW1516, a peroxisome proliferator-activated receptor δ (PPAR δ) agonist, is strictly prohibited in both horseracing and equestrian competitions. However, little is known about its metabolic fate in horses. To the best of our knowledge, this is the first reported metabolic study of GW1516 in equine urine. METHODS: Urine samples obtained from a thoroughbred after nasoesophageal administration with GW1516 were protein-precipitated and the supernatants were subsequently analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) with a Q-Exactive mass spectrometer. Monoisotopic ions of GW1516 and its metabolites were monitored from the full-scan mass spectral data of pre- and post-administration samples. A quantification method was developed and validated to establish the excretion profiles of GW1516, its sulfoxide, and its sulfone in equine urine. RESULTS: GW1516 and its nine metabolites [including GW1516 sulfoxide, GW1516 sulfone, 5-(hydroxymethyl)-4-methyl-2-(4-trifluoromethylphenyl)thiazole (HMTT), methyl 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylate (MMTC), 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylic acid (MTTC), and M1 to M4] were detected in post-administration urine samples. GW1516 sulfoxide and GW1516 sulfone showed the longest detection times in post-administration urine samples and were therefore recommended as potential screening targets for doping control purposes. Quantitative analysis was also conducted to establish the excretion profiles of GW1516 sulfoxide and GW1516 sulfone in urine. CONCLUSIONS: For the purposes of doping control of GW1516, the GW1516 sulfoxide and GW1516 sulfone metabolites are recommended as the target analytes to be monitored in equine urine due to their high specificities, long detection times (1 and 4 weeks, respectively), and the ready availability of their reference materials.

Label-free proteomics for discovering biomarker candidates of RAD140 administration to castrated horses.

Selective androgen receptor (AR) modulators (SARMs) are potent anabolic agents with a high potential of misuse in horseracing and equestrian sports. In this study, we applied label-free proteomics to discover plasma protein biomarkers in geldings (castrated horses) after administration with a popular SARM named RAD140. Tryptic peptides were prepared from plasma samples and analyzed by nano-flow ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (nano-UHPLC-HRMS/MS) using data-independent acquisition (DIA) method. Orthogonal projection on latent structure-discriminant analysis (OPLS-DA) has led to the development of a predictive model that could discriminate RAD140-administered samples from control samples and could also correctly classify 18 out of 19 in-training horses as control samples. The model comprises 75 proteins with variable importance in projection (VIP) score above 1. Gene Ontology (GO) enrichment analysis and literature review have identified upregulation of AR-regulated clusterin, and proteins associated with inflammation (haptoglobin, cluster of differentiation 14 [CD14], and inter-alpha-trypsin inhibitor heavy chain 4 [ITIH4]) and erythropoiesis (glycosylphosphatidylinositol-specific phospholipase D1 [GPLD1]) after RAD140 administration. Their changes were confirmed by selected reaction monitoring (SRM) experiments. Similar effects have been reported by the use of androgens and other SARMs. This is the first reported study that describes the use of a proteomic biomarker approach to detect horses that have been administered with RAD140 by applying label-free proteomic profiling of plasma samples. These results support the concept of a biomarker-driven approach to enhance the doping control of RAD140 and potentially other SARMs in the future.

Application of a non-target variable data independent workflow (vDIA) for the screening of prohibited substances in doping control testing.

A non-target variable Data Independent Acquisition (vDIA) workflow based on accurate mass measurements using a Q Exactive OrbiTrap is presented for the first time for equine doping control testing. The vDIA workflow uses a combination of MS1 events (1 to 2) and multiple vDIA events to cover the analytes of interest. The workflow basically captures a digital image of a sample allowing all relevant MS1 and MS2 data to be recorded. In theory, the workflow can accommodate an unlimited number of analytes as long as they are amenable to the sample extraction protocol and fall within the mass limits of the workflow. Additional targets fulfilling the above requirements can be added without changing any settings. The performance of the vDIA workflow was illustrated by applying it to two screening methods in horse urine, with one workflow covering 331 basic drugs and the other covering 45 quaternary ammonium drugs (QADs). Both screening methods have good detection sensitivity with 84% of the basic drugs having Limits of Detection (LoDs) of ≤ 1 ng/mL and 84% of the QADs having LoDs of ≤ 0.4 ng/mL. Other method characteristics including retention reproducibility, method precision and false hit rate will also be presented.

Quantitative analysis of paracetamol, metacetamol, and orthocetamol in equine urine from racehorses in Japan using liquid chromatography-electrospray ionization-tandem mass spectrometry.

Paracetamol is commonly used as an over-the-counter analgesic and antipyretic medication for humans, but not sold as a legitimate therapeutic medication for horses in Japan. However, paracetamol is commonly found in horses together with its two isomers, metacetamol and orthocetamol. We previously reported that paracetamol and orthocetamol were both present in selected feed consumed by Japanese racehorses. For the purpose of the doping control of paracetamol in local Japanese horses, we proposed establishing residue limits (Japanese residue limits, JRLs) to minimize the risk of reporting paracetamol from environmental contributions and to differentiate its presence from active administration. Recently, we proposed a preliminary JRL for paracetamol in equine plasma based on a population study of more than 300 Japanese racehorses. In this paper, we will present our studies on the urinary concentrations of paracetamol, metacetamol, and orthocetamol in postrace samples collected from 403 Japanese racehorses over a 1 year period, detected using liquid chromatography-electrospray ionization-tandem mass spectrometry. Our results revealed that the hydrolyzed urinary concentrations of paracetamol, metacetamol, and orthocetamol were in the range 15.7-2,360 ng/mL (median 363 ng/mL), 8.07-382 ng/mL (84.5 ng/mL), and 919-74,418 ng/mL (13,475 ng/mL), respectively. Based on our statistical model, the preliminary JRL of hydrolyzed paracetamol in equine urine was determined to be 7,400 ng/mL, at a risk factor of 1 in 10,000. Further investigations will be required to demonstrate the applicability and validity of our preliminary plasma and urine JRLs to local Japanese racehorses.

A high-throughput and broad-spectrum screening method for analysing over 120 drugs in horse urine using liquid chromatography-high-resolution mass spectrometry.

A high-throughput method has been developed for the doping control analysis of 124 drug targets, processing up to 154 horse urine samples in as short as 4.5 h, from the time the samples arrive at the laboratory to the reporting deadline of 30 min before the first race, including sample receipt and registration, preparation and instrument analysis and data vetting time. Sample preparation involves a brief enzyme hydrolysis step (30 min) to detect both free and glucuronide-conjugated drug targets. This is followed by extraction using solid-supported liquid extraction (SLE) and analysis using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). The entire set-up comprised of four sets of Biotage Extrahera automation systems for conducting SLE and five to six sets of Orbitrap for instrumental screening using LC-HRMS. Suspicious samples flagged were subject to confirmatory analyses using liquid chromatography-triple quadrupole mass spectrometry. The method comprises 124 drug targets from a spectrum of 41 drug classes covering acidic, basic and neutral drugs. More than 85% of the targets had limits of detection at or below 5 ng/mL in horse urine, with the lowest at 0.02 ng/mL. The method was validated for qualitative identification, including specificity, sensitivity, extraction recovery and precision. Method applicability was demonstrated by the successful detection of different drugs, namely (a) butorphanol, (b) dexamethasone, (c) diclofenac, (d) flunixin and (e) phenylbutazone, in post-race or out-of-competition urine samples collected from racehorses. This method was developed for pre-race urine testing in Hong Kong; however, it is also suitable for testing post-race or out-of-competition urine samples, especially when a quick total analysis time is desired.

Extraction of α-neurotoxins from equine plasma by receptor based affinity purification.

Venoms were first identified as potential doping agents by the racing industry in 2007 when three vials of cobra venom were seized during an inspection of a stable at Keeneland Racecourse in the USA. Venoms are a complex mixture of proteins, peptides, and other substances with a wide range of biological effects, including inhibiting the transmission of nervous and muscular impulses. As an example of this, cobratoxin, an α-neurotoxin found in cobra venom, is claimed to be an effective treatment for pain. Recent analysis of seized samples identified venom from two different species of snake. Proteomic analysis identified the first sample as cobra venom, while the second sample, in a vial labeled "Conotoxin", was identified as venom from a many banded krait. Cobratoxin, conotoxins, and bungarotoxins (a component of krait venom) are all α-neurotoxins, suggesting a common application for all three venom proteins as potential pain blocking medications. Using a peptide based on the nicotinic acetylcholine receptor, a one-step affinity purification method was developed for the detection of α-neurotoxins in plasma.

Tracking main environmental factors masking a minor steroidal doping effect using metabolomic analysis of horse urine by liquid chromatography-high-resolution mass spectrometry.

There is an urgent need to implement holistic and untargeted doping control protocols with improved discriminatory power, compared to conventional methods that only target doping agents. Metabolomics, which aims to characterize all metabolites present in biological matrices, could fulfill this need. In this context, the aim of this study was to evaluate the impact of environmental factors on the ability to obtain a metabolic signature of stanozolol administration in horse doping situation. Urine samples from 16 horses breeded in two different places were collected over a one-year period, before, during and seven months after the administration of stanozolol, a horse doping agent. Metabolomic analysis was performed using ultra-high pressure reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (MS). Results showed a major impact of the nutritional regimen, drug administration (for de-worming purpose) and breeding place on the metabolite profiles of horse urines, which hampered the detection of metabolic perturbations induced by stanozolol administration. After having used MS/MS experiments to characterize some MS features related to these environmental factors, we showed that highlighting and then removing the features impacted by these confounding factors before performing supervised multivariate statistical analyses could address this issue. In conclusion, adequate consideration should be given to environmental and physiological factors; otherwise, they can emerge as confounding factors and conceal doping administration.

A non-targeted LC-HRMS approach for detecting exposure to illegal veterinary treatments: The case of cephalosporins in commercial laying Hens.

Cephalosporins are of particular importance in human medicine and should be reserved for second-line curative treatment in the veterinary field to avoid any emerging antimicrobial resistance. Due to misuse of ceftiofur in the poultry sector in France, it is now recommended to completely stop using cephalosporins in this sector. Methods currently used for the control of veterinary practices are mostly based on liquid chromatography coupled to mass spectrometry in a targeted mode, including parent compounds and any major metabolites. The aim of the present study was to evaluate the relevance of untargeted metabolomic approaches to highlight a possible exposure of laying hens to cephalosporins using a predictive model including selected treatment biomarkers. An experimentation carried out on living animals involved the administration of cefquinome and ceftiofur. Three biological matrices-droppings, eggs and liver-were investigated. Metabolites were extracted and analysed by liquid chromatography coupled to high resolution mass spectrometry in a full scan mode. Metabolites impacted by the treatment were selected by using univariate and multivariate statistical analyses. Predictive models built from the potential biomarkers selected in the "droppings" matrix were validated and able to classify "treated" and "control" hens. PLS-DA and logistic regression models were compared and both models gave satisfactory results in terms of prediction. Results were of less interest for other matrices in which only biomarkers of exposure to cefquinome were detected.