JAVIERA CORNEJO, EKATERINA POKRANT, DANIELA ARAYA, ALDO MADDALENO, CAROLINA ARAYA-JORDÁN, HECTOR HIDALGO, BETTY SAN MARTIN.

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1 DEPLETION STUDY OF OXYTETRACYCLINE (OTC) AND ITS METABOLITE 4-EPI-OXYTETRACYCLINE RESIDUES (4-EPI-OTC) IN CLAWS OF BROILER CHICKENS BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (LC-MS/MS) JAVIERA CORNEJO, EKATERINA POKRANT, DANIELA ARAYA, ALDO MADDALENO, CAROLINA ARAYA-JORDÁN, HECTOR HIDALGO, BETTY SAN MARTIN. Financialsupport: U-Inicia VIDResearchProject

2 INTRODUCTION Oxytetracycline (OTC) is an antimicrobial from the tetracyclines family, authorized for therapeutic use in poultry to combat infectious diseases It is often administered orally through medicated water or feed in the case of outbreaks It is quickly absorbed by the gastrointestinal tract, being widely distributed throughout the organism and reaching high levels in kidneys and liver They may persist as chemical residues in food affecting their safety risk to consumers health.

3 INTRODUCTION Risks of tetracycline residues in food: Toxicological effects causing ossification and teething disorders. This has been reported especially in children, due to the chelating action of tetracyclines on the calcium ion. Allergic reactions in individuals who have been previously sensitized The introduction of these antimicrobial agents in production systems has created favourable conditions for the selection, propagation and persistence of antimicrobial resistant bacteria, capable of causing infections in animals and humans

4 Edible tissues of broiler chickens INTRODUCTION Enough information to evidence that antimicrobials after therapy completion may remain in tissues for varying times depending on the drug s elimination kinetics. Several authors have studied the OTC depletion profile and its metabolite 4-epi-OTC to establish the post-treatment residue levels in chickens edible tissues, such as muscle, liver and kidneys

5 Edible tissues of broiler chickens INTRODUCTION Scarce information in the literature about the behavior of antimicrobial concentrations in poultry by-products. Until today there are no studies on the chicken claws. This by-product may be destined to human consumption directly, or indirectly through claw-meal. Not considered an edible tissue. No regulations or MRLs established.

6 INTRODUCTION DURING THE LAST FEW YEARS, THE MARKET FOR THIS BY-PRODUCT HAS GROWN. IMPORT 2007 US$ 516 MILLiONS 106% MORE THAN PREVIUS YEAR EXPORTS MILLIONS OF METRIC TONS 5.6% INCREASE EXPORTS TONS US$ MILLIONS

7 Slide 6 C15 Esta diapo esta OK solo que esta en español, pero eso lo puedo cvambiar yo despues CLIENTE,

8 INTRODUCTION Chicken claws are basically bone and cartilage, so they may accumulate higher tetraciclines concentrations compared to other tissues due to they affinity for calcium. Medina et al. (2008) analysed the tetracycline content in bones from slaughterhouse swines and observed that 81% of the analysed samples were positive while 50% of the edible tissues samples were positive. U.

9 INTRODUCTION Körner et al. (2001) studied the incidence of tetracycline residues in commercial bone and meat meal samples. They found tetracyclines in all samples with concentrations of up to 2,295 µgkg-1. Odoreet al. (2015) analysedbones samples (breastbone, femur, tibia and fibula) from broilers treated with a therapeutic dose of OTC, by LC MS/MS. They found OTC and 4-epi-OTC levels as high as 1,286 µgkg-1 at day 10 post-treatment.

10 INTRODUCTION Chicken claws are basically bone and cartilage, so they may accumulate higher tetraciclinesconcentrations compared to other tissues due to they affinity for calcium. Medina et al. (2008) analysed the tetracycline content in bones from slaughterhouse swines and observed that 81% of the analysedsamples were positive while 50% of the edible tissues samples were positive. Körneret al. (2001) studied the incidence of tetracycline residues in commercial bone and meat meal samples. They found tetracyclines in all samples with concentrations of up to µgkg-1. Odoreet al. (2015) analysedbones samples (breastbone, femur, tibia and fibula) from broilers treated with a therapeutic dose of OTC, by LC MS/MS. They found OTC and 4-epi-OTC levels as high as µgkg-1 at day 10 post-treatment.

11 INTRODUCTION These results show the persistence of this drug in the studied tissues. It also gives evidence for the possible re-entry of veterinary drug residues into the food chain. Moreover, some authors state that due to OTC thermostability this drug is not affected by conventional cooking for human consumption, or by the industrial thermal processes performed in meals, including by-products CONSUME

12 INTRODUCTION Currently, there are no specific studies on broiler claws assessing the presence of antimicrobial residues. The pharmacokinetic characteristics of this antimicrobial would promote the accumulation of residues at higher concentrations during longer periods of time. OTC residues may remain in broiler claws evidencing the need of carrying out specific studies in this matrix. Considering the potential risk of a re-entrance of antimicrobials into the food chain through this by-product in the present study OTC residues depletion in broiler claws were studied for the first time therapeutically treated broiler chickens.

13 MATERIALS AND METHODS EXPERIMENTAL ANIMALS BROILER CHICKEN ROSS 308 GENETIC X 40 MALE/ ONE-DAY-OLD KEPT IN INDIVIDUAL CAGES AD LIBITUM ACCESS TO WATER AND NON- MEDICATED FEED 25 ±5 C AND 50 60% RELATIVE HUMIDITY THE CAGES HAD AN ELEVATED WIRE FLOOR

14 MATERIALS AND METHODS GROUP WITH AND WITHOUT TREATMENT X 32 GROUPA WITH TREATMENT 50 mg/kg -1 bwof OTC 10%. X 7 DAYS CONSECUTIVE ADMINISTRATED ORALLY ONCE A DAY THROUGH PREMIX POWDER X 8 WITHOUT TREATMENT GROUP B

15 MATERIALS AND METHODS NUMBER OF ANIMALS DETERMINED BY EMA GUIDELINES

16 MATERIALS AND METHODS TREATMENT DEPLETION STUDY GRUOP A X BREEDING TREATMENT BEGINS TREATMENT END 7 DAYS WDT END Day 3 Day 9 Day 15 Day 19 GRUOP B SLAUGHTER ANIMALS X 2 POST TREATMENT SLAUGHTER DAYS AND SAMPLING

17 MATERIALS AND METHODS SAMPLES COLLECTION

18 MATERIALS AND METHODS SAMPLE COLLECTION AND PROCESSING SAMPLES ALL SAMPLES WERE CHOPPED WITH SCISSORS HOMOGENIZED IN A FOOD PROCESSOR STORED IN FRESH BAGS AT -20 C UNTIL EXTRACTION

19 MATERIALS AND METHODS ANALYTICAL METHODOLOGY The method for the extraction of OTC and 4-epi-OTC developed for broiler claws was based on the methods described by Reveurs and Díaz (1994), Khong et al. (2005) and Castellari et al. (2009). Samples for the implemented analytical method were obtained from previously analysed commercial broiler claws. For the analysis and quantification of OTC and 4-epi-OTC in broiler claws, certified standard of purity were used (Dr. Ehren-storfer Gmbh). As internal standard (IS) an isotopically labelled tetracycline (TC-d6) of certified purity was obtained from the Toronto Research Chemicals (Canada).

20 MATERIALS AND METHODS ANALYTICAL METHODOLOGY SAMPLE ( g) EXTRACTION EDTA-MCILVAINE BUFFER AGITATED AND CENTRIFUGED SUPERNATANT OBTAINED LC VIAL RECONSTITUTION WITH 250 mlof MOBILE PHASE (OXALIC ACID 0.01 m/acetonitrile, 5:1) EVAPORATED UNDER A MILD NITROGEN FLUX (40-50 C) COLUMN ELUTION (WITH 1.0 m METHANOLIC OXALIC ACID) FILTRATION OF SUPERNATANT IN SPE SEP-PAK C18 COLUMN (PRECONDICIONED WITH HPLC WATER AND ACETONITRILE) PURIFICATION THROUGH GLASS WOOL

21 MATERIALS AND METHODS INSTRUMENTAL ANALYSIS LC-MS/MS analysis was carried with a liquid chromatograph (Agilent series 3200) coupled to a triple quadrupole mass spectrometer (API 4000, SCIEX). MONITORED ION MASSES. IONS PRECURSOR ION FRAGMENT ION OTC y 4-epi-OTC TC d-6 (IS)

22 RESULTS IMPLEMENTATION OF THE ANALYTICAL METHOD The analytical method was implemented and then optimized with the aim to improve the analyterecovery. The use of SPE sep-pakc18 instead of the SPE OASIS HLB gave a better linearity of the calibration curves. Using SPE sep-pak18 average R 2 in the three curves was for OTC and for 4-epi-OTC. Increasing centrifugation time allowed improvement of the recoveries of both analytes. When the OASIS HLB SPE columns was used average R 2 were and 0.908, respectively.

23 RESULTS IMPLEMENTATION OF THE ANALYTICAL METHOD Changes made for the improvement of analyterecovery Changes in methodology Reduction of sample size Increase in centrifugation time Addition of reagent tris (2-carbaxyethyl) phosphine hydrochloride 98% (TCEP-HCL) Centrifugation at 0 C Hexane addition SPE OASIS HLB cartridge SPE sep-pak C18 cartridge Results Improves matrix clean up Increases recovery No changes in extraction Improves sediment decanting Decreases recovery Decreases recovery Increases recovery

24 RESULTS Validation of the analytical method An internal protocol was used for the in-house validation. Estimation of linearity, repeatability, reproducibility, specificity and recovery (Commission Decision 2002/657/EC). LOD LOQ (FDA, VICH GL49). The method fulfil the acceptance criteria for the studied parameters. The method can be considered specific, since no interferences were observed at the retention times of the analytes.

25 RESULTS Summary of validation data for OTC and 4-epi-OTC in claws. Parameter Criteria Retention timecv -5% Linearity R Recovery (%) at LoDlevel Repeatability (%) at LoDCV > Repro. Reproducibility (%) at LODCV < 23% LoD (µg kg -1 ) LoQ (µg kg -1 ) OTC 0.32% % epi-OTC 2.41% %

26 RESULTS Validation of the analytical method An internal protocol was used for the inhouse validation. Parameter Criteria Retention timecv -5% Linearity R Recovery (%) at LoDlevel Repeatability (%) at LoDCV > Repro. Reproducibility (%) at LODCV < 23% LoD (µg kg -1 ) LoQ (µg kg -1 ) OTC 0.32% % epi-OTC 2.41% %

27 RESULTS Figure 1. Chromatograms representing pure drug injections: (a) OTC retention time (461.0/426.0) and (b) IS TC-d6 retention time (451.0/416.0). Chromatograms of claw blank samples injections: (c) no interferences at the OTC retention time (461.0/426.0) and (d) IS TC-d6 injection (451.0/416.0).

28 RESULTS Figure 2. (a) and (b) Chromatograms representative of pure drug injections. (a) 4-epi-OTC retentiontime (461.0/426.0). (b) IS TC-d6 retention time (451.0/416.0). (c) and (d) Chromatogram of claw blank samples injections. (c) No interferencesin 4-epi-OTC retention time (461.0/426.0). (d) IS TC-d6 injection (451.0/416.0).

29 C51 RESULTS in-house validation study ANALYTE RETENTION TIME The method fulfill the acceptance criteria for the validation parameters is valid to quantify these analytes on claws in a reliable and precise manner. The method can be considered specific, since no interferences were observed at the retention times of the analytes. PRECISION RECOVERY SPECIFICITY LOD LINEARITY LOQ

30 Slide 28 C51 CLIENTE,

31 RESULTS DETERMINATION OF THE CONCENTRATIONS OF OTC AND 4-EPI-OTC IN CLAWS The concentrations of OTC and 4-epi-OTC found in claws from broilers treated therapeutically were evaluated and quantified. Table 5 describes the average concentrations for OTC and 4-epi-OTC at each sampling moment. SLAUGHTER DAYS DAYSPOST TREATMENT DAYS OF LIFE NUMBER OF ANIMALS OTC AVERAGE CONCENTRATION (µg/kg) epi-otc AVERAGE CONCENTRATION (µg/kg) OTC + EPI OTC (µg/kg) M M M M

32 RESULTS DETERMINATION OF WDTS The withdrawal time for claws was established on the basis of the statistical method stipulated by the EMA Guidelines (95% tolerance and 95% confidence). The moment (day) at which concentrations are equal to or lower than the LoD (20 µg kg-1) was determined. Analytes concentrations were lower the LoD at 39 (38.6) days for OTC and 54 (53.5) days for 4-epi-OTC

33 DISCUSSION AND CONCLUSIONS Chicken claws are a matrix in which the antimicrobial presence has not been studied until now. Thus, it was necessary to implement, optimize and validate an analytical method for OTC and its metabolite appropriate for its purpose. Main challenges found during the development of the method were the great amount of fat, cartilage and bone difficult extraction and sample cleaning.

34 DISCUSSION AND CONCLUSIONS Depletion study in chicken claws evidence that this matrix can be a source of antimicrobial residues entry into food chain. In broiler chickens treated with a commercial formulation OTC 10%, residues can be found in claws for long periods of time, which can even surpass the average life of a broiler chicken (42 days approximately). In order to observe the behavior in the matrix for the first time, sampling started at day 3 post-treatment, plus 3 more sampling points after de drug WDT. OTC is capable to accumulate and remain in bones, since it binds to the matrix directly through a complex between the calcium ions in the bone and the four rings of the basic tetracicline structure.

35 DISCUSSION AND CONCLUSIONS The presence of residues from these antimicrobials may become a risk for public health due to their direct effects and through the development of antimicrobial resistance Research in the antimicrobial re-entrance paths into the food chain by this or other alternative matrices is needed. The present study provides scientific foundations for establishing future regulations and monitoring residues in this matrix. Edible tissues??

36 ACKNOWLEDGEMENTS Project research team FARMAVET Laboratory Ekaterina Pokrant Aldo Maddaleno

37 THANK YOU JAVIERA CORNEJO, MV., PhD