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

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.

HPLC-ESI-MS/MS assessment of the tetrahydro-metabolites of cortisol and cortisone in bovine urine: promising markers of dexamethasone and prednisolone treatment

“…However, the fragmentation of some analytes that had equal molecular mass (cortisone and PL, for example) was careful studied in order to enhance the capabilities of the method, avoiding the common transition. The m/z values of the selected parent and daughter ions used for MS detection were generally in agreement with those previously reported by Chiesa et al (2014). Special consideration was focused on fragmentation of the tetrahydro-metabolites, as there is limited number of publication that deal with the behaviour of their formic acid adducts in an ESI(-) source (Cuzzola et al 2009).…”

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

“…The CE was adjusted in the MRM mode for each transition monitored in order to reach the highest sensitivity and specificity for the two isomers. The Synergi Hydro RP column was our first choice as we used it successfully for anabolic hormone determination, [23,24,26] Also here, in the case of nandrolone, it showed good performances and satisfactory peak shapes and baseline separation. The m/z values of the selected precursor and fragment ions used for MS detection are generally in agreement with those previously reported in the literature, especially when it concerns quantifier ions for αand β-nandrolone (transition 275 → 109) and d3-β-nandrolone (transition 278 → 109).…”

“…[23,24] Principally, two enzymes, β-glucuronidase from Escherichia coli K 12 (EC 3.2.1.31) and β-glucuronidase/arylsulphatase from Helix pomatia, were considered in order to find the best solution that would accomplish our purpose. Initially, many efforts were made to develop unique deconjugation processes that would be suitable for either nandolone or ractopamine.…”

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

“…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  3 concentration levels  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.…”

“…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).…”