ABSTRACT
A reliable method using a QuEChERS approach and liquid chromatography coupled to Q-Orbitrap mass spectrometry was optimized and validated for the quantification of 20 growth promoters in bovine serum. The recoveries ranged from 91.4-114.1%, relative standard deviations varied between 0.3-4.0%, and CCα values were between 0.023-0.350 µg L-1. The developed method was applied in an in vivo study using steers, which were intramuscularly treated with commercial injections containing stanozolol. A rapid metabolization was observed, with a detection window ranging from 3 to 10 days. The stability of incurred stanozolol was confirmed after 240 days at -20 °C and also after 5 freeze-thaw cycles. To the best of our knowledge, this is the first work in which an in vivo study was performed to support the monitoring of stanozolol in bovine serum. In addition, the use of Q-Orbitrap high-resolution mass spectrometry allows for retrospective analysis from a surveillance perspective.
Subject(s)
Stanozolol , Chromatography, High Pressure Liquid/methods , Retrospective Studies , Mass Spectrometry/methods , Chromatography, LiquidABSTRACT
An in vivo study was performed in order to evaluate the depletion time of stanozolol and its main metabolites using naturally incurred urine sample collected after the administration of intramuscular injections in 12 steers. A stability study was also carried out to investigate the influence of the storage period and the freeze-thaw cycles. A fast parent drug metabolization was observed, because within 6 h after drug administration, the signal of the metabolite 16ß-hydroxystanozolol was predominant. After the second drug administration, a detection window of 17 days was obtained. The stability was studied using ANOVA, in which a storage condition of -20 °C proved stable during 240 days, which was also confirmed after 5 freeze-thaw cycles.
Subject(s)
Stanozolol , Animals , Cattle , Chromatography, High Pressure Liquid , Injections, Intramuscular , Stanozolol/urineABSTRACT
In this work multivariate strategies were employed in order to highlight new potential biomarkers of interest to detect the exogenous treatment of steers intramuscularly treated with boldenone undecylenate. Serum samples collected from treated (n = 4) and control (n = 8) crossbred animals of varying ages and weights were extracted using a simple sample preparation procedure based on salt assisted liquid-liquid extraction. Data acquisition was performed using liquid chromatography and Q-Exactive™ Orbitrap mass spectrometry. Data processing and treatment were performed using two non-targeted workflows: (1) Compound Discoverer software and (2) XCMS package on the open-source R software combined with MetaboAnalyst. Three potential biomarkers were highlighted taking into account the chromatographic shapes, the feature location on the generated s-plots, the fold change, the adjusted p values, the coefficient of variation in the QC samples and the area under the ROC curves. Predicted formulas based on mass accuracy, structural composition and spectra similarity were proposed. A robust statistical model to predict the boldenone treatment was further developed based on the weighted abundances of the selected biomarkers. The requirements for screening methods were successfully fulfilled, together with a wider detection window in comparison with the monitoring of the deconjugated metabolite boldenone, although biomarker identification studies are still ongoing.
Subject(s)
Chromatography, High Pressure Liquid , Animals , Biomarkers , Cattle , Chromatography, Liquid , Mass Spectrometry , Testosterone/analogs & derivativesABSTRACT
In this work, 37 growth promoters were quantitatively determined in bovine urine using a QuEChERS approach with acetonitrile, NaCl, and MgSO4:PSA for sample extraction. The analytes were separated and detected by liquid chromatography coupled to hybrid high-resolution mass spectrometry. The method was validated in accordance with the Decision 657/2002/EC guidelines, in which recoveries fell within the range 84-113%, relative standard varied between 2 and 32%, and detection limit between 0.1 and 2.5 µg L-1. An adequate performance was evidenced during a proficiency test evaluation, and the developed method has been applied to routine analysis of growth promoters in Brazil. A highlight is the easiness of sample extraction combined with a quantitative determination of forbidden drugs using high-resolution mass spectrometry, which enables retrospective analysis in a surveillance perspective.
Subject(s)
Chromatography, High Pressure Liquid , Animals , Brazil , Cattle , Chromatography, Liquid , Mass Spectrometry , Retrospective StudiesABSTRACT
This work involved a systematic comparison between serum and urine for the monitoring of anabolic androgenic steroids in livestock. Incurred samples were collected over 120 days from crossbred steers treated with intramuscular injections containing boldenone undecylenate. Independent high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods were used for the assessment of the respective detection windows, which were larger for serum samples. Both matrices presented adequate performance in terms of long-term stability, assessed using an isochronous approach during 196 days at -20 °C and for five freeze-thaw cycles. The effectiveness of the enzymatic hydrolysis reaction using Helix pomatia juice was also compared. The calculated concentrations in serum samples were not statistically influenced by the deconjugation reaction. On the other hand, urine hydrolysis conditions were studied using a 33 Box-Behnken Design, in which a central point condition led to a satisfactory deconjugation performance. It could be observed that serum exhibited equivalent or better performance than urine for most of the evaluated criteria; thus, its inclusion in the regulatory analysis of boldenone in cattle is supported.
Subject(s)
Anabolic Agents , Tandem Mass Spectrometry , Animals , Cattle , Chromatography, High Pressure Liquid , Hydrolysis , Testosterone/analogs & derivativesABSTRACT
Serum analysis has received much attention in regulatory analysis of food-producing animals, especially for anabolic steroids. The possibility of confirming the parent drugs with minimum metabolization enables the detection of intact steroid esters, whose identification represents unequivocal proof of drug administration. This work involved the development and validation of a quantitative LC-MS/MS method to determine 30 steroids and steroid esters in bovine serum. Sensitivity was improved using microwave-assisted chemical derivatization with methoxyamine hydrochloride. The validation was successfully conducted in accordance with the Decision 657/2002/EC guidelines. An in vivo experiment was performed on 12 crossbred steers in which two commercial formulations containing boldenone undecylenate and testosterone propionate were administrated via intramuscular injections. The samples were collected over a period of 120 days, in which both intact esters were identified within 11 days postadministration. 17ß-Boldenone was observed after 92 days for 2 steers and 56 days for the other animals. The applicability of a cut-off level to the ratio between 17ß-testosterone and epitestosterone was evaluated in an attempt to differentiate testosterone abuse from endogenous production. It could be observed that a calculated ratio above this level is strong evidence of drug administration, although a high false-negative rate was obtained.
Subject(s)
Cattle/blood , Chromatography, High Pressure Liquid/methods , Tandem Mass Spectrometry/methods , Testosterone Propionate/blood , Testosterone/analogs & derivatives , Anabolic Agents/blood , Animals , Drug Residues/chemistry , Male , Testosterone/bloodABSTRACT
Hair analysis has attracted great attention in the regulatory analysis of food-producing animals, particularly due to the wider detection window of veterinary drugs in this matrix and also the possibility of confirming parent drugs with minimum metabolization. This work involved the development and validation of a quantitative liquid chromatography-tandem mass spectrometry method to determine 25 steroids and steroid esters in bovine hair. Sensitivity was improved using a fast and effective microwave-assisted chemical derivatization with methoxyamine hydrochloride. The validation was conducted in accordance with the Decision 657/2002/EC guidelines. An animal experimentation procedure was performed on 12 bovine animals in which two commercial formulations containing boldenone undecylenate and testosterone propionate were administrated via intramuscular injections on the neck. The samples were collected for 78 days in which the detection of the administrated analytes was only observed near the application sites. For some of the monitored days, no analyte was detected on the neck area. Since the migration of the analytes was not observed in areas other than the application site, false-negative results should be carefully considered when monitoring animal hair samples.
Subject(s)
Chromatography, Liquid/methods , Hair/chemistry , Steroids/analysis , Tandem Mass Spectrometry/methods , Animals , Cattle , Chromatography, Liquid/veterinary , Esters/analysis , Esters/chemistry , Male , Microwaves , Steroids/chemistry , Tandem Mass Spectrometry/veterinary , Testosterone/analogs & derivatives , Testosterone/analysis , Testosterone Propionate/analysis , Veterinary Drugs/analysisABSTRACT
In this work, a GC-MS/MS method was developed for the determination of anabolic-agent residues in bovine urine. The optimized sample preparation was as follows: enzymatic hydrolysis by ß-glucuronidase-sulfatase enzyme from Helix pomatia for 16 h at 37.5 °C, liquid-liquid extraction with diethyl ether, solid-phase extraction with HLB and aminopropylsilane cartridges, and microwave-assisted derivatization using 25 µL of MSTFA/NH4I/ethanethiol and full microwave power for 2 min. The method was validated according to Decision 657/2002/EC, Codex Alimentarius, and Manual da Garantia da Qualidade Analítica guidelines. The acceptability criteria for quantitative analysis were met for α-ethinylestradiol, α-nandrolone, ß-estradiol, ß-zearalanol, ß-zearalenol, drostanolone, ethisterone, dienestrol, diethylstilbestrol, hexestrol, megestrol, methyltestosterone, and zearalenone. The analytes α-zearalenol, α-zearalanol, and norethandrolone were validated for qualitative analysis.
Subject(s)
Anabolic Agents/urine , Cattle/urine , Drug Residues/analysis , Gas Chromatography-Mass Spectrometry/methods , Lactones/urine , Steroids/urine , Stilbenes/urine , Tandem Mass Spectrometry/methods , Animals , Limit of Detection , MicrowavesABSTRACT
This work involved the optimization and validation of two methods according to the Commission Decision 2002/657/EC directives for determining fluoroquinolones residues in samples of poultry muscle and kidney: ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pipemidic acid and sarafloxacin. The extraction procedure was based on a QuEChERS approach, whose optimization employed a Box-Behnken 3(3) factorial design. A liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed for determining the twelve analytes using the multiple reaction monitoring mode (MRM). Accuracy, evaluated by recovery studies, varied from 88.8 to 112.2% for the selected levels with RSD values lower than 12.3%. The second validated method employed high resolution mass spectrometry (HRMS) performed in the single ion monitoring mode (SIM), determining nine among twelve analytes. The validation parameters were evaluated as satisfactory, with recoveries from 82.5 to 114.4% and RSD lower than 8.7%. Decision limits and detection capabilities for both methods were reported. The two methods were statistically compared using the Student's t test, at 95% confidence level, resulting in no significant difference.