Exploring methandienone metabolites generated via homogenized camel liver: Advancements for anti-doping applications through High Resolution-Liquid Chromatography Mass Spectrometry analysis.

RATIONALE: Anabolic steroids, also known as anabolic-androgenic steroids (AAS), encompass steroidal androgens such as testosterone, as well as synthetic counterparts with similar structures and effects. The misuse of AAS has increased over the years, leading to ethical and welfare concerns in sports. The World Anti-Doping Agency (WADA) and the International Federation for Equestrian Sports (FEI) have banned AAS in relevant sports. Methandienone is one of the most identified anabolic androgenic steroids in sports drug testing, Therefore, reliable detection methods are crucial for effective doping control and maintaining the integrity of the sports. METHODS: This study explores the use of homogenized camel liver for detecting methandienone metabolites in camels. The biotransformation pathways of methandienone in homogenized camel liver tissues are analyzed using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) to identify and characterize the phase I and phase II metabolites. Chromatographic separation was achieved using a Thermo-Hypersil C18 column. RESULTS: The study has identified 11 methandienone metabolites (M1-M11), this includes 10 phase I and one phase II metabolite. A glucuronic acid conjugate of methandienone was observed in this study, but no sulfonic acid conjugations were found. The metabolites and their possible chemical structures, along with their fragmentation patterns are confirmed using MSMS (MS2) experiments in data-independent acquisition (DIA) mode. CONCLUSIONS: These findings serve as a vital tool for the rapid detection of methandienone, combating its illicit use in camel racing. Comprehensive screenings covering both the parent drug and its metabolites are recommended to improve detection accuracy and ensure regulatory compliance in sports doping. Future research should explore methandienone's metabolite profile in administered camel samples.

Prediction model for anabolic androgenic steroid positivity in forensic autopsy cases - a new tool to the autopsy room.

Non-prescription use of anabolic androgenic steroids (AAS) is associated with an increased risk of premature death. However, these substances are seldom screened in connection with forensic cause-of-death investigation, unless the forensic pathologist specifically suspects use, often based on a positive AAS use history. Since AAS use is often concealed from others, this practice may lead to mistargeting of these analyses and significant underestimation of the true number of AAS positive cases undergoing forensic autopsy. Thus, more accurate diagnostic tools are needed to identify these cases. The main objective of this study was to determine, whether a multivariable model could predict AAS urine assay positivity in forensic autopsies. We analyzed retrospectively the autopsy reports of all cases that had been screened for AAS during forensic cause-of-death investigation between 2016-2019 at the Finnish Institute for Health and Welfare forensic units (n = 46). Binary logistic regression with penalized maximum likelihood estimation was used to generate a nine-variable model combining circumferential and macroscopic autopsy-derived variables. The multivariable model predicted AAS assay positivity significantly better than a "conventional" model with anamnestic information about AAS use only (area under the receiver operating characteristic curve [AUC] = 0.968 vs. 0.802, p = 0.005). Temporal validation was conducted in an independent sample of AAS screened cases between 2020-2022 (n = 31), where the superiority of the multivariable model was replicated (AUC = 0.856 vs. 0.644, p = 0.004). Based on the model, a calculator predicting AAS assay positivity is released as a decision-aiding tool for forensic pathologists working in the autopsy room.

Using Proteins As Markers for Anabolic Steroid Abuse: A New Perspective in Doping Control?

Drug toxicity is a major concern and has motivated numerous studies to elucidate specific adverse mechanisms, with acetaminophen being the favorite candidate in toxicology studies. Conversely, androgenic anabolic steroids (AASs) also represent a severe public health issue in sports for elite and non-elite athletes. Supraphysiological dosages of AASs are associated with various adverse effects, from cardiovascular to neurological repercussions including liver dysfunction. Yet, few studies have addressed the toxicity of anabolic steroids, and a significant amount of work will be needed to elucidate and understand steroid toxicity properly. This Perspective suggests ideas on how proteomics and liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) can contribute to (1) pinpoint serum proteins affected by substantial doses of anabolic steroids that would represent interesting novel candidates for routine testing and (2) provide additional knowledge on androgenic anabolic steroid toxicity to help raise awareness on the harmful effects.

Investigation of in vitro generated metabolites of LGD-4033, a selective androgen receptor modulator, in homogenized camel liver for anti-doping applications.

RATIONALE: The use of selective androgen receptor modulators (SARM) in sports is prohibited by the World Anti-Doping Agency (WADA) due to their potential as performance-enhancing drugs, offering an unfair advantage. LGD-4033 is a SARM known for its similarities to anabolic steroids and can be easily purchased online, leading to increased availability and misuse. Adverse analytical findings have revealed the presence of SARMs in dietary supplements. Although LGD-4033 misuse has been reported in human sports over the years, concerns also arise regarding its illicit use in animal sports, including camel racing. Although various studies have investigated the metabolism of LGD-4033 in humans, horse, and other species, there is limited research specifically dedicated to racing camels. METHODS: This study focuses on the in vitro metabolism of LGD-4033 in homogenized camel liver using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) to identify and characterize the metabolites. RESULTS: The findings indicated the presence of 12 phase I metabolites and 1 phase II metabolite. Hydroxylation was responsible for the formation of the main phase I metabolites that were identified. A glucuronic acid conjugate of the parent drug was observed in this study, but no sulfonic acid conjugate was found. The possible chemical structures of these metabolites, along with their fragmentation patterns, were identified using MS. CONCLUSIONS: These findings provide valuable insights into the metabolism of LGD-4033 in camels and aid in the development of effective doping control methods for the detection of SARMs in camel racing.

Identification and Synthesis of Selected In Vitro Generated Metabolites of the Novel Selective Androgen Receptor Modulator (SARM) 2f.

Among anabolic agents, selective androgen receptor modulators (SARMs) represent a new class of potential drugs that can exhibit anabolic effects on muscle and bone with reduced side effects due to a tissue-selective mode of action. Besides possible medical applications, SARMs are used as performance-enhancing agents in sports. Therefore, they are prohibited by the World Anti-Doping Agency (WADA) in and out of competition. Since their inclusion into the WADA Prohibited List in 2008, there has been an increase in not only the number of adverse analytical findings, but also the total number of SARMs, making continuous research into SARMs an ongoing topic in the field of doping controls. 4-((2R,3R)-2-Ethyl-3-hydroxy-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile (SARM 2f) is a novel SARM candidate and is therefore of particular interest for sports drug testing. This study describes the synthesis of SARM 2f using a multi-step approach, followed by full characterization using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR). To provide the first insights into its biotransformation in humans, SARM 2f was metabolized using human liver microsomes and the microsomal S9 fraction. A total of seven metabolites, including phase I and phase II metabolites, were found, of which three metabolites were chemically synthesized in order to confirm their structure. Those can be employed in testing procedures for routine doping controls, further improving anti-doping efforts.

Novel nandrolone aptamer for rapid colorimetric detection of anabolic steroids.

The illicit use of anabolic androgenic steroids (AAS) as performance-enhancing drugs remains a global issue threatening not only the credibility of competitive sports but also public health due to the well-documented adverse effects they elicit. AAS abuse is not restricted only to professional sports, but also extends to recreational athletes and adolescents as well as in livestock production as growth-promoting agents. Testosterone and nandrolone are among the AAS most frequently exploited. Gas chromatography-mass spectrometry is the reference method for AAS detection, but it is strictly laboratory-based and cannot be performed on-site. The great potential of aptamers in bioanalytical applications and specifically for the development of simple analytical tools suitable for on-site analysis has been extensively documented. In this report, we describe the selection and identification of aptamers binding nandrolone, exhibiting affinity dissociation constants in the low nanomolar range. A label-free colorimetric assay based on gold nanoparticles was developed using one of these novel aptamers for the detection of nandrolone and/or its metabolites. The assay could be deployed for the rapid, on-site, facile and cost-effective screening of samples and provide qualitative visual results with a red to purple/blue color change being indicative of a positive result.

Simplified screening approach of anabolic steroid esters using a compact atmospheric solid analysis probe mass spectrometric system.

Due to the absence of chromatographic separation, ambient ionization mass spectrometry had the potential to improve the throughput of control laboratories in the last decades and will soon be an excellent approach for on-site use as well. In this study, an atmospheric solids analysis probe (ASAP) with a single quadrupole mass analyzer has been evaluated to identify anabolic steroid esters rapidly. Sample introduction, applied scan time, and probe temperature were optimized for sensitivity. The in-source fragmentations of seventeen selected steroid esters, commonly found in illicit samples, were determined by applying different cone voltages (12, 20, 30, and 40 V). A spectral library was created for these steroid esters based on the four stages of in-source fragmentation spectra. The applicability of this method was demonstrated for the rapid identification of steroid esters in oily injection solutions, providing test results in less than 2 min.

Anabolic steroids and extreme violence: a case of murder after chronic intake and under acute influence of metandienone and trenbolone.

A 32-year-old male went to the police to claim he just killed his girlfriend by inflicting several stabs with a kitchen knife. He was very nervous and particularly aggressive. About 90 min after the assault, a blood specimen was collected with natrium fluoride as preservative. The blood was free of alcohol, pharmaceuticals and drugs of abuse, but tested positive by LC-MS/MS for metandienone (32 ng/mL) and trenbolone (9 ng/mL). The perpetrator admitted regular consumption of anabolic steroids to enhance his muscular mass, as he was a professional security agent. To document long-term steroid abuse, a hair specimen was collected 3 weeks after the assault, which tested positive for both drugs. Segmental analyses revealed in the proximal 1.5 cm segment, corresponding to the period of the assault, the simultaneous presence of metandienone (11 pg/mg) and trenbolone (14 pg/mg), while only metandienone (3 pg/mg) was identified in the distal 1.5 cm segment. As aggressiveness and violence can be associated with abuse of anabolic steroids, the aetiology of this domestic crime was listed to be due impulsive behaviour in a context of antisocial lifestyle.

Determination of anabolic androgenic steroids in dietary supplements and external drugs by magnetic solid-phase extraction combined with high-performance liquid chromatography-tandem mass spectrometry.

The widespread abuse of anabolic androgenic steroids by healthy people leads to the risk of major mood disorders and heart failure; thus, the determination of anabolic androgenic steroids is vital. In this study, 17 anabolic androgenic steroids in dietary supplements and external drugs were identified, and their concentration was determined. For this purpose, polyaniline-coated magnetic nanoparticles were prepared and then subjected to magnetic solid-phase extraction combined with high-performance liquid chromatography-tandem mass spectrometry. The experimental parameters of magnetic solid-phase extraction were studied in detail, and the optimal conditions were established. Under the optimal conditions, the limits of detection were in the range of 0.001-0.02 µg/L, with relative standard deviations of 5.52-11.6% (n = 7) for all the steroids, and the enrichment factors were in the range of 20.0-24.8. The developed method was then successfully applied for the determination of 17 anabolic androgenic steroids in real samples, and dehydroepiandrosterone (prasterone) was detected in a commercially available external drug.

Simple Synthesis of 17-ß-O-hemisuccinate of Stanozolol for Immunoanalytical Methods.

The use of doping in sports is a global problem that affects athletes around the world. Among the different methods developed to detect doping agents in biological samples, there are antibody-based methods that need an appropriate hapten design. Steroids with a hydroxyl group can be converted to the corresponding hemisuccinates. A novel approach to the synthesis of 17ß-O-hemisuccinate of the common doping agent stanozolol is described here. Acylation of stanozolol with methyl 4-chloro-4-oxobutyrate/4-dimethylaminopyridine, followed by mild alkaline hydrolysis with methanolic sodium hydroxide at room temperature, gave the simultaneous protection and deprotection of pyrazole-nitrogen atoms. The proposed new synthetic method allows the desired hemisuccinate derivative to be obtained in only two steps, and with a good total yield starting from stanozolol.

Human sports drug testing by mass spectrometry.

Since the installation of anti-doping rules and regulations and their international enforcement in the mid-1960s, mass spectrometry has been an integral part of doping control procedures. Although its utility was limited in the first decade, instrumental improvements and method optimizations have made mass spectrometry, in all its facets, an indispensable tool in modern sports drug testing. In this review, milestones in doping control analysis accomplished in Germany and reaching from the early developments to the current use of hyphenated mass spectrometric techniques concerning low- and high molecular mass analytes are presented. The considered drug classes include anabolic agents, peptidic drugs, nucleotide-derived therapeutics, approved and non-approved organic as well as inorganic analytes, and particular focus is put on drug class- and instrument-driven strategies. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 36:16-46, 2017.

Wastewater-based tracing of doping use by the general population and amateur athletes.

The present study investigates the applicability of the chemical analysis of wastewater to assess the use of doping substances by the general population and amateur athletes. To this end, an analytical methodology that can identify and quantify a list of 15 substances from the groups of anabolic steroids, weight loss products, and masking agents in wastewater has been developed. The method uses solid phase extraction to increase the detection sensitivity of the target analytes, expected to be present at very low concentrations (ng L-1 range), and decrease possible matrix interferences. Instrumental analysis is performed by liquid chromatography coupled to high-resolution mass spectrometry, allowing data acquisition in both full scan and tandem MS mode. The method has been successfully validated at two concentration levels (50 and 200 ng L-1) with limits of quantification ranging between 0.7 and 60 ng L-1, intra- and inter-day precision expressed as relative standard deviation below 15%, procedural recoveries between 60 and 160% and matrix effects ranging from 45 to 121%. The stability of the analytes in wastewater was evaluated at different storage temperatures illustrating the importance of freezing the samples immediately after collection. The application of the method to 24-h composite wastewater samples collected at the entrance of three wastewater treatment plants and one pumping station while different sport events were taking place revealed the presence in wastewater, and hence the use, of the weight loss substances ephedrine, norephedrine, methylhexanamine, and 2,4-dinitrophenol. The use of these stimulants was visible just prior and during the event days and in greater amounts than anabolic steroids or masking agents. Graphical abstract Chemical analysis of untreated wastewater reveals the use of prohibited doping substances during amateur sport event.

Mass spectrometric studies on selective androgen receptor modulators (SARMs) using electron ionization and electrospray ionization/collision-induced dissociation.

Selective androgen receptor modulators (SARMs) have been identified as a promising class of drug candidates potentially applicable to diverse pathological conditions commonly associated with significantly reduced muscle mass. Due to a suspected and meanwhile repeatedly proven misuse of SARMs in elite and amateur sport, sustaining constantly updated doping control analytical methods is critical for sports drug testing laboratories. These test methods predominantly utilize mass spectrometry-based instrumentations and, consequently, studies on the mass spectrometric behavior of new compounds and, where available, their metabolic products are vital for comprehensive doping controls. In this communication, the dissociation patterns of three new SARM drug candidates referred to as GSK2881078, PF-06260414, and TFM-4 AS-1 as observed under electron ionization as well as electrospray ionization/collision-induced dissociation are discussed. By means of high resolution/high accuracy tandem mass spectrometry employing quadrupole-orbitrap mass analyzers, information on precursor-product ion relationships and elemental compositions was obtained and subsequently utilized to suggest dissociation routes of the target compounds. This information can contribute to future studies concerning structure assignments of metabolites and accelerate the identification of related substances if distributed and/or illicitly used in the world of sport.

Appearance/Image- and Performance-Enhancing Drug Users: A Forensic Approach.

Image- and performance-enhancing drugs include a wide range of substances used to promote physical changes to enhance appearance. Anabolic androgen steroids are the most widely used image- and performance-enhancing drugs along with a wide variety of additional substances. The aim of this study was to identify the pathological changes pertaining to the cardiovascular system possibly involved in the death and characterize the substances associated with steroid use and their possible role in the death. A series of deaths involving image- and performance-enhancing drug users were selected. Nandrolone and testosterone were the most frequently identified steroids. The most commonly illegal drugs found were tetrahydrocannabinol, cocaine, and methadone. Antidepressants, opioids, benzodiazepines, and barbiturates were also found. These findings highlight that results obtained from postmortem investigations should be meticulously evaluated to understand the weight that each identified substance may have played in the death process and exclude any alternative causes of death.

Expanding analytical options in sports drug testing: Mass spectrometric detection of prohibited substances in exhaled breath.

RATIONALE: Continuously refining and advancing the strategies and methods employed in sports drug testing is critical for efficient doping controls. Besides improving and expanding the spectrum of target analytes, alternative test matrices have warranted in-depth evaluation as they commonly allow for minimal-/non-invasive and non-intrusive sample collection. In this study, the potential of exhaled breath (EB) as doping control specimen was assessed. METHODS: EB collection devices employing a non-woven electret-based air filter unit were used to generate test specimens, simulating a potential future application in doping controls. A multi-analyte sports drug testing approach configured for a subset of 12 model compounds that represent specific classes of substances prohibited in sports (anabolic agents, hormone and metabolic modulators, stimulants, and beta-blockers) was established using unispray liquid chromatography/tandem mass spectrometry (LC/MS/MS) and applied to spiked and elimination study EB samples. The test method was characterized concerning specificity, assay imprecision, and limits of detection. RESULTS: The EB collection device allowed for retaining and extracting all selected model compounds from the EB aerosol. Following elution and concentration, LC/MS/MS analysis enabled detection limits between 5 and 100 pg/filter and imprecisions ranging from 3% to 20% for the 12 selected model compounds. By means of EB samples from patients and participants of administration studies, the elimination of relevant compounds and, thus, their traceability in EB for doping control purposes, was investigated. Besides stimulants such as methylhexaneamine and pseudoephedrine, also the anabolic-androgenic steroid dehydrochloromethyltestosterone, the metabolic modulator meldonium, and the beta-blocker bisoprolol was detected in exhaled breath. CONCLUSIONS: The EB aerosol has provided a promising proof-of-concept suggesting the expansion of this testing strategy as a complement to currently utilized sports drug testing programs.

Detection and quantification of 12 anabolic steroids and analogs in human whole blood and 20 in hair using LC-HRMS/MS: application to real cases.

We developed and validated a method to detect and quantify 12 anabolic steroids in blood (androstenedione, dihydrotestosterone, boldenone, epitestosterone, mesterolone, methandienone, nandrolone, stanozolol, norandrostenedione, tamoxifene, testosterone, trenbolone) and eight more in hair samples (nandrolone phenylpropionate, nandrolone decanoate, testosterone propionate, testosterone benzoate, testosterone cypionate, testosterone decanoate, testosterone phenylpropionate, testosterone undecanoate) using liquid chromatography coupled to high-resolution mass spectrometry. This method used a benchtop Orbitrap mass spectrometer operating with an APCI probe under positive ionization mode. Analysis was realized in full scan experiment with a nominal resolving power of 140,000. After addition of the internal standard (testosterone-D3) and incubation in phosphate buffer pH = 5 for hair, 200 µL of blood and 30 mg of hair samples were extracted with heptane. LOQ and LOD were determined at 5 and 1 ng mL-1 in whole blood and 10 to 100 pg mg-1 and 2 to 20 pg mg-1 in hair according to the compounds, respectively. The method was linear in the 5-1000 ng mL-1 range in whole blood and between 10 or 100 pg mg-1 and 1000 pg mg-1 in hair with correlation coefficients >0.99, and intra- and inter-day accuracy and precision were <14.8% for all compounds except for some esters in hairs (<19.9%) probably due to an important matrix effect for these compounds. This sensitive and specific method to detect anabolic steroids has been successfully applied to two real cases, for which various anabolic steroids in whole blood, urine, and hair were identified and quantified.

Qualitative and Semiquantitative Analysis of Doping Products Seized at the Swiss Border.

BACKGROUND: Substances developed for therapeutic use are also known to be misused by athletes as doping agents and, outside of regulated sport, for image-enhancement. This has generated a market for counterfeit doping substances. Counterfeit doping agents may be of poor pharmaceutical quality and therefore constitute health risks to consumers. OBJECTIVES: This study aims to investigate the pharmaceutical quality of 1,190 doping products seized at the Swiss border. METHODS: Swiss customs authorities seize incoming shipments potentially containing doping agents. Qualitative and semiquantitative analyses were performed in order to test for prohibited doping substances. The main analytical methods utilized for characterizing confiscated compounds were liquid chromatography-high resolution mass spectrometry, polyacrylamide gel electrophoresis with subsequent in-gel tryptic digestion and identification of peptidic compounds using nanoliquid chromatography-tandem mass spectrometry, and electrochemiluminescence immuno assay. RESULTS: For 889 (75%) of the analyzed products, the label suggested the content of anabolic agents, for 146 samples (12%) peptide hormones or growth factors, and for 113 items (9%) antiestrogens, aromatase inhibitors or other metabolic modulators. For the majority of the investigated products, the pharmaceutical quality was an unsatisfactory standard: nonapproved substances were detected and less than 20% of the products contained the claimed substance in the respective amount. CONCLUSION: A comprehensive sample of confiscated doping products was analyzed, allowing for monitoring of developments regarding the use of doping substances in Switzerland and for anticipating future trends and challenges in sports drug testing. An alarming number of tested products was of substandard pharmaceutical quality.

Investigation of the selective androgen receptor modulators S1, S4 and S22 and their metabolites in equine plasma using high-resolution mass spectrometry.

RATIONALE: Selective androgen receptor modulators (SARMs) are prohibited in sports due to their performance enhancing ability. It is important to investigate the metabolism to determine appropriate targets for doping control. This is the first study where the equine metabolites of SARMs S1, S4 (Andarine) and S22 (Ostarine) have been studied in plasma. METHODS: Each SARM was administered to three horses as an intravenous bolus dose and plasma samples were collected. The samples were pretreated with protein precipitation using cold acetonitrile before separation by liquid chromatography. The mass spectrometric analysis was performed using negative electrospray, quadrupole time-of-flight mass spectrometry operated in MS(E) mode and triple-quadrupole mass spectrometry operated in selected reaction monitoring mode. For the quantification of SARM S1, a deuterated analogue was used as internal standard. RESULTS: The numbers of observed metabolites were eight, nine and four for the SARMs S1, S4 and S22, respectively. The major metabolite was formed by the same metabolic reactions for all three SARMs, namely amide hydrolysis, hydroxylation and sulfonation. The values of the determined maximum plasma concentrations were in the range of 97-170 ng/mL for SARM S1, 95-115 ng/mL for SARM S4 and 92-147 ng/mL for SARM S22 and the compounds could be detected for 96 h, 12 h and 18 h, respectively. CONCLUSIONS: The maximum plasma concentration of SARMs S1, S4 and S22 was measured in the first sample (5 min) after administration and they were eliminated fast from plasma. The proposed targets to be used in equine doping control are the parent compounds for all three SARMs, but with the metabolite yielding the highest response as a complementary target. Copyright © 2016 John Wiley & Sons, Ltd.

Development of a rapid method for the analysis of trenbolone, nortestosterone, and zeranol in bovine liver using liquid chromatography tandem mass spectrometry.

A rapid liquid chromatography tandem mass spectrometry method has been developed and validated for the determination of α-trenbolone, ß-trenbolone, α-nortestosterone, ß-nortestosterone, zeranol, and taleranol in bovine liver. The impact of liquid-liquid extraction with methyl tert-butyl ether and optimized solid phase extraction on silica cartridges significantly reduced effort and time of sample preparation. Electrospray ionization gives a significant signal increase compared with atmospheric pressure chemical ionization and atmospheric pressure photoionization. The HPLC gradient was optimized to separate isobaric analytes and matrix constituents from the hormone molecules. The optimized time and temperature of enzymatic hydrolysis of conjugated trenbolone was 4 h at 52 °C. The method validated in the range of 0.5-30 µg kg(-1) for α-trenbolone, ß-trenbolone, zeranol, taleranol, and 2-30 µg kg(-1) for α-nortestosterone, ß-nortestosterone. Combined uncertainty of measurements was in the range of 4%-23%. The matrix effect was negligible (1%-5%) for all analytes except of α-nortestosterone (19%). The developed method with changes concerning sample size and hydrolysis was also applied for the analysis of meat, serum, and urine samples. Graphical Abstract Determination of trenbolone, nortestosterone and zeranol in bovine liver.

Confirmation of protein biomarkers of corticosteroids treatment in veal calves sampled under field conditions.

In veal calf production, growth promoters are still illicitly used. Surveillance of misuse of such molecules is necessary to preserve human health. Methods currently adopted for their analysis are based on liquid chromatography-tandem mass spectrometry, but their efficacy can be affected by undetectable residual concentrations in biological matrices due to treatments at low-dosage or based on unknown anabolic compounds. The development of screening methods to identify the indirect biological effects of administration of growth promoters can improve the efficiency of drug residue monitoring. To this purpose, an integrated approach has been used to further validate the set of protein biomarkers defined in a previous controlled study to detect the use of corticosteroids through the changes caused in muscle protein expression. The thymus morphology of 48 samples collected under field conditions was evaluated to assess the presence of potential corticosteroids treatment. Animals were divided on the basis of their thymus characteristics in negative or suspected for illegal corticosteroids treatment. Drug residue analyses were performed on the liver, giving a satisfactory correlation with the histological examination (∼85%). Finally, the proteomics analysis of muscle protein extracts was carried out by 2D differential in gel electrophoresis, and proteins that were differentially expressed between the two animal groups (p value <0.01) were selected for MALDI-MS/MS analysis. This approach allowed us to identify 29 different proteins, and our findings indicate that the altered protein expression pattern can be used as an indirect method for the detection of illicit corticosteroids administration. A subset of the identified proteins was already reported in a previous controlled study, proving that these biomarkers can be used to develop a screening assay to improve the tools currently available for the detection of corticosteroids abuse in bovine meat production.