Gas chromatography coupled to mass spectrometry‐based metabolomic to screen for anabolic practices in cattle: identification of 5α‐androst‐2‐en‐17‐one as new biomarker of 4‐androstenedione misuse

Abstract

The use of anabolic steroids as growth promoters for meat-producing animals is banned within the European Union. However, screening for the illegal use of natural steroid hormones still represents a difficult challenge because of the high interindividual and physiological variability of the endogenous concentration levels in animals. In this context, the development of untargeted profiling approaches for identifying new relevant biomarkers of exposure and/or effect has been emerging for a couple of years. The present study deals with an untargeted metabolomics approach on the basis of GC-MS aiming to reveal potential biomarkers signing a fraudulent administration of 4-androstenedione (AED), an anabolic androgenic steroid chosen as template. After a sample preparation based on microextraction by packed sorbent, urinary profiles of the free and deglucurono-conjugates urinary metabolites were acquired by GC-MS in the full-scan acquisition mode. Data processing and chemometric procedures highlighted 125 ions, allowing discrimination between samples collected before and after an administration of 4-AED. After a first evaluation of the signal robustness using additional and independent non-compliant samples, 17 steroid-like metabolites were pointed out as relevant candidate biomarkers. All these metabolites were then monitored using a targeted GC-MS/MS method for an additional assessment of their capacity to be used as biomarkers. Finally, two steroids, namely 5α-androstane-3β,17α-diol and 5α-androst-2-en-17-one, were concluded to be compatible with such a definition and which could be finally usable for screening purpose of AED abuse in cattle.

Detection of 5α‐androst‐2‐en‐17‐one and variants: Identification of main urinary metabolites in human urine samples by GC‐MS and NMR

“…[13] At the end of 2015, an investigative documentary revealed that some North American professional athletes claimed to have consumed a designer steroid called D-2, which they believed to be undetectable. [18] Finally, the only existing link with DHEA is its formation following incubations of the sulfates of DHEA and androsterone with human axillary bacterial isolates. [15] In fact, 5α-androst-2-en-17-one (1) was described in the scientific literature as an aromatase inhibitor of the growth of hormone-dependent breast cancer cells, [16] which per se makes it a prohibited substance.…”

“…[17] Formed by the thermal degradation of primary metabolites epiandrosterone and androsterone sulfates during GC-MS analysis, 1 was proposed as a new biomarker of 4-androstenedione misuse in cattle. [18] Finally, the only existing link with DHEA is its formation following incubations of the sulfates of DHEA and androsterone with human axillary bacterial isolates. [19] With regard to the alleged anabolic activity, in 1963 Bowers et al described 'reasonable anabolic activity' for 2-ene and 3-ene 17β-ol steroids, particularly marked for the 17α-methylated compound (e.g.…”

Doping Control Using High and Ultra-High Resolution Mass Spectrometry Based Non-Targeted Metabolomics-A Case Study of Salbutamol and Budesonide Abuse

“…Up to present, this strategy has been applied in a variety of fields, including drug discovery [13,14], nutrition [15,16], toxicology [17], clinical trials [18] and more recently chemical submission [19]. It is of particular interest in areas like doping control as new approaches are needed to fight the ongoing development of performance-enhancing methods [20-23]. …”

Discovery of urinary biomarkers to discriminate between exogenous and semi-endogenous thiouracil in cattle: A parallel-like randomized design

“…Such a biomarker approach was already proven effective to unequivocally point out steroid treated animals, i.e. livestock being administered with prednisolone [ 18 ], 4-androstenedione [ 19 ], 17β-nandrolone laureate esters and 17β-estradiol 3-benzoate [ 20 ], or nadrolone [ 21 ]. Hereby, adequate analytical techniques, including nuclear magnetic resonance or full-scan mass spectrometry (time of flight, Orbitrap or Fourier transform ion cyclotron resonance), are most suited to perform global metabolome analyses [ 22 ].…”