Marta Leporati

A field survey on the presence of prednisolone and prednisone in urine samples from untreated cows

Prednisolone is a synthetic glucocorticoid widely employed in bovine clinical practice that may also be used illegally as a growth promoter. Recent in vitro and in vivo studies lend support to the hypothesis that prednisolone could be synthesised from cortisol in untreated cattle subjected to stressful events. To verify such a hypothesis, a field survey was conducted on urine samples collected from 131 guaranteed untreated cows and analysed for the presence of prednisolone and prednisone – in some instances also for cortisol and cortisone – with a validated LC/MS-MS method. None of the examined samples exhibited either prednisolone levels higher than the CCα limit (around 0.70 µg l−1) or prednisone, being therefore officially compliant for both analytes. Trace amounts of prednisolone, approximately estimated in the range 0.1–0.3 µg l−1 were found in only seven samples from cows also showing urinary cortisol and cortisone levels higher than those detected in negative specimens, as the result of a probable stress condition.

Determination by LC-MS/MS of colistins A and B in plasma and ultrafiltrate from critically ill patients undergoing continuous venovenous hemodiafiltration.

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.

Fit‐for‐purpose in veterinary drug residue analysis: Development and validation of an LC‐MS/MS method for the screening of thirty illicit drugs in bovine urine

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<R%<70 for 5 analytes, lower than 50% but reproducible for the remaining six analytes. The analyses were carried out using HPLC-ESI-MS/MS. The method sensitivity proved high enough to largely exceed the CCβ requirements of the Italian residue detection plan, ranging from 1 to 3 ng/mL (20 ng/mL for promazines). The present method allowed the simultaneous analysis of most drugs for which the European legislation prescribes official controls. Its practical applicability was verified on 494 real samples as an alternative to the traditional screening protocols based on multiple immunometric analysis, demonstrating high efficiency and comprehensive investigation capacity, allowing epidemiological assessment of the current trends in cattle breeding drug abuse. Among non-compliant results, nine borderline cases of growth-promoters illegal treatments, making use of long-term low-dosage administrations and typically yielding urine residues below the cut-off value for immunochemical methods, were detected by using the present LC-MS/MS method.

Determination of prednisolone metabolites in beef cattle

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.

Profile of the urinary excretion of prednisolone and its metabolites in finishing bulls and cows treated with a therapeutic schedule

BackgroundPrednisolone 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, 20β-dihydroprednisolone, 20β-dihydroprednisone, 6β-hydroxyprednisolone, cortisol, and cortisone were determined using a validated LC/MS-MS method.ResultsThe 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.ConclusionsThe 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.

Development and validation of an UHPLC–MS/MS method for β2-agonists quantification in human urine and application to clinical samples

A fast analytical method for the simultaneous detection of 24 β2-agonists in human urine was developed and validated. The method covers the therapeutic drugs most commonly administered, but also potentially abused β2-agonists. The procedure is based on enzymatic deconjugation with β-glucuronidase followed by SPE clean up using mixed-phase cartridges with both ion-exchange and lipophilic properties. Instrumental analysis conducted by UHPLC-MS/MS allowed high peak resolution and rapid chromatographic separation, with reduced time and costs. The method was fully validated according ISO 17025:2005 principles. The following parameters were determined for each analyte: specificity, selectivity, linearity, limit of detection, limit of quantification, precision, accuracy, matrix effect, recovery and carry-over. The method was tested on real samples obtained from patients subjected to clinical treatment under chronic or acute therapy with either formoterol, indacaterol, salbutamol, or salmeterol. The drugs were administered using pressurized metered dose inhalers. All β2-agonists administered to the patients were detected in the real samples. The method proved adequate to accurately measure the concentration of these analytes in the real samples. The observed analytical data are discussed with reference to the administered dose and the duration of the therapy.

Determination of Anticoagulant Rodenticides and α-Chloralose in Human Hair. Application to a Real Case.

Anticoagulant rodenticides are the largest group of poisons used to kill harmful rodents. Their fundamental mode of action consists in the inhibition of the vitamin K epoxide reductase, which causes blood-clotting alteration, ultimately leading to hemorrhagic events as the cause of death. In this study, we developed an UHPLC-MS-MS for the simultaneous determination of 10 anticoagulant hydroxycoumarine rodenticides, plus α-chloralose in human hair, with the scope of detecting potential trace of chronological poison exposure in clinical and forensic cases. The method was fully validated and applied to a case of intentional poisoning perpetrated by administration of difenacoum and α-chloralose to a 97-year-old woman, who was hospitalized because of severe symptoms, including drowsiness, convulsions, pallor and hematoma. Hair sample from the victim was segmentally analyzed. Difenacoum was detected in the proximal 3-cm hair segment at the concentration of 2.9 pg/mg. To our knowledge, this is the first study to report that exposure to difenacoum is detectable in real hair samples. The other target analyte found in the hair sample was α-chloralose, which was detected in the 0-3 cm segment at the concentration of 85 pg/mg. The two subsequent and consecutive segments (3-6 cm and 6-9 cm) showed only traces of difenacoum (below LOQ) and low but quantifiable concentrations of α-chloralose (29 and 6 pg/mg, respectively). Therefore, hair segmental analysis allowed us to conclude that the victim was repeatedly exposed to two poisons in the period corresponding to the first segment of hair.