Withdrawal Times of Oxytetracycline and Tylosin in Eggs of Laying Hens After Oral Administration

1017

Journal of Food Protection, Vol. 77, No. 6, 2014, Pages 1017–1021 doi:10.4315/0362-028X.JFP-13-440 Copyright G, International Association for Food Protection

Research Note Withdrawal Times of Oxytetracycline and Tylosin in Eggs of Laying Hens after Oral Administration

RUBE´ N MUN˜ OZ,1 JAVIERA CORNEJO,2 ALDO MADDALENO,1 CAROLINA ARAYA-JORDA´ N,1 DANIELA IRAGU¨ EN,1 NICOLA´ S PIZARRO,1 AND BETTY SAN MARTI´N1*

1 2 Laboratory of Veterinary Pharmacology and Department of Animal Preventive Medicine, Faculty of Veterinary and Animal Sciences, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/6/1017/1687041/0362-028x_jfp-13-440.pdf by guest on 30 September 2021 Universidad de Chile, Avenida Santa Rosa 11735, La Pintana, Santiago, Chile

MS 13-440: Received 3 October 2013/Accepted 3 January 2014

ABSTRACT Antimicrobials administered to laying hens may be distributed into egg white or yolk, indicating the importance of evaluating withdrawal times (WDTs) of the pharmaceutical formulations. In the present study, oxytetracycline and tylosin’s WDTs were estimated. The concentration and depletion of these molecules in eggs were linked to their pharmacokinetic and physicochemical properties. Twenty-seven Leghorn hens were used: 12 treated with oxytetracycline, 12 treated with tylosin, and 3 remained as an untreated control group. After completion of therapies, eggs were collected daily and drug concentrations in egg white and yolk were assessed. The yolk was used as the target tissue to evaluate the WDT; the results were 9 and 3 days for oxytetracycline and tylosin, respectively. In particular, oxytetracycline has a good oral bioavailability, a moderate apparent volume of distribution, a molecular weight of 460 g/mol, and is lightly liposoluble. Tylosin, a hydrosoluble compound, with a molecular weight of 916 g/mol, has a low oral bioavailability and a low apparent volume of distribution, too. Present results suggest that the WDTs of the studied antimicrobials are strongly influenced by their oral bioavailability, the distribution, and the molecular weight and solubility, and that these properties also influence the distribution between the egg yolk and white.

Nowadays, good practices in the use of antimicrobial lipid-soluble drugs (2, 20, 21), thus defining the pharma- agents in food-producing animals has become an issue of cokinetic behavior of the drug and, consequently, its special concern, aiming to reduce or prevent antimicrobial presence in the different eggs components. Other research- resistance and avoid the presence of these drug residues in ers conclude that the main factor that determines the drug food for human consumption (6). In the case of poultry, the presence in egg yolk or white is the egg-forming physiology antimicrobial agents are the main therapeutic tool used for (14). According to these authors, the lipid-soluble antimi- the control of bacterial diseases (11). Therapeutic doses of crobial agents can be detected for longer periods of time these drugs administered to laying hens can lead to the after therapy withdrawal, consequently producing longer presence of the parent drug and/or its metabolites in eggs. WDTs than water-soluble antimicrobial agents. Some other To ensure the safety of these products, only eggs produced authors point out that the distribution of the antimicrobial after completion of an adequate withdrawal period can be agents to egg components is a multifactorial phenomenon, used for human consumption (24). Thus, many authors have which can also involve animal physiology factors, such as studied and established the withdrawal times (WDTs) for organ perfusion, tissue composition, and pH, among others. several veterinary antimicrobials in eggs, considering the This complex mix of factors makes it difficult to predict the egg as a whole edible product (4, 8, 23, 31) or egg behavior of drug residues in eggs (12, 24, 25, 32). compartments considered as separate matrices (9, 27, 32, Tylosin, a macrolide antibiotic, and oxytetracycline, from 33). Different authors have studied factors that determine the tetracycline family, are two antibiotics used in poultry for the antimicrobial agents’ distribution between egg yolk and disease treatment. Even though both drugs are widely used, white; however, currently, there is no consensus. they differ in their action range and in their pharmacokinetic It has been stated that drug concentration in each egg and physicochemical characteristics. Oxytetracycline has a compartment (yolk and white), mostly depends upon the molecular weight of 460 g/mol, has a mild liposolubility, and physicochemical properties of the drug, such as the presents a moderate oral bioavailability, with an intermediate molecular weight and solubility. Different authors empha- apparent volume of distribution (1, 3). In contrast, tylosin has size that water-soluble drugs have a higher affinity for the a molecular weight of 916 g/mol, is hydrosoluble, has a low egg white, while the yolk has a higher concentration of oral bioavailability, and its apparent volume of distribution is lowincomparisontooxytetracycline(23, 26). * Author for correspondence. Tel: z56 02 9785610; Fax: z56 02 According to the safety assessment of tylosin and 9785613; E-mail: [email protected]. oxytetracycline residues in eggs, the Codex Alimentarius (7) 1018 MUN˜ OZ ET AL. J. Food Prot., Vol. 77, No. 6 assigned a maximum residue limit (MRL) of 400 mg/kg to Chemical reagents and standards. Oxytetracycline and oxytetracycline and a MRL to tylosin of 300 mg/kg in eggs. tylosin purity standards were used, provided by Dr. Ehrenstorfer However, the European Union (17) has set a MRL of GmbH (Augsburg, Germany). The analytical grade citric acid, 200 mg/kg for both antimicrobial agents in this product. high-performance liquid chromatography (HPLC) grade water, Even when the MRLs for these antimicrobial agents are analytical grade anhydrous sodium sulfate, HPLC grade acetonitrile, and analytical grade oxalic acid were purchased from Merck known, the available information in the literature regarding (Darmstadt, Germany). HPLC grade methanol was purchased from the distribution of these drugs in egg yolk and white and the Fisher Scientific (Fair Lawn, NJ). WDTs for these drugs in eggs before being available for human consumption is limited. The goal of the present study Standard and work solutions. Standard solutions of was to evaluate the depletion of two antibiotics from oxytetracycline were prepared in aqueous mobile phase at different families, oxytetracycline and tylosin, in egg white 4,000 ng/ml and standard solutions of tylosin in HPLC grade water and yolk and to compare the behavior of these two at 10,000 ng/ml. Both solutions were stored at 4 ¡ 2uC in the dark for no longer than 3 months. Matrix-matched calibration curves were

antimicrobials in both eggs compartments. For this purpose, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/6/1017/1687041/0362-028x_jfp-13-440.pdf by guest on 30 September 2021 the concentrations of these drugs in egg white and yolk were spiked with the standard solution immediately prior to extraction. evaluated after therapy withdrawal. Following Regulation Sample preparation. Eggs were analyzed individually each 37/2010/EC (17) recommendations, the concentrations of day. The egg white and egg yolk were separated for analysis of the the parent compound oxytetracycline and its 4-epimer were components. Oxytetracycline extraction was carried out according assessed in the case of oxytetracycline depletion. For to the method described by Cristofanie et al. (10). Briefly, 10 g of tylosin, the concentrations of the metabolite tylosin A were yolk or white was homogenized, and 20 ml of McIlvaine-EDTA determined. buffer (pH 4 ¡ 0.1) was added. The sample was vortexed for 1 min, sonicated for 5 min, and centrifuged at 3,130 | g for MATERIALS AND METHODS 15 min. The supernatant was filtered through glass wool. A solid- Facilities. The present study was conducted in the Laboratory phase extraction column Sep-Pak C18 (Waters Corporation, of Veterinary Pharmacology of the Veterinary and Animal Milford, MA) was used for the clean-up process, and elution was Sciences Faculty, University of Chile, accredited under the performed with 0.01 M oxalic acid in methanol. The fluid was ¡ International Organization for Standardization 17025 standards. dried under nitrogen stream at 40 0.5uC, and the residue was dissolved with 500 ml of mobile phase. An aliquot of 100 ml Pharmaceutical formulations. Two commercially available was injected into the chromatographic system. The detection limit formulations were used: a pharmaceutical formulation of hydro- was set at 30 mg/kg in both egg components. chloride oxytetracycline 80% powder for the oxytetracycline study Tylosin extraction was carried out according to the method and a 10% powder formulation for the tylosin experiment. Prior to described by Civitareale et al. (5). Five milliliters of phosphate the study, the content of oxytetracycline and tylosin were verified buffer (pH 2.5), 5 ml of methanol, and 15 ml of HPLC grade water in the solutions. For this purpose, standard calibration curves of were added to 5 g of egg yolk or white. Samples were agitated for certified standards of these drugs were performed. 5 min, sonicated for 5 min, and centrifuged at 3,130 | g for 10 min. The supernatant was drawn through a SCX Aromatic Sulfonic Acid Animals. Twenty-seven 20-week-old Leghorn hens were extraction column (Bakerbond spe, Deventer, The Netherlands). The used; none of the hens had previous exposure to oxytetracycline extraction columns were set up with 5 ml of methanol and 5 ml of and tylosin. This was confirmed by the chromatographic analysis phosphate buffer (pH 4.0). The elution was carried out with 2 ml of of white and yolk, demonstrating the absence of drugs in eggs. methanol. Each elute fraction was collected and dried under a stream Animals were maintained in batteries (25 ¡ 5uC temperature, 50 of nitrogen at 45 ¡ 0.5uC. The residue was dissolved with 100 mlof to 60% relative humidity) with ad libitum access to water and no mobile phase and injected into chromatographic system. The medicated feed. detection limit was set at 100 mg/kg in both egg compartments. The diet was formulated according to the requirements outlined by the National Research Council (28), and the Liquid chromatography. Both antimicrobial agents were ingredients were previously analyzed to ensure antimicrobial agent analyzed using an Elite LaChrom HPLC system coupled to a diode absence. Experimental animals were kept in conditions in array detector (DAD; Hitachi High Technologies America, Inc.) agreement with animal welfare guidelines outlined by the Bioethics operated at a wavelength of 360 mn. An XTerra RP18 analytical Committee of the Veterinary Sciences Faculty of the University of column (inside diameter, 100 by 4.6 mm) was used for Chile and recommendations of the European Council Directive oxytetracycline chromatographic separation. The mobile phase 2007/43 (18). consisted of a mixture of 13% acetonitrile and 87% oxalic acid. Hens were randomly allocated in one of three experimental The chromatography was conducted at 35uC at a flow rate of groups: group A and group B (n ~ 12) were treated with 40 mg/kg 0.7 ml/min and at wavelength of 360 nm. of body weight of oxytetracycline for 10 days and 35 mg/kg of A Chromolith RP18 analytical column (100 by 4.6 mm) and a body weight of tylosin for 7 days, respectively. Group C (n ~ 3) Chromolit Guard Cartridge precolumn (Merck, Darmstadt, Ger- remained as untreated control hens. Treatments were given via a many) were used for tylosin chromatographic separation. The gastric tube to ensure total dose intake. The number of animals per mobile phase was a mixture of 19% acetonitrile, 19% water, and treated group was selected according to the European Union 62% 0.02 M dibutyl ammonium phosphate solution. Chromatog- standards for WDTs, established by the European Agency for the raphy was conducted at 30uC, with a flow rate of 1.0 ml/min and at Evaluation of Medicinal Products (15), to obtain at least 10 eggs wavelength of 287 nm. per sampling time. When the drug administration period was completed, eggs were collected daily for a 15-day period, labeled, Determination of WDTs. Oxytetracycline and tylosin and stored at 220uC until analysis. concentrations in the incurred samples were calculated using the J. Food Prot., Vol. 77, No. 6 WITHDRAWAL TIMES OF OXYTETRACYCLINE AND TYLOSIN IN EGGS 1019

TABLE 1. Daily average concentrations of oxytetracycline and tylosin in egg yolk and white after completion of treatment in laying hens

Concn ¡ SD (mg/kg)

Oxytetracycline Tylosin

Posttreatment days Yolk White Yolk White

1 601.9 ¡ 312 NDa 134.7 ¡ 19.5 80.1 ¡ 61.1 2 406.2 ¡ 283 109.9 ¡ 13.5 114.4 ¡ 43.1 57.5 ¡ 46.1 3 386.1 ¡ 183.8 95.2 ¡ 63.8 109.7 ¡ 3ND 5 226.3 ¡ 175.5 39.9 ¡ 30.9 70.5 ¡ 32.1 ND 6 131.9 ¡ 41.8 38.4 ¡ 13.3 NEb NE 7 128.2 ¡ 95.7 ND 55.2 ¡ 16.3 ND 8 78.5 ¡ 32.2 ND ND ND 9 58.1 ¡ 18.5 ND ND ND Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/6/1017/1687041/0362-028x_jfp-13-440.pdf by guest on 30 September 2021 10 53.1 ¡ 8.5 ND ND ND 11 42.2 ¡ 24.4 ND ND ND 12 37 ¡ 14.5 ND ND ND a ND, nondetected. b NE, nonestimated. equation from the regression analysis of the matrix-matched regression analyses of the yolk depletion are shown in calibration curves (r . 0.97) at different concentrations to avoid Figure 1. Considering the MRLs and the guidelines of the extrapolations. Evaluation of Medicinal Products (15), the WDT for The recommendations of the Evaluation of Medicinal oxytetracycline was determined to be 9 days. In the case Products (15) and the MRL of the European Union were of tylosin, even when the concentrations on the first day considered to determine the WDT. The WDTs were estimated posttreatment were below the MRL, the WDT was 3 days from the linear regression analysis of log-transformed tissue concentration and was determined at the time when the 95% upper when the 95% upper one-sided tolerance limit was below one-sided tolerance limit was below the MRL with 95% the MRL with 95% confidence. confidence. DISCUSSION RESULTS Following the Codex Alimentarius recommendations Prior to the WDT calculation for each antimicrobial (6), analytical methods for validation and calibration of agent, the selected analytical methods were validated in both equipment involved in the study are necessary for egg components, white and yolk, to demonstrate that the guaranteeing the quality of the laboratory results. Prior to methods performance criteria were suitable for the detection the study, the analytical methods selected to quantify of the target compounds in the selected component. For oxytetracycline and tylosin concentrations in egg yolk and oxytetracycline, from day 1 to day 5 posttreatment, white were validated following European Commission concentrations above the MRL in the egg yolk (200 mg/ Decision 2002/657/EC guidelines (16), and all validation kg) were found. From day 1 posttreatment, concentrations parameters met the criteria set in this guideline. These were below the MRL in the egg white. Results are shown in analytical methods have been previously used by other Table 1. For tylosin, from day 1 posttreatment, concentra- authors to determine drug residues in eggs (5, 10, 21). tions in egg yolk and white were below the set MRL According to the obtained results, egg yolk was (200 mg/kg). Results are shown in Table 1. considered the target tissue for the estimation of oxytetra- The matrix-matched calibration curves were used for cycline and tylosin WDTs. The selection of yolk as target drug quantification in experimental samples. The linear tissue for the studied drugs was decided because both

FIGURE 1. Determination of oxytetracycline and tylosin withdrawal times (WDT) in egg yolk after oral administration of these antimicrobial agents. (A) Oxytetracycline (r ~ 20.753); (B) tylosin (r ~ 20.806). Each point represents an individual concentration of drug measured per day. (A) Linear regression line; (B) 95% tolerance limit with 95% confidence. The arrow indicates the time in which the WDT was calculated, according to maximum residual limits (MRL). 1020 MUN˜ OZ ET AL. J. Food Prot., Vol. 77, No. 6 antimicrobial agents deplete more slowly in this egg concentrate in the egg yolk. They attribute this difference component. Previous studies also consider the egg yolk as to unspecified multifactorial causes. the main compartment for quantifying drug residues in eggs For tylosin, even though it presented the same (19, 29, 30, 32), while some other authors have used the distribution as oxytetracycline for both egg components, whole egg as target tissue (4, 8, 31). However, the use of its pharmacokinetics properties and its molecular weight both components as one matrix can be considered as a drug resulted in the lower transfer concentrations of this dilution factor. In this study, yolk and white were analyzed compound to the analyzed eggs. Other researchers have separately to evaluate differences in the concentration of the also detected low transfer ratios of tylosin to the eggs (20, selected antimicrobials in both egg compartments. 23, 32), while oxytetracycline concentrations were over the The higher concentration reached by oxytetracycline in MRL for the first 5 days posttreatment. egg yolk in relation to tylosin concentrations, can be The present study confirmed the presence of antimi- attributed to different factors. The pharmacokinetic charac- crobial residues in eggs after oral therapy with oxytetracy- teristics of both antimicrobials differ such that absorption cline and tylosin in laying hens and established the WDTs Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/6/1017/1687041/0362-028x_jfp-13-440.pdf by guest on 30 September 2021 and bioavailability of oxytetracycline is higher when given for both studied drugs in egg white and yolk. Additionally, orally. The low binding of oxytetracycline to plasmatic this is the first study in which concentrations of both drugs proteins and its moderate apparent volume of distribution, were analyzed in egg yolk and white separately, using liquid contrasts with tylosin characteristics. Other factors that chromatography coupled to a diode arrange detector (22). should be considered are the physicochemical properties of This information points out the rising need of surveillance the drugs: the molecular weight in oxytetracycline is almost programs to monitor drug residues in different edible animal half of tylosin’s molecular weight, facilitating passing products in developing economies. through the tissue membranes of the former. In addition, the liposolubility of oxytetracycline favors this drug REFERENCES reaching the peripheral tissues with high lipid content, in 1. Agwuh, K., and A. MacGowan. 2006. Pharmacokinetics and comparison with tylosin, a hydrosoluble molecule (1, 3, 23, pharmacodynamics of the tetracyclines including glycylcyclines. 26). J. Antimicrob. Chemother. 58:256–265. Another factor that determines the antimicrobial 2. Alaboudi, A., E. Basha, and I. Musallam. 2013. 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