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

Chiesa, Luca Maria; Pavlović, Radmila; Dusi, Guglielmo; Pasquale, Elisa; Casati, Anna; Panseri, Sara; Arioli, Francesco

doi:10.1016/j.talanta.2014.07.035

Cited by 11 publications

(13 citation statements)

References 13 publications

(14 reference statements)

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“…The validation protocols, made accordingly 2002/657/EC (European Union, 2002), and the obtained parameters are described in detail in previous studies (Chiesa et al, 2014(Chiesa et al, , 2015 and reported here in a summarized way: linear matrix calibration curves built for each analyte (0.05-0.1-0.2 ng mL À1 for all analytes in urine, and 0.3-0.6-0.9 ng ml À1 for anabolic steroids and 0.1-0.2-0.3 ng mL À1 for corticosteroids in bile) (6 samples Â 3 concentration levels Â 3 series = 54 analyses for each matrix). Intra-day and inter-day repeatability (Thompson, 2000), representing precision, were calculated using one-way analysis of variance (ANOVA), expressed as CVs, and were below 15.8% and 19.9% in bile and 17.2% and 21.8% in urine, for all analytes.…”

Section: Methods Validationmentioning

confidence: 99%

“…The bile and urine extractions and analyses were performed as previously described by Chiesa et al (2014) and Chiesa et al (2015). Briefly, a 5 mL centrifuged bile or urine sample spiked with internal standards (2 ng mL À1 ), was adjusted, when necessary, to pH = 7.5-8.8 with NaOH 0.1 N; the sample was loaded into an IAC column that was previously washed (5 mL ethanol:water; 70:30, v/v) and equilibrated (3 Â 5 mL wash buffer).…”

Section: Sample Extraction and Lc-ms/ms Analysismentioning

confidence: 99%

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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

et al. 2015

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Section: Methods Validationmentioning

confidence: 99%

Section: Sample Extraction and Lc-ms/ms Analysismentioning

confidence: 99%

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

et al. 2015

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“…Urine extraction and analysis were performed as previously described by Chiesa et al (2015). Briefly, a 5 mL centrifuged urine sample spiked with internal standards (2 ng mL -1 ) and adjusted to pH 8 with 0.1 N NaOH was loaded into a previously washed IAC column (5 mL ethanol: water; 70:30, v/v) and equilibrated (3 x 5 mL wash buffer).…”

Section: Sample Preparation Extraction Lc-ms/ms Analysis Validatiomentioning

confidence: 99%

“…Figure 2 shows the reconstructed chromatograms with the ion spectra of β-bold glucuronide and sulfate in the solvent. The validation protocol, performed according to the European Commission Decision 2002/657/EC, is described by Chiesa et al (2015). The analytes were quantified through a matrix calibration curve prepared with seven levels and three replications in the range 0.1 -5.0 ng mL -1 .…”

Section: Sample Preparation Extraction Lc-ms/ms Analysis Validatiomentioning

confidence: 99%

“…Moreover, steroid conversion or degradation and artefact formation(Pozo et al 2008;Gomez et al 2014) may occur.As an alternative to the indirect detection (i.e. after enzymatic hydrolysis) of β-bold conjugates, we have developed and validated a method which uses immunoaffinity purification coupled to LC-MS/MS in order to perform direct analysis of the glucuronide and sulfate forms of β-bold (European Commission 2002;Chiesa et al 2015). In contrast to the present study, in that study the free forms of α-and β-bold, as well as α-bold conjugates and androstadienedione (ADD) were also considered.In order to verify the possible endogenous origin of β-bold II phase metabolites in young bulls, we carried out a study on urine samples from 56 animals collected at different times at the farm, where the animals were under veterinary control, and at the slaughterhouse.…”

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confidence: 99%

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

Chiesa

Pasquale

Panseri

et al. 2015

Food Additives & Contaminants: Part A

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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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Evaluation of nandrolone and ractopamine in the urine of veal calves: liquid chromatography‐tandem mass spectrometry approach

Chiesa

Panseri

Cannizzo

et al. 2016

Drug Testing and Analysis

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Research articles LC-MS/MS technology allows the direct detection of glucuronoconjugated metabolites. PI scan, NL scan and SRM methods, have demonstrated to be useful for the detection of unknown unpredicted and predicted glucuronide MTD metabolites in metabolic studies. Thirteen glucuronide metabolites were observed, and one of them was resistant to enzymatic hydrolysis, not detectable using the traditional GC-MS strategies. Sport supplements may contain steroids never approved for therapeutic use. In this study, 18 sport supplements purchased online underwent organic solvent extraction and subsequent isolated steroid tested for intrinsic androgen bioactivity. Many were found to be strong androgens. These potent pharmacologically active steroids are being used without regulatory control or consumer awareness of their potential adverse effect. Avidin-biotin technology was used for the implementation of an enzyme-linked immunosorbent assay (AB-ELISA) as a sensitive method for the detection of anabolic androgenic steroids (AAS) present in dietary supplements. Assembled AB-ELISAs were characterized and used for detection of various AAS in 49 samples of counterfeit dietary supplements. The concordance between UHPLC-MS and the CR corrected data from AB-ELISA indicated the potential of this method even to quantification of some steroids. Nandrolone (anabolic steroid) and ractopamine (β-adrenergic agonist) are used quite frequently in food-producing livestock, but little is known about their synergic action when they are administered together. The objective of this study was to determine levels after experimental treatment of bovines with both of the above-mentioned drugs. For this purpose two specific analytical methods based on liquid chromatography-tandem mass spectrometry were developed. The use of tandem yeast and mamma-lian cell in vitro androgen bioassays to detect androgens in internet-sourced sport supplements 545-552 We evaluated the effectiveness of drugs testing using hair and nails and the feasibility of determining when drugs were ingested by measuring the time courses of drug concentrations in hair and toenails after single administrations of various drugs. For most basic compounds, the maximum concentrations in hair segments tended to be higher than those in nails. Nails were an effective specimen to detect neutral and weakly acidic compounds. In this work, we aimed to develop a new, simple and confident sensory platform for cocaine detection. For the fabrication of this test system, surface of μ-well plates was modified with quantum dots (QDs) and then cocaine-binding aptamer modified gold nanoparticles (Apt-AuNP) were covalently attached to the QDs. Detectable signals were obtained owing to the target-induced conformational change of aptamer which gives rise to close interaction of QDs with their quencher, AuNPs. Hence, decrease in fluorescence intensity as a cocaine response was recorded. The developed test system-called as Aptamer Folding based Sensory Device, (AFSD)-was evaluated in terms of main a...

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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

Cited by 11 publications

References 13 publications

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

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

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

Evaluation of nandrolone and ractopamine in the urine of veal calves: liquid chromatography‐tandem mass spectrometry approach

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