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Leporati M.,Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria | Capra P.,Istituto Zooprofilattico Sperimentale del Piemonte | Brizio P.,Istituto Zooprofilattico Sperimentale del Piemonte | Ciccotelli V.,Istituto Zooprofilattico Sperimentale del Piemonte | And 3 more authors.
Journal of Separation Science | Year: 2012

A selective and sensitive method for screening 31 analytes (nine corticosteroids, eight β-agonists, seven anabolic steroids, six promazines and zeranol) in bovine urine was validated according to 2002/657/EC guidelines. Upon optimization of sample treatment conditions, the extraction was performed by diethylether at pH 9, after deconjugation. Extraction yields (R%) proved higher than 70% for 19 analytes, 50

Capra P.,Istituto Zooprofilattico Sperimentale del Piemonte | Ciccotelli V.,Istituto Zooprofilattico Sperimentale del Piemonte | Vincenti M.,Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria | Vincenti M.,University of Turin
Analytica Chimica Acta | Year: 2011

An analytical, pharmacokinetic and histopathologic investigation was conducted by two experimental trials on beef cattle in order to determine fate and effects of dexamethasone and prednisolone, administered to distinct cattle groups at low dosage for long periods of time. In trial 1, eighteen Charolaise beef cattle, male, 17-22-months-old, were divided in three groups: to group A (n=6) dexamethasone-21-sodium-phosphate 0.7mgday-1 per os for 40 days was administered; group B (n=6) was orally treated with prednisolone 15mgday-1 for 30 days, while group C (n=6) served as negative control. Urine was collected at days 0, 7, 15, 25 and 47 from groups A and C, and at days 0, 8, 18 and 42 from group B. In trial 2, sixteen Friesian cattle, male, 10-17-months-old, were randomly divided into two groups: group D (n=8) was administered prednisolone 30mgday-1 per os for 35 days, while group K (n=8) served as control. In both trials, the animals were slaughtered after a 6-days drug withdrawal and thymus and livers were collected and properly stored until the analysis was performed. Quantitative determinations of dexamethasone, prednisolone and its main metabolite, prednisone, in urine and liver samples were conducted by HPLC-MS/MS, after the analytical procedure was optimized and fully validated. The method validation included the assessment of specificity, linearity, precision, trueness, robustness, CCα and CCβ values. By a morphological point of view, severe atrophy of thymus parenchyma was observed in group A, together with a significant (P< 0.005) reduction of the mean thymus weight (217 ± 94. g), while group B (646 ± 215. g) presented normal thymus features and weights (group C, 415 ± 116. g). Accordingly, no differences were found in trial 2 for groups D (727 ± 275. g) and K (642 ± 173. g).Average dexamethasone concentrations in group A urine samples ranged from 1.4 to 3.0μgL-1 during the treatment, while no residue was detected in the urine samples collected 6-7 days after the end of the treatment. Low amounts of dexamethasone (<1μgL-1) were detected in liver samples of group A. All average prednisolone concentrations in group B urine samples (sum of conjugate and free form) turned out to be below 1.0μgL-1 during the treatment, despite the much higher concentration administered (15-30mgday-1) with respect to dexamethasone in group A (0.7mgday-1). No prednisolone residues were found in the urine and liver samples taken at the slaughterhouse. The absence of any prednisolone residue in the urine samples of control group animals supports the theory that the origin of this molecule is fundamentally exogenous, at least for this cattle category maintained under unstressing conditions. Remarkable findings are represented by the absence of thymus atrophy in the prednisolone treated animals and the extremely low residue concentrations found in urine during the treatment. Both findings reveal that the detection of illegal growth-promoting treatments with this drug is difficult. © 2010 Elsevier B.V.

Leporati M.,Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria | Bergoglio M.,University of Turin | Capra P.,University of Bologna | Bozzetta E.,University of Bologna | And 3 more authors.
Journal of Mass Spectrometry | Year: 2014

β2-agonists are often abused in cattle breeding because of their effects on animal growth and meat properties. The use of β2-agonists as growth promoters is forbidden in the European Union (Council Directive 96/23/EC classifies them into group A of Annex I), due to their toxicity and carcinogenic properties, as for anabolic steroids, which are often administered in combination with β2-agonists, to promote the storage of proteins and increase muscle size. A unique confirmatory liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative detection of 13 β2-agonists and anabolic steroids plus the qualitative identification of other three analytes in bovine hair was developed and validated, according to Decision 2002/657/CE. Hair samples were washed with dichloromethane, digested within a NaOH solution and subjected to liquid-liquid extraction. The analysis was performed by high performance liquid chromatography coupled to a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. The absence of matrix interferents, together with good repeatability of both retention times and relative abundances of diagnostic transitions, allowed the correct identification of all analytes. The quantitative calibrations obtained from spiked blank hair samples proved linear in the range tested. CCα and CCβ ranged from 0.5 ng/g to 30 ng/g. Intralaboratory reproducibility (CV%) ranged between 5.0 and 17.7 and trueness between 96%±7% and 105%±8%. The applicability of the method to real positive samples was demonstrated for both β2-agonists and anabolic steroids. 17α-boldenone was found in most (70%) hair samples obtained from untreated animals, supporting the hypothesis of endogenous production of this steroid. Copyright © 2014 John Wiley & Sons, Ltd.

Mariano F.,Nefrologia | Leporati M.,Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria | Vincenti M.,Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria | Vincenti M.,University of Turin | Biancone L.,University of Turin
Journal of Nephrology | Year: 2015

Background: Colistin pharmacokinetics data are scarce regarding patients undergoing renal replacement therapy (RRT), or even absent as in patients treated with sorbent technologies potentially capable of removing colistin by extensive absorption on many polymeric materials. Methods: Twelve septic shock patients with acute kidney injury (AKI) undergoing RRT [continuous venovenous hemodiafiltration (CVVHDF) n = 7, coupled-plasma filtration adsorption-HF (CPFA-HF) n = 4, hemoperfusion n = 1] treated with colistin methanesulfonate at a dose of 4.5 × 106 U bid were studied. Colistin A (Col-A) and colistin B (Col-B) concentrations on plasma and effluent at time 0, 0.2, 1, 3, 6, 12, 24 and 48 h were determined by the liquid chromatography-tandem mass spectrometry method. Results: With CVVHDF the sieving coefficient was lower for Col-A, peaked early (0.40 for Col-A at 10 min, and 0.59 for Col-B at 3 h) and declined after 48 h (0.22 and 0.30 for Col-A and Col-B, respectively). Colistin’s filter clearance showed a similar pattern, with the highest clearance value of 18.7 ml/min for Col-B at 1 h. With CPFA-HF after the cartridge the Col-A and Col-B levels were negligible (<0.2 mg/l) or not detectable. The sum of the effluent and cartridge clearances reached values of 30 and 40 ml/min for Col-A and Col-B, respectively. With hemoperfusion the postcartridge concentrations for Col-A and Col-B were about 30 % lower than those determined precartridge. Conclusions: During CPFA-HF and CVVHDF, the extent of colistin removal is high, and patients should receive an unreduced dosage. However, due to risk of accumulation in long-term administration colistin plasma levels determination is recommended. © 2014, Italian Society of Nephrology.

Leporati M.,Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria | Capra P.,Istituto Zooprofilattico Sperimentale del Piemonte | Cannizzo F.T.,University of Turin | Biolatti B.,University of Turin | And 3 more authors.
Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment | Year: 2013

Prednisolone is a synthetic corticosteroid acting on both hydrosaline balance and metabolism that is liable to fraudulent administration to meat-producing animals for growth-promoting purposes. Its use outside strict therapeutic control and prescription is banned by the European legislation, but official controls are hampered by its negligible direct excretion into the urinary matrix. Recent studies reported on a potential endogenous origin of prednisolone in animals subjected to stressful conditions, accounting for its occasional detection in control urines. The objective of the present study was the identification and quantification of prednisolone urinary metabolites to be used as illicit treatment biomarkers in place of the parent drug. An LC-MS/MS screening was conducted on urine samples collected from a bullock intramuscularly administered with prednisolone acetate by using a therapeutic protocol (2 × 0.52 mg kg-1 at 48-hour interval). Four prednisolone metabolites were identified: 20β-dihydroprednisolone, 20α-dihydroprednisolone, 6β-hydroxyprednisolone and 20β-dihydroprednisone; the first was detected at relatively high concentrations. An existing quantitative LC-MS/MS method was expanded and revalidated to include these metabolites. The new analytical method proved sensitive (LODs: 0.35-0.42 ng mL-1) and specific and was applied to urine samples collected from eight beef cattle subjected to low-dosage oral administration of prednisolone acetate for a 35-day period, as in standard growth-promoting treatments. 20β-Dihydroprednisolone was detected in all urine samples collected during the treatment, at relatively high concentration (1.2-27 ng mL-1), whereas the prednisolone concentration was virtually negligible (<0.7 ng mL-1). 20β-Dihydroprednisolone was no longer present in almost all samples collected 6 days after the end of the treatment, but trace amounts of this metabolite were found in two urine samples from control animals. 20β-Dihydroprednisolone is proposed as an effective biomarker to test illegal growth-promoting treatments with prednisolone in meat cattle, alternatively to the parent drug. © 2013 Copyright Taylor & Francis.

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