Croubels, Siska

De Backer, Patrick

Devreese, Mathias

Gorissen, Brecht

Reyns, Tim

Van Loco, Joris

English

Ghent University Academic Bibliography

    DETECTION AND QUANTIFICATION OF SELECTED VETERINARY ANTIMICROBIALS IN POULTRY EXCRETA  B. Gorissen1,2, T. Reyns1, M. Devreese2, P. De Backer2, J. Van Loco1, S. Croubels2  1Scientific Institute of Public Health, Department of Food, Medicines and Consumer Safety, Service of Chemical Residues and Contaminants, Rue Juliette Wytsmanstraat 14, 1050 Brussels, Belgium     2Ghent University, Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Salisburylaan 133, 9820 Merelbeke, Belgium                                    WIV-ISP | Rue Juliette Wytsmanstraat 14 | 1050 Brussels | Belgium T +32 2 642 53 56 | M +32 485 95 99 72 | e-mail: Brecht.Gorissen@wiv-isp.be | www.wiv-isp.be                                        Acknowledgements: this study was financially supported by the Federal Public Service of Health, Food Chain Safety and Environment (contract RT 12/06 AMPOULDETECT)         Background  Due to the risk of antimicrobial resistance development, the Belgian Royal Decree concerning the eradication of Salmonella (C – 2007/22784) prohibits treatment of poultry with antimicrobials against zoonotic Salmonella spp. Nowadays, the unauthorized use of antimicrobials is determined through analysis of tissue samples of sacrificed animals. There is a need to develop a more animal-friendly method to detect illicit use of antimicrobials. Objectives  Development and validation of an UHPLC-MS/MS method for the quantification of residues of β-lactams (amoxicillin (AMO) and phenoxymethylpenicillin), fluoroquinolones (enrofloxacin, difloxacin (DFX) and flumequine), sulfonamides (sulfachloropyridazine (SCP), sulfadiazine and sulfaclozine) in combination with trimethoprim (TMP), tetracyclines (chlortetracycline and doxycycline (DOX)) and for qualitative detection of polymyxins (colistin A) in samples of poultry manure.  Conduction of an animal experiment to gain insight into  excretion of the selected compounds. The developed  method was applied in the determination of target  compounds in derived samples. • A quick and simple UHPLC-MS/MS method for determination of antimicrobials in non-invasive samples of poultry manure was developed and validated. • The applicability of the developed method was tested on biological samples in a preliminary animal experiment; all studied compounds were successfully extracted (Fig. 2). Colistin A was detected until 1,5 days after the last treatment (LOD: 50 ng/g). • The method is an animal-friendly alternative, suitable for high-throughput analysis in official control programmes to tackle the resistance development of Salmonella spp. Conclusions    Animal experiment  Twelve laying hens were equally divided into one control group and five experimental groups. For each antimicrobial class, one active compound was selected and administered by route of drinking water. Between every sample point, floor bedding, feed (and, during administration, medicated drinking water) were refreshed.  D1-7 acclimatisation time zero sample on day 7  D8-12 administration  sampling of manure every 12h  D13-19 wash-out period sampling of manure every 12h Background and objectives Animal experiment Sample preparation Results + 100 µL IS working solution + 10 mL acetonitrile/McIlvaine buffer pH 3.6     (75/25, v/v)  Rotation, ultrasonication and centrifugation            Ultracentrifugation Filtration using Whatman™ PVDF 0.2 µm filter        Acquity UPLC® BEH C18 (100 x 2.1 mm i.d., 1.7µm)                      0.1% HCOOH in water (phase A) and 0.1% HCOOH in acetonitrile (phase B)                                                          Xevo™ TQ-S triple quadrupole (Waters Corp., Milford, MA, USA)      Figure 2. Results of an excretion study performed in laying hens, after oral administration of   either DFX, SCP/TMP combination, DOX or AMO  Figure 1. MS/MS chromatograms for a blank poultry manure sample spiked at 50 ng/g (LOQ)   1,6                                       2,5                                        3,4                                         time (min) 1,6                                       2,5                                        3,4                                        time (min)                 colistin A (390.8>379.2) amoxicillin (366>114)                     flumequine (261.6>243.9) chlortetracycline (479.1>443.8)     phenoxymethylpenicillin (351>160)   sulfachloropyridazine           (285.1>155.8) difloxacin (400.8>356.9)               sulfaclozine (285.7>130) enrofloxacin (360.4>316)    trimethoprim (291>230)   doxycycline (445.3>427.9) sulfadiazine (250.3>155.8) 

Detection and quantification of selected veterinary antimicrobials in poultry excreta

B. Gorissen

T. Reyns

M. Devreese

P. De Backer

J. Van Loco

S. Croubels

Sciensano Publications Repository

Determination of selected veterinary antimicrobials in poultry excreta

DETERMINATION OF SELECTED VETERINARY ANTIMICROBIALS IN POULTRY EXCRETA B. GORISSEN*1, T. REYNS1, M. DEVREESE2, P. DE BACKER2, J. VAN LOCO1, AND S. CROUBELS2 1: Scientific Institute of Public Health, Department of Food, Medicines and Consumer Safety, Service of Chemical Residues and Contaminants, Brussels, Belgium 2: Ghent University, Department of Pharmacology, Toxicology and Biochemistry, Laboratory of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Merelbeke, Belgium  The most important source of Salmonella infection is through consumption of contaminated poultry products in men. Due to the risk of resistance development, the Royal Decree concerning the eradication of Salmonella (C – 2007/22784) prohibits treatment of poultry with antimicrobials against zoonotic Salmonella. An analytical method using UPLC-MS/MS for the determination of antimicrobial residues in samples of poultry manure was developed including beta-lactams (amoxicillin, phenoxymethylpenicillin), fluoroquinolones (enrofloxacin, difloxacin, flumequine), sulfonamides in combination with trimethoprim (sulfachloropyridazine, sulfadiazine, sulfaclozine), tetracyclines (chlortetracycline, doxycycline) and polymyxins (colistin). Extraction of samples was carried out by means of ultrasonication and vortex mixing using a combination of acetonitrile and McIlvaine buffer as the extraction solution, followed by centrifugation and filtration for subsequent analysis. During validation the performance characteristics of the developed method were determined in accordance with Commission Decision 2002/657/EC.  An animal experiment with twelve laying hens was conducted to gain insight into excretion of the selected compounds in faeces and urine. For each antimicrobial class, one active compound was selected and administered by route of drinking water. Samples were collected every twelve hours before, during and after treatment. The developed method was applied in the determination of target compounds in these samples.     *: Corresponding author: e-mail: brecht.gorissen@wiv-isp.be  

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Detection and quantification of selected veterinary antimicrobials in poultry excreta

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Ghent University Academic Bibliography

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Ghent University Academic Bibliography