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F. Arioli - One of the best experts on this subject based on the ideXlab platform.
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Pseudoendogenous presence of β-Boldenone sulphate and glucuronide in untreated young bulls from the food chain
'Informa UK Limited', 2015Co-Authors: L. Chiesa, Francesca Tiziana Cannizzo, Bartolomeo Biolatti, E. Pasquale, S. Panseri, R. Pavlovic, F. ArioliAbstract: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 α-Boldenone conjugates, is considered fully compliant up to 2 ng ml(-1). Greater attention has been placed on β-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 β-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 β-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 β-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 β-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
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detection of Boldenone its conjugates and androstadienedione as well as five corticosteroids in bovine bile through a unique immunoaffinity column clean up and two validated liquid chromatography tandem mass spectrometry analyses
Analytica Chimica Acta, 2014Co-Authors: L M Chiesa, Radmila Pavlovic, Sara Panseri, C Sgoifo A Rossi, Maria Nobile, F. ArioliAbstract:Abstract The presence of β-Boldenone II phase metabolites and prednisolone in urine samples, owing to endogenous or natural origin or illicit treatment, is under debate within the European Union. The detection of β-Boldenone conjugates, α-Boldenone conjugates at concentrations higher than 2 ng mL −1 and prednisolone above the cut-off level of 5 ng mL −1 in urine have been, until now, critical in deciding if illegal drug use has occurred. The use of urine sometimes is not entirely satisfactory, especially when the drug is administrated at low doses or when its metabolic conversion is very fast. This subsequently would hamper its detection in urine. The introduction of a new, advantageous matrix where the illicit treatment can be investigated would be highly appreciated. In this study, we have developed and validated a simple and unique immunoaffinity clean-up procedure, which was applied to bovine bile samples, followed by two different analytical liquid chromatography-electrospray-tandem mass spectrometry methods. The first method tests androstadienedione, α- and β-Boldenone sulphate, glucuronate and related free forms, while the other method assays prednisolone, prednisone, dexamethasone, cortisone, and cortisol. The methods were validated according to European Commission Decision 2002/657/EC. The evaluated parameters were linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit and detection capability. The decision limits (CCα) were between 0.38 and 0.45 ng mL −1 for anabolic steroids, and 0.13 and 0.15 ng mL −1 as far as corticosteroids were concerned. Intra- and inter-day repeatability was below 15.8 and 19.9% for all analytes, respectively. The methods were applied to the analysis of some bile samples collected from untreated young bulls in order to investigate the presence of the studied steroids in this matrix.
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neoformation of Boldenone and related steroids in faeces of veal calves
Food Additives and Contaminants Part A-chemistry Analysis Control Exposure & Risk Assessment, 2006Co-Authors: G Pompa, F. Arioli, M L Fracchiolla, C Sgoifo A Rossi, A L Bassini, S Stella, P A BiondiAbstract:Abstract Conflicting findings regarding the Boldenone content of bovine faeces suggest it may be synthesized de novo in emitted faeces. We tested this hypothesis by analysing uncontaminated urine, fresh and various forms of dried faeces from 10 calves (not given Boldenone) by liquid chromatography/tandem mass spectrometry for 17α- and 17β-Boldenone (α and β BOL); 1,4-androstadiene-3,17-dione (ADD); 4-androstene-3,17-dione (AED), testosterone (T) and epitestosterone (ET). Urine contained no α BOL, β BOL or ADD. The analysed substances were variably present in the rectal faeces, and at generally higher levels in faeces scraped from skin or stall floor. In pooled rectal faeces naturally dried for 13 days, α BOL, ADD, AED and ET levels were extremely high (much higher than accounted for by increases due to drying), and β BOL and T were absent. It is concluded that de novo synthesis of α BOL and metabolites occurs naturally in bovine faeces and only uncontaminated urine should be analysed for illegal Boldenone.
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evidence for false positive results for Boldenone testing of veal urine due to faecal cross contamination during sampling
Food Additives and Contaminants Part A-chemistry Analysis Control Exposure & Risk Assessment, 2004Co-Authors: C Sgoifo A Rossi, F. Arioli, A L Bassini, L M Chiesa, V Dellorto, M Montana, G PompaAbstract:European Directive 96/22/EC, which controls veterinary residues in animals, does not permit the presence of synthetic growth promoters in products of animal origin or in livestock. Boldenone is categorized in class A3 (growth promoters — steroids) and is thus a banned substance. Testing of veal urine for banned substances is part of the European Union statutory programme for animals going into the food chain. In relation to this monitoring, three studies were conducted to investigate the apparent presence of the banned growth promoter Boldenone in veal urine, which was suspected as being caused by interference from faecal contamination of the sample. In the first study, urine samples were collected at different times (time 0 and after 30 min) using (1) a conventional zoonotechnical apron and (2) a technique designed specifically to avoid faecal contamination (‘kettle’). This resulted in samples that were, respectively, positive and negative for the presence of α-Boldenone (α-BOL). In a second study, urine...
Francesco Arioli - One of the best experts on this subject based on the ideXlab platform.
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Suitability of bovine bile compared to urine for detection of free, sulfate and glucuronate Boldenone, androstadienedione, cortisol, cortisone, prednisolone, prednisone and dexamethasone by LC–MS/MS
Food Chemistry, 2015Co-Authors: Luca Maria Chiesa, Radmila Pavlovic, Sara Panseri, Maria Nobile, Daniele Vigo, Francesco ArioliAbstract:Abstract The administration of Boldenone and androstadienedione to cattle is forbidden in the European Union, while prednisolone is permitted for therapeutic purposes. They are pseudoendogenous substances (endogenously produced under certain circumstances). The commonly used matrices in control analyses are urine or liver. With the aim of improving the residue controls, we previously validated a method for steroid analysis in bile. We now compare urine (a ‘classic’ matrix) to bile, both collected at the slaughterhouse, to understand whether the detection of steroids in the latter is easier. With the aim of having clearer results, we tested the presence of the synthetic corticosteroid dexamethasone. The results show that bile does not substantially improve the detection of Boldenone, or its conjugates, prednisolone and prednisone. Dexamethasone, instead, was found in 10 out of 53 bovine bile samples, but only in one urine sample from the same animals. Bile could constitute a novel matrix for the analysis of residues in food-producing animals, and possibly not only of synthetic corticosteroids.
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Pseudoendogenous presence of β-Boldenone sulphate and glucuronide in untreated young bulls from the food chain.
Food Additives and Contaminants Part A-chemistry Analysis Control Exposure & Risk Assessment, 2015Co-Authors: Luca Maria Chiesa, Radmila Pavlovic, Elisa Di Pasquale, Sara Panseri, Francesca Tiziana Cannizzo, Bartolomeo Biolatti, Francesco ArioliAbstract: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 α-Boldenone conjugates, is considered fully compliant up to 2 ng ml−1. Greater attention has been placed on β-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 β-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 β-Boldenone. We assessed the occurrence of these molecules in young bull urine, with the aim of understanding whether th...
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Detection of Boldenone, its conjugates and androstadienedione, as well as five corticosteroids in bovine bile through a unique immunoaffinity column clean-up and two validated liquid chromatography–tandem mass spectrometry analyses
Analytica Chimica Acta, 2014Co-Authors: Luca Maria Chiesa, Radmila Pavlovic, Sara Panseri, Maria Nobile, C.a. Sgoifo Rossi, Francesco ArioliAbstract:Abstract The presence of β-Boldenone II phase metabolites and prednisolone in urine samples, owing to endogenous or natural origin or illicit treatment, is under debate within the European Union. The detection of β-Boldenone conjugates, α-Boldenone conjugates at concentrations higher than 2 ng mL −1 and prednisolone above the cut-off level of 5 ng mL −1 in urine have been, until now, critical in deciding if illegal drug use has occurred. The use of urine sometimes is not entirely satisfactory, especially when the drug is administrated at low doses or when its metabolic conversion is very fast. This subsequently would hamper its detection in urine. The introduction of a new, advantageous matrix where the illicit treatment can be investigated would be highly appreciated. In this study, we have developed and validated a simple and unique immunoaffinity clean-up procedure, which was applied to bovine bile samples, followed by two different analytical liquid chromatography-electrospray-tandem mass spectrometry methods. The first method tests androstadienedione, α- and β-Boldenone sulphate, glucuronate and related free forms, while the other method assays prednisolone, prednisone, dexamethasone, cortisone, and cortisol. The methods were validated according to European Commission Decision 2002/657/EC. The evaluated parameters were linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit and detection capability. The decision limits (CCα) were between 0.38 and 0.45 ng mL −1 for anabolic steroids, and 0.13 and 0.15 ng mL −1 as far as corticosteroids were concerned. Intra- and inter-day repeatability was below 15.8 and 19.9% for all analytes, respectively. The methods were applied to the analysis of some bile samples collected from untreated young bulls in order to investigate the presence of the studied steroids in this matrix.
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Determination of α- and β-Boldenone sulfate, glucuronide and free forms, and androstadienedione in bovine urine using immunoaffinity columns clean-up and liquid chromatography tandem mass spectrometry analysis.
Talanta, 2014Co-Authors: Luca Maria Chiesa, Radmila Pavlovic, Guglielmo Dusi, Elisa Di Pasquale, Alessio Casati, Sara Panseri, Francesco ArioliAbstract:Abstract The debate about the origin s of Boldenone in bovine urine is ongoing for two decades in Europe. Despite the fact that its use as a growth promoter has been banned in the European Union (EU) since 1981, its detection in bovine urine, in the form of α-Boldenone conjugate, is considered fully compliant up to 2 ng mL−1. The conjugated form of β-Boldenone must be absent. In recent years, the literature about Boldenone has focused on the identification of biomarkers that can indicate an illicit treatment. β-Boldenone sulfate is a candidate molecule, even if the only studies currently available have taken place in small populations. In this study, a method for the determination of sulfate and glucuronate conjugates of β-Boldenone was developed and validated according to the European Commission Decision 2002/657/EC and applied to α-Boldenone sulfate and glucuronide, α- and β-Boldenone free forms and androstadienedione (ADD), too. The clean-up with immunoaffinity columns enabled the direct determination of the conjugates and free forms and allowed specific and sensitive analyses of urine samples randomly selected to verify this method. The decision limits (CCα) ranged between 0.07 and 0.08 ng mL−1, the detection capabilities (CCβ) between 0.08 and 0.1 ng mL−1. Recovery was higher than 92% for all the analytes. Intra-day repeatability was between 5.8% and 17.2%, and inter-day repeatability was between 6.0% and 21.8% for the studied free and conjugated forms. This method has been developed as a powerful tool with the aim to study the origin of Boldenone in a trial on a significant number of animals.
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Neoformation of Boldenone and related steroids in faeces of veal calves
Food Additives and Contaminants, 2006Co-Authors: G Pompa, Francesco Arioli, M L Fracchiolla, A L Bassini, S Stella, Carlo A. Sgoifo Rossi, Pierantonio BiondiAbstract:Conflicting findings on the Boldenone content of bovine faeces suggests it may be synthesised de novo in emitted faeces. We tested this hypothesis by analysing uncontaminated urine, fresh and various forms of dried faeces from 10 calves (not given Boldenone) by liquid chromatography/tandem mass spectrometry for 17alpha- and 17beta-Boldenone; 1,4-androstadiene-3,17-dione (ADD); 4-androstene-3,17-dione (AED), testosterone (T) and epitestosterone (ET). Urine contained no alpha BOL, beta BOL or ADD. Analysed substances were variably present in rectal faeces, and at higher levels in faeces scraped from skin or the stall floor. In pooled rectal faeces naturally dried for 13 days, alpha BOL, ADD, AED and ET levels were extremely high (much higher than accounted for by increase due to drying), while beta BOL and T were absent. De novo synthesis of alpha BOL and metabolites occurs naturally in bovine faeces and only uncontaminated urine should be analysed for illegal Boldenone.
Guglielmo Dusi - One of the best experts on this subject based on the ideXlab platform.
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Determination of α- and β-Boldenone sulfate, glucuronide and free forms, and androstadienedione in bovine urine using immunoaffinity columns clean-up and liquid chromatography tandem mass spectrometry analysis.
Talanta, 2014Co-Authors: Luca Maria Chiesa, Radmila Pavlovic, Guglielmo Dusi, Elisa Di Pasquale, Alessio Casati, Sara Panseri, Francesco ArioliAbstract:Abstract The debate about the origin s of Boldenone in bovine urine is ongoing for two decades in Europe. Despite the fact that its use as a growth promoter has been banned in the European Union (EU) since 1981, its detection in bovine urine, in the form of α-Boldenone conjugate, is considered fully compliant up to 2 ng mL−1. The conjugated form of β-Boldenone must be absent. In recent years, the literature about Boldenone has focused on the identification of biomarkers that can indicate an illicit treatment. β-Boldenone sulfate is a candidate molecule, even if the only studies currently available have taken place in small populations. In this study, a method for the determination of sulfate and glucuronate conjugates of β-Boldenone was developed and validated according to the European Commission Decision 2002/657/EC and applied to α-Boldenone sulfate and glucuronide, α- and β-Boldenone free forms and androstadienedione (ADD), too. The clean-up with immunoaffinity columns enabled the direct determination of the conjugates and free forms and allowed specific and sensitive analyses of urine samples randomly selected to verify this method. The decision limits (CCα) ranged between 0.07 and 0.08 ng mL−1, the detection capabilities (CCβ) between 0.08 and 0.1 ng mL−1. Recovery was higher than 92% for all the analytes. Intra-day repeatability was between 5.8% and 17.2%, and inter-day repeatability was between 6.0% and 21.8% for the studied free and conjugated forms. This method has been developed as a powerful tool with the aim to study the origin of Boldenone in a trial on a significant number of animals.
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development and validation of a liquid chromatography tandem mass spectrometry method for the separation of conjugated and unconjugated 17α and 17β Boldenone in urine sample
Analytica Chimica Acta, 2007Co-Authors: Mara Gasparini, Walter Assini, Eros Bozzoni, Nadia Tognoli, Guglielmo DusiAbstract:Abstract Natural occurrence or illegal treatment of Boldenone (BOLD) presence in cattle urine is under debate within the European Union. Separation of conjugated and unconjugated forms of 17α-Boldenone (α-BOLD) and 17β-Boldenone (β-BOLD) and presence of related molecules as androsta-1,4-diene-3,17-dione (ADD) appear critical points for the decision of an illegal use. The aim of this study is a new analytical approach of BOLD and ADD confirmation in cattle urine. The separation between conjugated and unconjugated forms of BOLD was obtained by a preliminary urine liquid–liquid extraction step with ethyl acetate. In this step the organic phase extracts only unconjugated BOLD and ADD, while BOLD in conjugated form remain in urine phase. Afterwards the urine phase, contains conjugated BOLD, was subjected to an enzymatic deconjugation. Solid-phase extraction (OASIS-HLB Waters) was used for the purification and concentration of analytes in organic and urine phases and liquid chromatography ion electrospray tandem mass spectrometry (LC–MS–MS) was applied for the confirmation of BOLD and ADD, using deuterium-labelled 17β-Boldenone (BOLD-d3) as internal standard. The method was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/CE. The results obtained demonstrate that the developed method show very high specificity, precision, trueness and ruggedness. Decision limits (CCα) smaller than 0.5 ng mL−1 were obtained for each analyte.
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Development and validation of a liquid chromatography–tandem mass spectrometry method for the separation of conjugated and unconjugated 17α- and 17β-Boldenone in urine sample
Analytica Chimica Acta, 2006Co-Authors: Mara Gasparini, Walter Assini, Eros Bozzoni, Nadia Tognoli, Guglielmo DusiAbstract:Abstract Natural occurrence or illegal treatment of Boldenone (BOLD) presence in cattle urine is under debate within the European Union. Separation of conjugated and unconjugated forms of 17α-Boldenone (α-BOLD) and 17β-Boldenone (β-BOLD) and presence of related molecules as androsta-1,4-diene-3,17-dione (ADD) appear critical points for the decision of an illegal use. The aim of this study is a new analytical approach of BOLD and ADD confirmation in cattle urine. The separation between conjugated and unconjugated forms of BOLD was obtained by a preliminary urine liquid–liquid extraction step with ethyl acetate. In this step the organic phase extracts only unconjugated BOLD and ADD, while BOLD in conjugated form remain in urine phase. Afterwards the urine phase, contains conjugated BOLD, was subjected to an enzymatic deconjugation. Solid-phase extraction (OASIS-HLB Waters) was used for the purification and concentration of analytes in organic and urine phases and liquid chromatography ion electrospray tandem mass spectrometry (LC–MS–MS) was applied for the confirmation of BOLD and ADD, using deuterium-labelled 17β-Boldenone (BOLD-d3) as internal standard. The method was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/CE. The results obtained demonstrate that the developed method show very high specificity, precision, trueness and ruggedness. Decision limits (CCα) smaller than 0.5 ng mL−1 were obtained for each analyte.
A. Terracciano - One of the best experts on this subject based on the ideXlab platform.
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Detection of Boldenone and its major metabolites by liquid chromatography—tandem mass spectrometry in urine samples
Analytica Chimica Acta, 2005Co-Authors: Francesca Buiarelli, G.p. Cartoni, F. Coccioli, L. Giannetti, M. Merolle, Bruno Neri, A. TerraccianoAbstract:Boldenone is an androgenic anabolic steroid (AAS) intensively used for growth promoting purposes in animals destined for meat production and as a performance enhancer in athletics. Therefore its use is officially banned either in animals intended for consumption or in humans. Because most anabolic steroids are completely metabolized and usually no parent steroid is excreted, metabolite identification is crucial to detect the illegal use of anabolic steroids either in humans or in livestock. The aim of this work is the investigation of 17-Boldenone and its main metabolites, 17-sulphate, 17-glucuronide, 5-androst-1-en17-ol-3-one and 17-Boldenone, in human and bovine urine developing a multiresidue analysis. After solid phase extraction of urine samples, detection is carried out by high performance liquid chromatography–tandem mass spectrometry in multiple reaction monitoring. The average recovery for all the investigated compounds is above 70%. The developed method is very easy, quick and highly specific. Linearity, precision, decision limit and detection capability were also evaluated. © 2005 Elsevier B.V. All rights reserved.
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detection of Boldenone and its major metabolites by liquid chromatography tandem mass spectrometry in urine samples
Analytica Chimica Acta, 2005Co-Authors: Francesca Buiarelli, G.p. Cartoni, F. Coccioli, L. Giannetti, M. Merolle, Bruno Neri, A. TerraccianoAbstract:Boldenone is an androgenic anabolic steroid (AAS) intensively used for growth promoting purposes in animals destined for meat production and as a performance enhancer in athletics. Therefore its use is officially banned either in animals intended for consumption or in humans. Because most anabolic steroids are completely metabolized and usually no parent steroid is excreted, metabolite identification is crucial to detect the illegal use of anabolic steroids either in humans or in livestock. The aim of this work is the investigation of 17-Boldenone and its main metabolites, 17-sulphate, 17-glucuronide, 5-androst-1-en17-ol-3-one and 17-Boldenone, in human and bovine urine developing a multiresidue analysis. After solid phase extraction of urine samples, detection is carried out by high performance liquid chromatography–tandem mass spectrometry in multiple reaction monitoring. The average recovery for all the investigated compounds is above 70%. The developed method is very easy, quick and highly specific. Linearity, precision, decision limit and detection capability were also evaluated. © 2005 Elsevier B.V. All rights reserved.
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Detection of Boldenone and its major metabolites by liquid chromatography—tandem mass spectrometry in urine samples
Analytica Chimica Acta, 2005Co-Authors: Francesca Buiarelli, G.p. Cartoni, F. Coccioli, L. Giannetti, M. Merolle, Bruno Neri, A. TerraccianoAbstract:Boldenone is an androgenic anabolic steroid (AAS) intensively used for growth promoting purposes in animals destined for meat prodn. and as a performance enhancer in athletics. Therefore its use is officially banned either in animals intended for consumption or in humans. Because most anabolic steroids are completely metabolized and usually no parent steroid is excreted, metabolite identification is crucial to detect the illegal use of anabolic steroids either in humans or in livestock. The aim of this work is the investigation of 17.beta.-Boldenone and its main metabolites, 17.beta.-sulfate, 17.beta.-glucuronide, 5.beta.-androst-1-en-17.beta.-ol-3-one and 17.alpha.-Boldenone, in human and bovine urine developing a multiresidue anal. After solid phase extn. of urine samples, detection is carried out by high performance liq. chromatog.-tandem mass spectrometry in multiple reaction monitoring. The av. recovery for all the investigated compds. is above 70%. The developed method is very easy, quick and highly specific. Linearity, precision, decision limit and detection capability were also evaluated
Radmila Pavlovic - One of the best experts on this subject based on the ideXlab platform.
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Suitability of bovine bile compared to urine for detection of free, sulfate and glucuronate Boldenone, androstadienedione, cortisol, cortisone, prednisolone, prednisone and dexamethasone by LC–MS/MS
Food Chemistry, 2015Co-Authors: Luca Maria Chiesa, Radmila Pavlovic, Sara Panseri, Maria Nobile, Daniele Vigo, Francesco ArioliAbstract:Abstract The administration of Boldenone and androstadienedione to cattle is forbidden in the European Union, while prednisolone is permitted for therapeutic purposes. They are pseudoendogenous substances (endogenously produced under certain circumstances). The commonly used matrices in control analyses are urine or liver. With the aim of improving the residue controls, we previously validated a method for steroid analysis in bile. We now compare urine (a ‘classic’ matrix) to bile, both collected at the slaughterhouse, to understand whether the detection of steroids in the latter is easier. With the aim of having clearer results, we tested the presence of the synthetic corticosteroid dexamethasone. The results show that bile does not substantially improve the detection of Boldenone, or its conjugates, prednisolone and prednisone. Dexamethasone, instead, was found in 10 out of 53 bovine bile samples, but only in one urine sample from the same animals. Bile could constitute a novel matrix for the analysis of residues in food-producing animals, and possibly not only of synthetic corticosteroids.
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Pseudoendogenous presence of β-Boldenone sulphate and glucuronide in untreated young bulls from the food chain.
Food Additives and Contaminants Part A-chemistry Analysis Control Exposure & Risk Assessment, 2015Co-Authors: Luca Maria Chiesa, Radmila Pavlovic, Elisa Di Pasquale, Sara Panseri, Francesca Tiziana Cannizzo, Bartolomeo Biolatti, Francesco ArioliAbstract: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 α-Boldenone conjugates, is considered fully compliant up to 2 ng ml−1. Greater attention has been placed on β-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 β-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 β-Boldenone. We assessed the occurrence of these molecules in young bull urine, with the aim of understanding whether th...
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detection of Boldenone its conjugates and androstadienedione as well as five corticosteroids in bovine bile through a unique immunoaffinity column clean up and two validated liquid chromatography tandem mass spectrometry analyses
Analytica Chimica Acta, 2014Co-Authors: L M Chiesa, Radmila Pavlovic, Sara Panseri, C Sgoifo A Rossi, Maria Nobile, F. ArioliAbstract:Abstract The presence of β-Boldenone II phase metabolites and prednisolone in urine samples, owing to endogenous or natural origin or illicit treatment, is under debate within the European Union. The detection of β-Boldenone conjugates, α-Boldenone conjugates at concentrations higher than 2 ng mL −1 and prednisolone above the cut-off level of 5 ng mL −1 in urine have been, until now, critical in deciding if illegal drug use has occurred. The use of urine sometimes is not entirely satisfactory, especially when the drug is administrated at low doses or when its metabolic conversion is very fast. This subsequently would hamper its detection in urine. The introduction of a new, advantageous matrix where the illicit treatment can be investigated would be highly appreciated. In this study, we have developed and validated a simple and unique immunoaffinity clean-up procedure, which was applied to bovine bile samples, followed by two different analytical liquid chromatography-electrospray-tandem mass spectrometry methods. The first method tests androstadienedione, α- and β-Boldenone sulphate, glucuronate and related free forms, while the other method assays prednisolone, prednisone, dexamethasone, cortisone, and cortisol. The methods were validated according to European Commission Decision 2002/657/EC. The evaluated parameters were linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit and detection capability. The decision limits (CCα) were between 0.38 and 0.45 ng mL −1 for anabolic steroids, and 0.13 and 0.15 ng mL −1 as far as corticosteroids were concerned. Intra- and inter-day repeatability was below 15.8 and 19.9% for all analytes, respectively. The methods were applied to the analysis of some bile samples collected from untreated young bulls in order to investigate the presence of the studied steroids in this matrix.
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Detection of Boldenone, its conjugates and androstadienedione, as well as five corticosteroids in bovine bile through a unique immunoaffinity column clean-up and two validated liquid chromatography–tandem mass spectrometry analyses
Analytica Chimica Acta, 2014Co-Authors: Luca Maria Chiesa, Radmila Pavlovic, Sara Panseri, Maria Nobile, C.a. Sgoifo Rossi, Francesco ArioliAbstract:Abstract The presence of β-Boldenone II phase metabolites and prednisolone in urine samples, owing to endogenous or natural origin or illicit treatment, is under debate within the European Union. The detection of β-Boldenone conjugates, α-Boldenone conjugates at concentrations higher than 2 ng mL −1 and prednisolone above the cut-off level of 5 ng mL −1 in urine have been, until now, critical in deciding if illegal drug use has occurred. The use of urine sometimes is not entirely satisfactory, especially when the drug is administrated at low doses or when its metabolic conversion is very fast. This subsequently would hamper its detection in urine. The introduction of a new, advantageous matrix where the illicit treatment can be investigated would be highly appreciated. In this study, we have developed and validated a simple and unique immunoaffinity clean-up procedure, which was applied to bovine bile samples, followed by two different analytical liquid chromatography-electrospray-tandem mass spectrometry methods. The first method tests androstadienedione, α- and β-Boldenone sulphate, glucuronate and related free forms, while the other method assays prednisolone, prednisone, dexamethasone, cortisone, and cortisol. The methods were validated according to European Commission Decision 2002/657/EC. The evaluated parameters were linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit and detection capability. The decision limits (CCα) were between 0.38 and 0.45 ng mL −1 for anabolic steroids, and 0.13 and 0.15 ng mL −1 as far as corticosteroids were concerned. Intra- and inter-day repeatability was below 15.8 and 19.9% for all analytes, respectively. The methods were applied to the analysis of some bile samples collected from untreated young bulls in order to investigate the presence of the studied steroids in this matrix.
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Determination of α- and β-Boldenone sulfate, glucuronide and free forms, and androstadienedione in bovine urine using immunoaffinity columns clean-up and liquid chromatography tandem mass spectrometry analysis.
Talanta, 2014Co-Authors: Luca Maria Chiesa, Radmila Pavlovic, Guglielmo Dusi, Elisa Di Pasquale, Alessio Casati, Sara Panseri, Francesco ArioliAbstract:Abstract The debate about the origin s of Boldenone in bovine urine is ongoing for two decades in Europe. Despite the fact that its use as a growth promoter has been banned in the European Union (EU) since 1981, its detection in bovine urine, in the form of α-Boldenone conjugate, is considered fully compliant up to 2 ng mL−1. The conjugated form of β-Boldenone must be absent. In recent years, the literature about Boldenone has focused on the identification of biomarkers that can indicate an illicit treatment. β-Boldenone sulfate is a candidate molecule, even if the only studies currently available have taken place in small populations. In this study, a method for the determination of sulfate and glucuronate conjugates of β-Boldenone was developed and validated according to the European Commission Decision 2002/657/EC and applied to α-Boldenone sulfate and glucuronide, α- and β-Boldenone free forms and androstadienedione (ADD), too. The clean-up with immunoaffinity columns enabled the direct determination of the conjugates and free forms and allowed specific and sensitive analyses of urine samples randomly selected to verify this method. The decision limits (CCα) ranged between 0.07 and 0.08 ng mL−1, the detection capabilities (CCβ) between 0.08 and 0.1 ng mL−1. Recovery was higher than 92% for all the analytes. Intra-day repeatability was between 5.8% and 17.2%, and inter-day repeatability was between 6.0% and 21.8% for the studied free and conjugated forms. This method has been developed as a powerful tool with the aim to study the origin of Boldenone in a trial on a significant number of animals.