Potential of Treatment-Specific Protein Biomarker Profiles for Detection of Hormone Abuse in Cattle

Targeted protein biomarker profiling is suggested as a fast screening approach for detection of illegal hormone treatment in meat production. The advantage of using biomarkers is that they mark the biological response and, thus, are responsive to a panel of substances with similar effects. In a preliminary feasibility study, a 4-plex protein biomarker flow cytometric immunoassay (FCIA) previously developed for the detection of recombinant bovine somatotropin (rbST) was applied to cattle treated with steroids, such as estradiol, dexamethasone, and prednisolone. Each treatment resulted in a specific plasma biomarker profile for insulin-like growth factor-1 (IGF-1), IGF binding protein 2, osteocalcin, and anti-rbST antibodies, which could be distinguished from the profile of untreated animals. In summary, the 4-plex biomarker FCIA is, apart from rbST, also capable of detecting treatment with other growth-promoting agents and therefore clearly shows the potential of biomarker profiling as a screening method in veterinary control. It is proposed to perform additional validation studies covering high numbers of treated and untreated animals to support inclusion or adaptation of protein biomarker approaches in future monitoring regulations

Bovine teeth as a novel matrix for the control of the food chain: liquid chromatography-tandem mass spectrometry detection of treatments with prednisolone, dexamethasone, estradiol, nandrolone and seven beta(2)-agonists

Veterinary drugs usually have rapid clearance rates in the liver and kidney, hampering their detection in conventional matrices such as the liver or urine. Pharmacological principles such as esterification may be applied to facilitate the administration of veterinary drugs and increase drug half-life. Prednisolone, whose therapeutic administration is regulated for food producing animals in the EU, is available in its acetate form as well as nandrolone, a banned anabolic steroid, which may be obtained as nandrolone phenylpropionate and estradiol as a benzoyl ester. While the distribution and accumulation of lipophilic and hydrophilic substances in human teeth have been well documented, studies on residues in bovine teeth are lacking. We hypothesised that analysis of bovine teeth could be used to detect both regulated and banned veterinary drugs. Steroids may be illegally used as growth promoters in food producing animals, alone or combined with \u3b22-agonists; therefore, we developed, and validated, in accordance with the Commission Decision 2002/657/EC, two analytical confirmatory LC-MS/MS methods to detect these classes of compounds following a unique liquid extraction procedure. Finally, we analysed teeth from three male Friesian veal calves treated with intramuscular estradiol benzoate, oral prednisolone acetate or intramuscular nandrolone phenylpropionate in combination with oral ractopamine, respectively, and from seven bovines from the food chain. Teeth from treated animals were positive for their respective drugs, with the exception of nandrolone phenylpropionate. One sample from a food chain bovine was positive for isoxsuprine, one of the seven \u3b22-agonists studied. Non-esterified forms of the steroids were not found. These results demonstrate that bovine teeth are a suitable matrix for the determination of pseudoendogenous substances or illicit administration of veterinary drugs

A liquid chromatography-tandem mass spectrometry method for the detection of antimicrobial agents from seven classes in calf milk replacers: validation and application

Calf milk replacers are low-cost feeds that contain available, digestible protein. During their reconstitution, however, the addition of drugs, such as antibiotics, could make them a very simple route for illicit treatment for therapeutic, preventive, or growth-promoting purposes. We developed an HPLC-MS/MS method, preceded by a unique extraction step, able to identify 17 antibiotics from seven classes (penicillins, tetracyclines, fluoroquinolones, sulfonamides, cephalosporins, amphenicols, and lincosamides) in this matrix. Prior to solid phase extraction (SPE), the sample underwent deproteinization and defatting. The method was fully validated according to Commission Decision 2002/657/EC. Decision limits (CC\u3b1) and detection capability (CC\u3b2) were in the ranges of 0.13-1.26 and 0.15-1.47 ng/mL, respectively. Thirty-eight samples were finally analyzed, showing the occasional presence of marbofloxacin (six samples) and amoxicillin (one sample)

Determination of cortisol, cortisone, prednisolone and prednisone in bovine urine by liquid chromatography\u2013electrospray ionisation single quadrupole mass spectrometry

A quantitative LC-ESI single quadrupole MS method for the determination of cortisol (F), cortisone (E), prednisolone (PL), and prednisone (PN) in bovine urine has been developed and validated. After adding flumethasone as internal standard, the samples were subjected to filtration, deconjugation, and solid-phase extraction, while the chromatographic separation was achieved using a Restek Ultra II Allure Biphenyl column with isocratic mobile phase. The analytes were detected after negative electrospray ionization using SIM mode. In order to obtain spectra with maximum intensities of at least one of the three characteristic ions, (M+formate), (MH), and [(MH) -CH2O], an individual optimization of MS parameters for each corticosteroid was set up. MS data was acquired in the three-ion selected monitoring mode and the ion ratios between chosen diagnostic ions were used in order to increase the specificity. Calibration graphs were linear and the intra-day and intermediate precision was estimated as RSD values which were less than 17%. For F and E, obtained values indicated negligible absolute matrix effects (103% and 98%, respectively). The method was applied to real samples, and basal levels of F and E were preliminarily evaluated, while PL and PN were not detected

Pseudoendogenous presence of β-boldenone sulphate and glucuronide in untreated young bulls from the food chain

The administration of boldenone (bold) to bovines, either for growth promotion or therapeutic purposes, has been banned in the EU since 1981. It is, however, a pseudoendogenous hormone, thus its detection in bovine urine, in the form of \u3b1-boldenone conjugates, is considered fully compliant up to 2 ng ml(-1). Greater attention has been placed on \u3b2-boldenone, the anabolic active epimer, whose conjugated form must be absent in urine. Recently, the identification of a biomarker representing unquestionable evidence of illicit treatment with bold or its precursor androstadienedione has been a major topic in the literature regarding the detection of residues in bovine urine, and \u3b2-boldenone sulphate is a candidate molecule. In this study, we used a method previously validated according to the European Commission Decision 2002/657/EC for the determination of sulphate and glucuronide conjugates of \u3b2-boldenone. We assessed the occurrence of these molecules in young bull urine, with the aim of understanding whether they could be of endogenous origin, and to check for a possible relationship with particular environmental and stress conditions. Urine samples from 56 young bulls were collected after transport stress, under non-stressful conditions and after transport and slaughter stress. Histopathological investigation of the hormone target organs, i.e. the bulbourethral and prostate glands, was also performed. The results indicate an inverse relationship between the presence and concentration of \u3b2-boldenone sulpho- and gluco-conjugates in urine, and stress conditions, expressed by the absence of detection at the slaughterhouse. No significant macroscopic and histologic lesions were detected. Our study indicates that \u3b2-boldenone sulphate could be a biomarker of treatment only at the slaughterhouse, while at the farm, in untreated animals (i.e. after a five-month period under the control of Official Veterinarians), sulphate and glucuronide metabolites were found with a frequency of 78% and 46%, respectively, showing the endogenous origin of boldenone

Detection of selected corticosteroids and anabolic steroids in calf milk replacers by liquid chromatography-electrospray ionisation - Tandem mass spectrometry

The use of corticosteroids and anabolic steroids in food producing animals is regulated or banned in the European Union (EU). However, their use as growth promoters cannot be excluded. Milk replacers, considered by EU legislation as feeds, may be a good way of administration of these compounds. In order to improve the control of growth promoter utilization in animal husbandry and preventing possible consequences to animal welfare, we developed a method for multiresidue analysis of prednisolone, prednisone, dexamethasone, cortisone, cortisol, 17\u3b1- and 17\u3b2-boldenone and their precursor androstadienedione (ADD), testosterone, epitestosterone, 17\u3b1- and 17\u3b2-nandrolone, and trenbolone in powdered milk for calves. All analytes were extracted, after a common deproteinization and defatting sample pre-treatment, by a unique immunoaffinity column and analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) in both positive and negative electrospray ionization (ESI) modes. The method was validated according to the criteria of the Commission Decision 2002/657/CE. The analytical limits were from 0.39 to 0.73 ng mL-1 for the decision limit (CC\u3b1) and 0.46-0.99 ng mL-1 for detection capability (CC\u3b2). The analysis of 50 samples of milk replacers for calves, always revealed the presence of cortisol and cortisone (average concentrations 2.56 and 1.06 ng mL-1, respectively), frequently testosterone and epitestosterone (1.24 and 0.63 ng mL-1, respectively), occasionally \u3b2-nandrolone (0.82 ng mL-1) and prednisolone (0.41 ng mL-1). The other anabolic steroids were never found

Use of NMR metabolomic plasma profiling methodologies to identify illicit growth-promoting administrations

Detection of growth-promoter use in animal production systems still proves to be an analytical challenge despite years of activity in the field. This study reports on the capability of NMR metabolomic profiling techniques to discriminate between plasma samples obtained from cattle treated with different groups of growth-promoting hormones (dexamethasone, prednisolone, oestradiol) based on recorded metabolite profiles. Two methods of NMR analysis were investigated-a Carr-Purcell-Meiboom-Gill (CPMG)-pulse sequence technique and a conventional H-1 NMR method using pre-extracted plasma. Using the CPMG method, 17 distinct metabolites could be identified from the spectra. H-1 NMR analysis of extracted plasma facilitated identification of 23 metabolites-six more than the alternative method and all within the aromatic region. Multivariate statistical analysis of acquired data from both forms of NMR analysis separated the plasma metabolite profiles into distinct sample cluster sets representative of the different animal study groups. Samples from both sets of corticosteroid-treated animals-dexamethasone and prednisolone-were found to be clustered relatively closely and had similar alterations to identified metabolite panels. Distinctive metabolite profiles, different from those observed within plasma from corticosteroid-treated animal plasma, were observed in oestradiol-treated animals and samples from these animals formed a cluster spatially isolated from control animal plasma samples. These findings suggest the potential use of NMR methodologies of plasma metabolite analysis as a high-throughput screening technique to aid detection of growth promoter use

A RIKILT yeast estrogen bioassay (REA) for estrogen residue detection in urine of calves experimentally treated with 17ß-estradiol

17ß-Estradiol is one of the most powerful sex steroids illegally used in bovine production. The objective of this study was to evaluate the application and the specificity of the RIKILT yeast estrogen bioassay (REA) for the detection of molecules with estrogenic activities in the urine of calves experimentally treated with anabolics. Four groups of six calves each received an injection of 17ß-estradiol intramuscularly (group B), androsterone and gliburide (group A), and testosterone (group C) molecules at different dosage for 40 days. Group D was the control. The ability of the REA test to detect estrogenic activity in urine samples from all animals was assessed. All estrogen-treated animals (group B) showed as being positive up to 7 days after administration of the highest dosage of 17ß-estradiol, while the other three groups showed as being negative. The identity of estrogenic molecules in the urine of group B (17ß-estradiol, 17-estradiol) was confirmed by gas chromatography-mass spectrometry (GC/MS). This is the first time the REA test has been applied to detect 17ß-estradiol in the urine of calves treated with the hormone in vivo. The technique may offer an advantageous laboratory method for the veterinary surveillance of illegal steroid use

Presence of β-boldenone sulphate and glucuronide in urine of untreated beef cattle

The use of the anabolic steroids in livestock breeding is prohibited by the EU legislation. As regards \u3b2-boldenone, the presence of its conjugates at any levels in urine is a proof of illegal treatment. A debate on the biomarker indicating the illicit use of boldenone is nevertheless in progress. The sulphate and glucuronide metabolites of \u3b2-boldenone were determined in urine from untreated, and under veterinary control, Friesian and Limousine beef cattle. A new method for the direct determination in urine of the II phase metabolites of \u3b2-boldenone by HPLC-MS/MS was used after extraction trough immunoaffinity columns.The preliminary data show the presence of the sulphate and glucuronide forms in the 75% and 67% percent of urine samples collected at the farm from the studied animals, respectively, and its absence in those from slaughterhouse. These results suggest that presence of \u3b2-boldenone conjugate, as both sulphate and glucuronide, in bovine urine cannot be always considered a biomarker of illicit treatment but that a threshold level should be set. These preliminary data need to be strengthened through a wider study including negative and experimentally treated animals