Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria

Orbassano, Italy

Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria

Orbassano, Italy

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Leporati M.,Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria | Bua R.O.,University of Turin | Stella M.,Centro Grandi Ustionati | Biancone L.,University of Turin | And 2 more authors.
Therapeutic Drug Monitoring | Year: 2014

BACKGROUND:: Colistin is a 50-year-old antibiotic, the use of which was ceased in the 70s and recently resumed as a "salvage therapy" against multidrug-resistant gram-negative bacteria, such as Pseudomonas aeruginosa and Acinetobacter baumannii. The narrow therapeutic range of colistin makes the choice of its correct dosage crucial, and monitoring of blood concentration is occasionally necessary for critically ill patients, including intensive care patients subjected to continuous renal replacement therapy. METHODS:: Two LC-MS/MS methods were developed and fully validated for the quantitative determination of colistins A and B in plasma and dialysis ultrafiltrate (UF) samples, ultimately arising from 4 patients undergoing continuous venovenous hemodiafiltration (CVVHDF). RESULTS:: The developed methods proved to be both specific and selective. They showed good fit and linearity over the entire range of interest. Trueness and accuracy proved satisfactory. Both methods have excellent intraassay precision (percent coefficient of variations were lower than 10%) and limit of detection values in the range 20-100 ng/mL, about 1-2 orders of magnitude below the concentrations commonly detected in real samples. The mean sieving coefficient (SC) values, measured after 10 minutes of CVVHDF, were 0.42 for colistin A and 0.48 for colistin B. SC values proved to be quite stable for 24 hours, but then declined to 0.24 for colistin A and 0.32 for colistin B, respectively, after 48 hours. At the median blood flow and effluent flow rate of 120 and 28 mL/min, clearance values for colistin B were higher than 15 mL/min. During the entire duration of CVVHDF sessions, the SC and clearance values for colistin A were significantly lower than colistin B. CONCLUSIONS:: Two simple methods for the simultaneous determination of colistins A and B have been developed and validated. Their application in the clinical setting demonstrates that CVVHDF treatment lasting 48 hours produces a relatively constant and efficient removal of the drug. © 2014 by Lippincott Williams & Wilkins.


Capra P.,Instituto Zooprofilattico Sperimentale del Piemonte | Ciccotelli V.,Instituto 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.


PubMed | Nefrologia, University of Turin and Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria
Type: Journal Article | Journal: Journal of nephrology | Year: 2015

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.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 10(6) 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.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). Colistins 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.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.


Leporati M.,Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria | Bergoglio M.,University of Turin | Capra P.,Instituto Zooprofilattico Sperimentale del Piemonte | Bozzetta E.,Instituto Zooprofilattico Sperimentale del Piemonte | 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.


Leporati M.,Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria | Capra P.,Instituto 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.


Leporati M.,Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria | Capra P.,Instituto Zooprofilattico Sperimentale Del Piemonte | Brizio P.,Instituto Zooprofilattico Sperimentale Del Piemonte | Ciccotelli V.,Instituto 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


Nebbia C.,University of Turin | Capra P.,Instituto Zooprofilattico Sperimentale del Piemonte | Leporati M.,Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria | Girolami F.,University of Turin | And 4 more authors.
BMC Veterinary Research | Year: 2014

Background: Prednisolone was one of the first glucocorticoids to be synthesised, but it is still widely applied to cattle. Illegal uses of prednisolone include its uses for masking a number of diseases before animal sale and, at lower dosages for extended periods of time, for the improvement of feed efficiency and carcass characteristics. Since occasional presence of prednisolone has been detected at trace level in urine samples from untreated cattle, the Italian Ministry of Health introduced a provisional limit of 5 ng/mL to avoid false non-compliances. However, this limit proved ineffective in disclosing prednisolone misuse as a growth-promoter. In the present study, prednisolone acetate was administered to finishing bulls and cows according to a therapeutic protocol (2 × 0.4-0.5 mg/kg bw i.m. at 48 h interval) to further verify the practical impact of this cut-off limit and develop sound strategies to distinguish between exogenous administration and endogenous production. Urinary prednisolone, prednisone, 20β-dihydroprednisolone, 20α-dihydroprednisolone, 20β-dihydroprednisone, 6β-hydroxyprednisolone, cortisol, and cortisone were determined using a validated LC/MS-MS method. Results: The urinary excretion profile showed the simultaneous presence of prednisolone, 20β-dihydroprednisolone, and prednisone, the latter at lower concentrations, up to 33 days after the first dosing. Higher analyte levels were detected in bulls even after correction for dilution in the urine. Prednisolone concentrations below 5 ng/ml were determined in half of the samples collected at 19 days, and in all the samples obtained 26 and 33 days after the first administration. No measurable concentrations of prednisolone or its metabolites were found in the samples collected before the treatment, while cortisol and cortisone levels lower than the respective LOQs were observed upon treatment. Conclusions: The present study confirms the criticism of the coarse quantitative approach currently adopted to ascertain illegal prednisolone administration in cattle. As previously shown for growth-promoting treatments of meat cattle, the simultaneous determination of urinary prednisolone, prednisone, 20β-dihydroprednisolone, along with cortisol and cortisone, may represent a more reliable approach to confirm the exogenous origin of prednisolone. Such a strategy would facilitate unequivocal detection of animals treated with prednisolone acetate using a therapeutical protocol, even 3 to 4 weeks after the treatment. © 2014 Nebbia et al.; licensee BioMed Central Ltd.


PubMed | Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria
Type: Journal Article | Journal: Journal of separation science | Year: 2012

A selective and sensitive method for screening 31 analytes (nine corticosteroids, eight -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


PubMed | Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria
Type: Journal Article | Journal: Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & 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 (20.52mgkg(-1) at 48-hour interval). Four prednisolone metabolites were identified: 20-dihydroprednisolone, 20-dihydroprednisolone, 6-hydroxyprednisolone and 20-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: 0.35-0.42ngmL(-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. 20-Dihydroprednisolone was detected in all urine samples collected during the treatment, at relatively high concentration (1.2-27ngmL(-1)), whereas the prednisolone concentration was virtually negligible (<0.7ngmL(-1)). 20-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. 20-Dihydroprednisolone is proposed as an effective biomarker to test illegal growth-promoting treatments with prednisolone in meat cattle, alternatively to the parent drug.


PubMed | Centro Regionale Antidoping E Of Tossicologia Alessandro Bertinaria
Type: Journal Article | Journal: Journal of mass spectrometry : JMS | Year: 2014

(2) -agonists are often abused in cattle breeding because of their effects on animal growth and meat properties. The use of (2) -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 (2) -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 (2) -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. CC and CC ranged from 0.5ng/g to 30ng/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 (2) -agonists and anabolic steroids. 17-boldenone was found in most (70%) hair samples obtained from untreated animals, supporting the hypothesis of endogenous production of this steroid.

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