Financial support: U-Inicia VID Research Project

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.

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 out-breaks

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.

INTRODUCTION

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

• Edible tissues of broiler chickens

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

INTRODUCTION

Enough information to evidence that antimicrobials

after therapy completion may remain in tissues for varying times depending on the drug’s elimination kinetics.

• 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.

INTRODUCTION

EXPORTS

2014

IMPORT

2007US$ 516

MILLiONS

EXPORTS

2013

106%MORE THAN

PREVIUS YEAR

US$19.194MILLIONS

2.72MILLIONS OF METRIC TONS

14.990TONS

5.6%INCREASE

DURING THE LAST FEW YEARS, THE MARKET FOR THIS BY-PRODUCT HAS GROWN.

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C15 Esta diapo esta OK solo que esta en español, pero eso lo puedo cvambiar yo despues CLIENTE, 18-5-2016

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.

INTRODUCTION

Körner et al. (2001) studied the incidence of tetracyclineresidues in commercial bone and meat meal samples. They found tetracyclines in all samples with concentrations of up to 2,295 µg kg-1.

Odore et al. (2015) analysed bones 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 µg kg-1 at day 10 post-treatment.

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.

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 µg kg-1.

Odore et al. (2015) analysed bones 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 µg kg-1 at day 10 post-treatment.

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

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.

MATERIALS AND METHODS

EXPERIMENTAL ANIMALS

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

BROILER CHICKEN ROSS 308 GENETIC

X 40MALE/ ONE-DAY-OLD

MATERIALS AND METHODS

GROUP WITH AND WITHOUT TREATMENT

WITH TREATMENT•ADMINISTRATED ORALLY ONCE A DAY •THROUGH PREMIX POWDERX 32

GROUPA

X 8GROUP B

WITHOUT TREATMENT

50mg/kg-1

bw of OTC 10%.

X 7 DAYS

CONSECUTIVE

NUMBER OF ANIMALS DETERMINED BY EMA GUIDELINES

MATERIALS AND METHODS

MATERIALS AND METHODS

TREATMENT

DEPLETION STUDY

1 14 15 21 22 28 40BREEDING TREATMENT BEGINS TREATMENT END 7 DAYS WDT END

Day 3 Day 9 Day 15 Day 19

POST TREATMENT SLAUGHTER DAYS AND SAMPLING

GRUOP A

GRUOP B

X 8

X 2SLAUGHTER ANIMALS

MATERIALS AND METHODS

SAMPLES COLLECTION

SAMPLE COLLECTION AND PROCESSING

ALL SAMPLES WERE CHOPPED WITH SCISSORS

SAMPLES HOMOGENIZED IN A FOOD PROCESSOR

STORED IN FRESH BAGS AT -20° C UNTIL EXTRACTION

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 analysedcommercial 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).

MATERIALS AND METHODS

MATERIALS AND METHODS

SAMPLE(5 0.05 g)

EXTRACTIONEDTA-MCILVAINE BUFFER

AGITATED AND CENTRIFUGED SUPERNATANT OBTAINED

PURIFICATION THROUGH GLASS WOOL

FILTRATION OF SUPERNATANT IN SPE SEP-PAK C18 COLUMN (PRECONDICIONED WITH HPLC WATER AND ACETONITRILE)

COLUMN ELUTION(WITH 1.0 m METHANOLIC OXALIC ACID)

EVAPORATED UNDER A MILD NITROGEN FLUX (40-50°C)

RECONSTITUTION WITH 250 mL OF MOBILE PHASE (OXALIC ACID 0.01 m/ACETONITRILE, 5:1)

LC VIAL

ANALYTICAL METHODOLOGY

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).

IONS PRECURSOR ION FRAGMENT ION

OTC y 4-epi-OTC 461.0 426.0

461.0 381.0

TC d-6 (IS) 451.0 416.0

MONITORED ION MASSES.

RESULTS

The analytical method was implemented and then optimized with the aim to improve the analyte recovery.

IMPLEMENTATION OF THE ANALYTICAL METHOD

When the OASIS HLB® SPE columns was used average R2 were 0.869 and 0.908, respectively.

The use of SPE sep-pak C18 instead of the SPE OASIS HLB® gave a better linearity of the calibration curves. Using SPE sep-pak 18 average R2 in the three curves was 0.991 for OTC and 0.983 for 4-epi-OTC.

Increasing centrifugation time allowed improvement of the recoveries of both analytes.

RESULTS

Changes in methodology Results

Reduction of sample size Improves matrix clean up

Increase in centrifugation time Increases recovery

Addition of reagent tris (2-carbaxyethyl) phosphine hydrochloride 98% (TCEP-HCL) No changes in extraction

Centrifugation at 0° C Improves sediment decanting

Hexane addition Decreases recovery

SPE OASIS HLB cartridgeSPE sep-pak C18 cartridge

Decreases recoveryIncreases recovery

Changes made for the improvement of analyte recovery

IMPLEMENTATION OF THE ANALYTICAL METHOD

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.

RESULTS

ParameterCriteria

Retention time CV -5%

LinearityR2≥0.95

Recovery (%) at LoD level

Repeatability (%) at LoD CV > Repro.

Reproducibility (%) at LOD CV < 23%

LoD(µg kg-1)

LoQ(µg kg-1)

OTC 0.32% 0.9608 97% 13.8 16 20 22

0.9932

0.9930

4-epi-OTC 2.41% 0.9611 116% 10.3 15 20 22

0.9927

0.9801

Summary of validation data for OTC and 4-epi-OTC in claws.

RESULTS

Validation of the analytical method

RESULTS

ParameterCriteria

Retention time CV -5%

LinearityR2≥0.95

Recovery (%) at LoD level

Repeatability (%) at LoD CV >

Repro.

Reproducibility (%) at LOD CV < 23%

LoD(µg kg-1)

LoQ(µg kg-1)

OTC 0.32% 0.9608 97% 13.8 16 20 22

0.9932

0.9930

4-epi-OTC 2.41% 0.9611 116% 10.3 15 20 22

0.9927

0.9801

• An internal protocol was used for the in-

house validation.

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).

RESULTS

RESULTS

Figure 2. (a) and (b) Chromatograms representative of pure drug injections. (a) 4-epi-OTC retention time (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 interferences in 4-epi-OTC retention time (461.0/426.0). (d) IS TC-d6 injection (451.0/416.0).

• in-house validation study

• 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.

ANALYTERETENTION TIME

SPECIFICITY

LOD

LOQLINEARITY

RECOVERY

PRECISION

RESULTS

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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.

RESULTS

SLAUGHTER DAYS

DAYS POST TREATMENT

DAYS OF LIFE

NUMBER OF ANIMALS

OTC AVERAGE CONCENTRATION

(µg/kg)

epi-OTC AVERAGE CONCENTRATION

(µg/kg)

OTC + EPI OTC (µg/kg)

M1 3 24 8 1835 1162 2997

M2 9 30 6 984 370 1354

M3 15 36 7 157 154 311

M4 19 40 7 20 84 104

RESULTS

• The withdrawal time for claws was established on the basis of the statistical method stipulated by the EMA Guidelines (95% tolerance and 95% confidence).

DETERMINATION OF WDTS

• 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

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.

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.

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??

• Project research team

• FARMAVET Laboratory

• Ekaterina Pokrant

• Aldo Maddaleno

ACKNOWLEDGEMENTS

THANK YOU

JAVIERA CORNEJO, MV., PhDjacornej@uchile.cl