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P112 (117) THE DEPLETION OF DOXYCYCLINE RESIDUES IN POULTRY TISSUES 1

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Nora Mestorino , Martín Daniele , Martín Dadé , Andrea Buchamer , Valeria Vedovato and Laura Marchetti 1

Laboratory of Pharmacological and Toxicological Studies (LEFyT), Faculty of Veterinary Science, Universidad Nacional de La Plata, 60 and 118, 1900 La Plata, Argentina ([email protected]) Abstract Doxycycline (DOX), tetracycline of second generation, is active against Gram+ and Gram- bacteria, aerobic and anaerobic. Although there are few pharmacokinetic studies in chickens, it is frequently used for colibacillosis treatment, salmonellosis, staphylococcal infections, avian mycoplasmosis and chlamydia. Our objective was to evaluate the withdrawal time (WT) of DOX formulation at 25% in edible tissues, after PO use in 40 healthy broilers (30-35 days of age). DOX was administered -1 through medicated drinking water for 5 days at 10 mg kg (N = 36). Four untreated animals were reserved as controls. Six animals per group were sacrificed by exsanguination, after 24 h until 9 d post treatment when control animals were sacrificed as well. Muscle, liver, kidney and skin/fat samples were obtained. DOX was determined by HPLC with UV detection. DOX concentrations were determined in all tissues examined; generally falling below the MRL at 7 d after administration is terminated. The calculated WTs were 6.58, 8.18, 8.69 and 6.96 d for muscle, liver, kidney and skin/ fat, respectively. After -1 DOX administration at a rate of 10 mg kg for 5 days through the drinking water, the WT must be 9 d before treated chickens can be consumed. Introduction Doxycycline (DOX) is a semi-synthetic bacteriostatic tetracycline and a broad-spectrum antibiotic against Gram-negative and Gram-positive aerobic and anaerobic bacteria, Rickettsiae, Chlamydiae, Mycoplasmas and some protozoa (Anadón et al., 1994; Prats et al., 2005). Pharmacokinetics properties of doxycycline are superior over older tetracyclines in terms of higher lipid solubility, complete absorption, better tissue distribution, longer elimination half-life and lower affinity for calcium (Goren et al., 1998). The in vitro antimicrobial activity of doxycycline is more effective than other tetracyclines for the treatment of respiratory, urinary and gastrointestinal tract diseases (Abd El-Aty et al., 2004). Although there are few pharmacokinetic studies in chickens, it is frequently used for the treatment of colibacillosis, salmonellosis, staphylococcal infections, avian mycoplasmosis and chlamydiasis (Anadón et al., 1994). The misuse of DOX, illegal administration to animals, overdosing, and not obeying withdrawal periods can lead to accumulation of residues of this antibiotic in edible animal tissues. Due to persistence of DOX residues in tissues and for consumer’s -1 health protection, the European Commission had set a maximum residue limits (MRLs) for this compound at 100 μg kg for -1 -1 muscle, 300 μg kg for liver and skin/fat, and 600 μg kg for kidney. According to Commission Regulation (EU 37/2010) and EMA Summary Report Doxycycline 2, quantification of DOX in animal tissues requires a determination of only the parent compound as the residue marker. In contrast to other tetracyclines for which the MRLs are defined as a sum of the parent drug and its 4-epimer (EU N° 37/2010, EMEA/MRL/270/97, 1997), DOX is not appear with a corresponding 4-epimer. The antibiotics residue levels reached in organs and the rate of their depletion from tissues depend on the method of administration, animal species, as well as dose and specific given drug (Kung & Wanner, 1994). The differences in the antibiotic concentration and time of its depletion may be also influenced by differences in drinking water intake by animals. The purpose of the present study was to evaluate the withdrawal time (WT) for DOX formulation at 25% in edible tissues, after its PO use in broilers. Materials and Methods Study Design Treatment and Administration -1 The experiment was conducted with 40 five-week-old chickens; 36 chickens were treated with 10 mg kg bw of DOX (25%) once a day for five consecutive days through the drinking water. Solution was prepared by dilution of 400 g of the medicament in 1,000 mL water. The birds were kept in a special space designed for performing experiments on animals. Prior to the treatment, chickens were deprived of water. Antibiotic-free feed was available ad libitum. The chickens treated with DOX were euthanized 24 h, 2d, 3d, 5d, 7d and 9d after final drug administration (six animals at each time point) and muscle (breast), liver, skin plus fat and kidney were collected. Four chickens used as controls were euthanized before the experiment commenced, and the same tissues samples were collected from these animals. All samples were stored separately at -20°C until analysis. The animal experiment protocol was in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (Federation of Animal Science societies -FASS-).

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Reagents Doxycycline (DOX) standard was obtained from Sigma-Aldrich Chemical Company (USA). Acetonitrile and methanol were from JT Baker. Oxalic acid dehydrate, trichloroacetic acid and sodium sulphate anhydrous were from Fluka (USA). Solid-phase extraction (SPE) columns (Strata, C18, 100 mg, 1 mL) and analytical column (Luna C18) were obtained from Phenomenex (USA). Doxycycline was formulated as a 25% experimental water soluble powder (DOX 25 g, Tartaric acid 5g and Lactose sqt 100 g). Standard solutions -1 Stock standard solution (1 mg mL ) was prepared by weighing 10.0 ± 0.1 mg of standard and dissolving it in 10 mL methanol. -1 The stock was stored at -20°C in amber glass, and was stable for six months. Secondary standard solutions (100 μg mL , -1 10 μg mL ) prepared in methanol by diluting suitable aliquots of stock standard were stable for one month and were stored at 2-8°C in amber glass. Working standard solutions in mobile phase were prepared on the day of analysis. Extraction and clean-up A portion of 0.4 g of tissue (problem or spiked) was homogenized with 1.4 mL of McIlvaine buffer-EDTA, shaken at high speed and centrifuged at 2,500 g at 4°C for 15 min. The upper layer (supernatant S1) was transferred into a new tube. The extraction was repeated thrice. The supernatants S2, S3 and S4 were combined with S1. The mixture was vortexed for 30 s and centrifuged again for 10 min at 2,500 g at 4°C. Clean-up The supernatant mix (S1-S2-S3-S4) was transferred to SPE C18 cartridges, which were preconditioned with 3 mL methanol and 2 mL ultrapure water. The tube reservoir mix supernatants were washed with 1 mL McIlvaine buffer-EDTA and 1 mL water, after percolation of the whole solution; the columns were washed with these solutions (under vacuum). After drying for 2 min, the doxycycline was eluted with 4 mL of methanol 0.01 M oxalic acid pH 2.0. The cleaned extracts were evaporated to dryness in nitrogen evaporator at 40°C. The dried residues were reconstituted in 200 µL mobile phase. After vortexing and centrifugation, 100 µL were injected into the chromatographic system. LC-UV analysis The instrumental analysis was performed using Gilson HPLC system equipped with isocratic pump, autosampler, column oven, and UV/Vis detector (λ = 346 nm), controlled by Unipoint Workstation software. Chromatographic analyses were performed on Luna (Phenomenex) C18 column (5 μm, 150 mm x 4.6 mm) with mobile phase consisting of water-acetonitrile -1 with 0.02 M oxalic acid and 0.5 mM EDTA (72:28, v/v) at 1.2 mL min flow rate. The column oven temperature was controlled at 30°C. Method validation The following parameters were evaluated for the analysis of each matrix: linearity (concentrations of DOX ranging between -1 -1 0.1 and 6.0 µg mL - µg g ), precision and accuracy, limit of quantitation, limit of detection and selectivity. The standard calibration curve was prepared by the injection of standard solutions on seven levels, namely 0.1, 0.2, 0.5, 1.0, -1 -1 2.0, 4.0 and 6.0 µg mL or µg g ). The correlation coefficient was evaluated. -1

Samples of tissues were spiked with the DOX working solution to levels corresponding to 0.2, 0.5, 1.0 and 2.0 µg g . The six spiked samples with DOX were analysed within three different days. Based on these spiked samples replicates, the precision (repeatability and reproducibility) of the method was determined. The mean accuracy (% recovery) was evaluated by comparing the concentrations in the spiked samples with known amounts of analytes to the concentrations in standard solution which should be within the range 85-115%, while the variation in precision should be ≤ 20%. The limit of detection (LOD) was estimated through the analysis of 20 aliquots of control tissue (free of DOX). The noise of the base-line was measured; the average and the standard deviation were calculated. The LOD corresponded to three times SD (signal-to-noise ≥ 3:1) and the limit of quantitation (LOQ) corresponded to ten times this SD (signal-to-noise ≥ 10:1). Withdrawal time Numerous experimental designs and statistical approach are used to establish the withdrawal time. The EMEA recommends the use of a linear regression analysis of the logarithmic transformed data as the choice method (EMEA, 2002). The withdrawal time is determined as the time when the one-sided, 95% upper tolerance limit of the regression line with a 95% confidence level is below the MRL. Doxycycline concentrations in function of time found in muscle, kidney, liver and skin/fat were plotted and analysed with the program WT version 1.4 in order to recommend a withdrawal time for this experimental formulation.


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Results The development and validation were successfully accomplished. This method performed accurately and reproducibly over a -1 -1 range of 0.1 to 6.0 µg mL or µg g for DOX. Precision of the system -1 One standard solution was prepared containing 0.2 µg mL of DOX and the precision of the system was evaluated after the placement of twenty (20) injections in the chromatographic system. In this manner the efficiency of the column and of the system were evaluated. After twenty injections a coefficient of variation (CV) of 5.2% was determined. Assay linearity -1 This assay exhibited a linear dynamic range between 0.1 and 6.0 µg mL . A linear relationship was obtained across one dynamic range (r values ranged from 0.992 to 0.9997). Specificity Six different samples from control tissue (free of DOX) and six tissue samples fortified with DOX were analysed by HPLC and the corresponding chromatograms were compared. No matrix interferences were observed in the chromatograms of the samples with the same retention time as doxycycline. The chromatographic analysis time was short; DOX eluted at 4 min as a sharp and symmetrical peak with no interfering peaks. Limit of detection (LOD) -1 The limits of detections (LODs) were 0.040, 0.024, 0.024 and 0.010 µg g for DOX in kidney, skin/fat, muscle and liver respectively. Limit of quantitation (LOQ) -1 The LOQs were 0.095, 0.074, 0.054 and 0.100 µg g for DOX in kidney, skin/fat, muscle and liver, respectively. Intra-day and inter-day accuracy and precision The method for the analysis of tissue samples was thoroughly validated and the results are presented in Table 1. To assess the inter-day (over three days) assay accuracy and precision, six sets of tissue samples were prepared containing DOX at 0.2, -1 0.5, 1.0 and 2.0 µg g . The inter-day variation in accuracy (recovery) and precision were assessed. The mean accuracy (recovery) should be within the range 85-115 % and the variation in precision should be ≤ 20%. To determine the intra-day accuracy and precision, six replicates at four concentrations were analysed along with duplicate standard calibration curves prepared from two separate stock solutions (Table 1). Table 1. Validation results for the analysis of DOX in chickens tissues

matrix

r

µg g Accuracy (%), n=6 Precision (%), n=6

Intra-day

Inter-day (over 3 days)

Muscle

0.995 -1 (0.2-2.0 µg g )

0.2 0.5 1.0 2.0

99.9 89.7 110 97.8

5.4 5.5 4.5 3.3

99.96 91.2 108 96.8

5.0 4.6 2.3 2.5

Liver

0.992 -1 (0.2-2.0 µg g )

0.2 0.5 1.0 2.0

96.4 93.1 82.3 86.4

8.6 3.0 0.62 7.4

97.8 101.6 90.7 93.96

5.0 6.6 8.3 7.4

Kidney

0.993 -1 (0.2-2.0 µg g )

0.2 0.5 1.0 2.0

97.5 94.0 106 97.8

3.5 5.7 2.1 1.1

104 94.4 98.5 98.8

13 2.7 8.4 1.0

Skin/Fat 0.995 -1 (0.2-2.0 µg g )

0.2 0.5 1.0 2.0

89.1 98.9 105 98.4

0.00 0.03 0.07 0.10

86.4 99.0 99.5 96.1

2.7 3.0 5.8 2.6

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Accuracy (%) Precision (%)


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Doxycycline tissue concentrations DOX was determined in all tissues examined; generally falling below the MRL at 7 d after the end of the administration. The mean ± SD DOX tissue concentrations after the PO administration of doxycycline to chickens in muscle, liver, kidney and skin/fat tissues are presented in Figure 1. After DOX administration to chickens at the dose of 10 mg kg-1 for five consecutive days through the drinking water, the highest content was found in the kidney and liver at one day after the treatment was -1 -1 completed. The maximum determined concentration in the kidney was 6.5 μg g and in the liver was 4.79 μg g . Whereas in -1 -1 muscle and skin/fat the detected values were lower, with maximum concentration of 1.46 μg g and 1.23 μg g , respectively. Subsequently a rapid decrease in DOX concentration was observed in all tissues. At 7 d after treatment was completed, the DOX concentration in muscle was above LOQ of the used method.

Figure 1. Mean tissue concentrations of doxycycline in chickens slaughtered at day 1, 2, 3, 5, 7 and 9 after oral administration of DOX 25% -1 (dose of 10 mg kg body weight during 5 days).

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B

C

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Figure 2: Plot of the withdrawal times calculation for DOX in chicken muscle (A), kidney (B), skin/fat (C) and liver (D) at the time when the -1 one-sided 95% upper tolerance limit is below the EU MRL for doxycycline after its oral administration (5 doses of 10 mg kg body weight of DOX 25%). [Residue marker: doxycycline].

Taking into account the MRLs for chickens and considering that the marker residue is doxycycline, the calculated withdrawal times 6.58, 8.18, 8.69 and 6.96 d for muscle, liver, kidney and skin / fat, respectively (Figure 2A, 2B, 2C and 2D).


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Discussion Doxycycline is known as strong lipophilic compound, well absorbed after oral administration, and showing high tissue binding (Jerome del Castillo, 2006). The route of drug administration plays an important role in the effectiveness of the treatment, as well as in the distribution of antibiotics to tissues (Laczay et al., 2001). The antibiotics’ residue levels reached in organs and the rate of their depletion from tissues depend on the method of administration, animal species, as well as dose and specific formulation drug given. The differences in the antibiotic concentration and time of its depletion may be also influenced by the differences in the intake of drinking water by animals and water quality. Studies on tissue concentrations after different drug formulation administration are essential to control antibiotic residues in food animal products, i.e. that an appropriate withdrawal time is recommended. The data obtained after DOX administration with water shows that at the beginning after treatment, DOX reached high concentrations in all edible tissues. One day after administration of the last dose, DOX concentration rapidly decreased in all assayed tissues. Then, the residues decreased gradually and only trace concentrations were observed on day 9 post-treatment. Conclusions The development and validation of the analytical method were successfully accomplished. This method performed accurately -1 -1 and reproducibly over a range of 0.1 to 6.0 µg mL or µg g for DOX. -1

Our results demonstrate that DOX oral administration at the 10 mg kg for five days jointly with drinking water require withdrawal time of nine days in order to respect the MRL fixed for edible part of broilers. Acknowledgements Mr. Pedro Zeinsteger is acknowledged for checking the manuscript. References − − − − − − − − − − −

Abd El-Aty AM, Goudaha A, Zhoub HH. Pharmacokinetics of doxycycline after administration as a single intravenous bolus and intramuscular doses to non-lactating Egyptian goats. Pharmacol Res. 2004;49:487-91. Anadón A., Martínez-Larrañaga MR., Díaz MJ., Bringas P., Fernández MC., Fernández-Cruz ML., Iturbe J., Martínez MA. Pharmacokinetics of doxycycline in broiler Chickens. Avian Pathol. 1994;23:79-90. Commission Regulation EU No 37/2010. Off J Eur Comm. 2010;L15:1–72. EMEA Summary Report Doxycycline (2), European Medicines Agency Veterinary Medicines and Inspections, Committee for Veterinary Medicinal Products. Doxycycline – Summary Report (2). EMEA/MRL/270/97, 1997. EMEA. Updated Application Software relating to Note for Guidance on Approach towards Harmonisation of Withdrawal Periods for Meat. EMEA/CVMP/563/02, London, UK. 2002. Geflugelkunde 1994;58:84–88. Goren E, De Jong WA, Doornenbal P, Laurense T. Therapeutic efficacy of doxycycline hyclate in experimental Escherichia coli infection in broilers. Vet Q. 1998;10:48-52. Jerome del Castilo RE. Tetracyclines. In: Antimicrobial Therapy in Veterinary Medicine, edited by S. Giguere, J.F. Prescott, J.D. Baggot, R.D. Walker, P.M. Dowling, New York, 2006, pp. 31–239. Kung K, Wanner M. Pharmacokinetics of doxycycline in turkeys and comparison between feed and water medication. Archiv Laczay P, Semjen G, Lehel J, Nagy G. Pharmacokinetics and bioavailability of doxycycline in fasted and nonfasted broiler chicken. Acta Vet Hung. 2001;49:31–7. Prats C, Elkorchi G, Giralt M, Cristofol C, Pena J, Zorrilla I, Saborit J, Perez B. PK and PK/PD of doxycycline in drinking water after therapeutic use in pigs. J Vet Pharmacol Ther. 2005;28:525-30.


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