Oral Fluid as a Biological Material for Antemortem Detection of

Food Chem. , 2017, 65 (2), pp 494–500. DOI: 10.1021/acs.jafc.6b05205. Publication Date (Web): January 2, 2017. Copyright © 2017 American Chemical ...
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Oral Fluid as a Biological Material for Antemortem Detection of Oxytetracycline in Pigs by Liquid Chromatography−Tandem Mass Spectrometry Anna Gajda,*,† Artur Jablonski,‡ Tomasz Bladek,† and Andrzej Posyniak† †

Department of Pharmacology and Toxicology, National Veterinary Research Institute, Partyzantów 57, 24-100 Pulawy, Poland Department of Swine Diseases, National Veterinary Research Institute, Partyzantów 57, 24-100 Pulawy, Poland

‡

ABSTRACT: The presence of antibiotic residues in pig tissues requires a search for new methods for their antemortem detection. To find an alternative for postmortem pig carcass analysis, an oral fluid was tested. To prove the suitability of oral fluid for the detection of antibiotics administered by injection, oxytetracycline was chosen. Research was conducted on two groups of animals: group 1, 100% treated; and group 2, 50% treated and 50% untreated. Oxytetracycline was assayed by a high-performance liquid chromatography−tandem mass spectrometry method. The antibiotic was detectable 2 h post administration in group 1 and group 2 at the concentrations of 10653 ± 1421 μg/kg and 7457 ± 1145 μg/kg, respectively. At withdrawal period (21st day), oxytetracycline concentrations in oral fluid (30.8 ± 9.4 μg/kg in group 1 and 11.6 ± 5.6 μg/kg in group 2) were similar to those determined in muscle (34.5 ± 8.2 μg/kg). The concentrations of oxytetracycline in liver and kidney were 76.8 ± 22 μg/kg and 204 ± 49 μg/kg, respectively. The results of this study indicate that oral fluid analysis can be used for antemortem oxytetracycline detection in pigs, even if the half of animals in one pen are treated. KEYWORDS: pigs, oral fluid, oxytetracycline, detection, LC−MS/MS

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INTRODUCTION Intensive and massive pig production often contributes to excessive administration of antibacterials in veterinary medicine. The misuse and failures to follow the label directions of antibiotics, as well as withdrawal inadequacy, can lead to residues occurrence in products of animal origin. The residue of drugs may result in many biological adverse effects and allergic reactions in consumers.1,2 Moreover, low level doses of antibiotic consumed for a long period can cause the spread of drug-resistant bacteria and bacterial resistance acquisition in humans.3 For consumer health protection, the European Union has established the maximum residue limits (MRLs) in pig muscle, liver, and kidney for most of the drugs used in veterinary medicine. Different antibiotics have been employed in pig housing, such as β-lactams, macrolides, sulfonamides fluoroquinolones, and tetracyclines. Among them, tetracyclines have been detected worldwide both in tissues samples,4−6 as well as in manure samples and wastewater effluents from animal husbandry.7−9 To avoid an inappropriate usage of veterinary medicinal products in animals, as well as to eliminate veterinary drugs abuse, a special control in each European country has been established. Under this monitoring program, numerous compounds (including tetracyclines) are analyzed. Oxytetracycline, 1 (Figure 1), a member of tetracycline group, is one of the most commonly used antibiotics in the treatment of many bacterial diseases in swine. However, there are many reported and confirmed incidents of oxytetracycline residues presence in pig muscle, liver, and kidney, resulting from overdosing this commonly administered compound. The MRL level for oxytetracycline in pigs has been established as 100 μg/kg in muscle, 300 μg/kg in liver, and 600 μg/kg in kidney. The incidence of oxytetracycline residues are included © XXXX American Chemical Society

in European Union reports for the monitoring of veterinary medicinal product residues from European countries in 2012− 2014.4−6 Additionally, the RASFF food and feed safety alerts results confirm the many incidents of oxytetracycline residues > MRL values. In the years 2014−2015, 26 violations of oxytetracycline concentrations in food products were reported. Consolidation of pig production requires ensuring adequate conditions of animals maintenance with high health conditions, consistent with the guidelines for animal welfare. At the same time, antibiotics control in animals, especially in the case of illegal administration, is an important element providing the high quality of pig breeding as well as consumer protection. The postmortem detection and the presence of antibiotic residues in pork tissues above MRL values very often lead to recall and destruction of significant quantities of meat, which may contribute to economic losses. Therefore, there is a strong need to find and implement new methods for antemortem detection of antibiotics in animals, while minimizing the interference in animal welfare. Generally, the primary material for the control of antibiotics presence in pigs are tissue samples, collected from animals at the slaughterhouse. In residues monitoring programs, muscle, kidney, and liver are the target tissues. However, postmortem analysis does not provide the opportunity of monitoring the usage and administration of antibiotics on the farm during the animal breeding. In antemortem drugs analysis, the blood can be used as a diagnostic biological matrix; however, the main Received: Revised: Accepted: Published: A

November 19, 2016 December 28, 2016 January 2, 2017 January 2, 2017 DOI: 10.1021/acs.jafc.6b05205 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. Chemical structure of 4-epi-oxytetracycline 1, oxytetracycline 2, and demeclocycline 3.

noninvasive antibiotic detection in pig populations. A single injection of oxytetracycline at labeled dose was used in the experiment. The ability of oxytetracycline detection was tested on a group with half of the animals treated in one pen. Additionally, to demonstrate and confirm the utility of oral fluid as a biological material for oxytetracycline control, the concentrations in edible tissues at the withdrawal period of administered medicinal product (21st day) were investigated and the relation between oxytetracycline content in oral fluid and tissues was determined. The levels of oxytetracycline and 4epi-oxytetracycline in oral fluid bulk samples and the persistence of antibiotic in pig fluid were evaluated. To determine the oxytetracycline and 4-epi-oxytetracycline concentration, a suitable, sensitive and labor-effective LC−MS/MS method was developed.

problem with this method of control is to obtain blood samples from animals. Blood sampling is connected with certain disadvantages for the animals and the staff responsible for material collection. Plasma is collected from each animal individually and animals are exposed to the high stress associated with such sampling. It can lead to a significant health status decrease of animals and further daily weight gain reduction can occur, which are reflected in economic losses. For pigs, which are particularly sensitive species for stressful situations, blood sampling is especially dangerous.10 Therefore, the safety of animals, stress minimization, as well as time saving and facility in material collection are the most important factors limiting the different sampling techniques. In veterinary applications, urine and milk are also used in drugs analysis, but milk samples are limited to cows only and urine is very difficult to sample from living pigs. In monitoring control programs of veterinary drug residues, urine is used for the determination of forbidden compounds only, belonging to group A.11 In the antemortem control of drugs, drinking water and feed can be also analyzed. However, this way of checking can be helpful in finding the origin of contamination, not in evaluation of their presence in animals. The literature describes the detection of antibodies and pathogens in oral fluid (mixed saliva) of domesticated animals.12,13 Literature data also report a high probability of drug transmission to oral fluid after their application.14−16 However, the information about veterinary drugs analysis in oral fluid of animals are very poor. A suitable analytical method is an essential tool for determination of drugs in analyzed matrix. No liquid chromatography−tandem mass spectrometry (LC−MS/MS) method for oxytetracycline determination in oral fluid after intramuscular administration has been reported. According to Commission Regulation (EU) No. 37/2010, MRL values for oxytetracycline in animal tissues has been set as the sum of parent compound with its metabolite, so the quantitative determination of oxytetracycline requires inclusion of 4-epi-oxytetracycline, 2 (Figure 1).17 The major challenge in the analysis of oxytetracycline residues is the separate of oxytetracycline and 4-epi-oxytetracycline. These two components have the same molecular mass and elemental composition, with the only difference being the orientation of the −N(CH3)2 function at the 4-position. The difference in retention time, combined with the difference in ion ratio between two product ions in the MS/MS spectra of oxytetracycline and 4-epi-oxytetracycline, enabled their subsequent identification and quantitative analysis on the MS detector. To prevent positive oxytetracycline residues in pig, an alternative for postmortem antibiotics control in tissues was investigated. The aim of this study was to prove and demonstrate that oral fluid collected from pigs after intramuscular (i.m.) drug administration is a suitable matrix for

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MATERIALS AND METHODS

Animals and Dosing. In total, 78 healthy, 14 week old Danbred Race pigs were used in the experiment. The study was approved by the local regulatory authorities and the Ethic Animal Experiments Inspectorate (Lublin, Poland). Pigs were housed at a farrow-to-finish farm. Animals were kept at 20−25 °C. Drinking water and antibioticsfree feed were available ad libitum throughout the study. All pigs used in this study were tested negative for oxytetracycline presence in oral fluid, prior to the study. The study used three pens arranged in a 5 m × 5 m format with shared flooring. Animals were divided into two experimental groups (2 pens), each group kept in separate pen (26 pigs per group). Group 1, 100% of the pigs in one pen (26 animals) were administered one intramuscular injection of oxytetracycline (Tetravet L.A. Injectable Solution, Ceva Sante Animale, Libourne, France) at a dose of 20 mg/kg body weight into the right side of the neck. Group 2, 50% of the pigs in one pen (13 animals) were administered one intramuscular injection of oxytetracycline (Tetravet L.A. Injectable Solution, Ceva Sante Animale, Libourne, France) at a dose of 20 mg/kg body weight into the right side of the neck and 13 animals in that group were no treated with oxytetracycline. For the control purposes, 26 animals were used. Sample Collection. Oral fluid was collected from all three pens by hanging a cotton rope on the pen gate, away from water and feed, with a separate rope in each pen. The rope was attached to the pen barrier for 30 min, with the end located at the height of a pig’s sternum (30 cm from the ground). For this period pigs chewed and moistened the rope. After this time, the portion of the rope saturated with oral fluid was put into a plastic bag and fluid was squeezed from the rope until it accumulated in the bottom of the bag. Next, the corner of the bag was cut away and the fluid was drained into a 50 mL plastic tube and stored frozen at −20 °C until analysis. Oral fluid was taken at specified time intervals. It was collected at time zero (prior to antimicrobial treatment) and 2, 6, 12, 24, 48, 72 h, as well as 8, 14, and 21 days post oxytetracycline injection. Oral fluid sampling was carried out in duplicate (n = 2) at each time. Additionally, oral fluid samples were collected from pigs not treated with oxytetracycline (control group). At the withdrawal period (21st day), six animals treated with oxytetracycline were killed by i.v. injection of sodium pentobarbital (Morbital) (Biowet, Pulawy, Poland), followed by exsanguinations. Before euthanasia, sedation of animals was performed by a previously B

DOI: 10.1021/acs.jafc.6b05205 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry i.m. injection of azaperone. Samples of muscle, liver, and kidney were collected from each animal (n = 6) individually, in a clean plastic bag and the samples were stored at −20 °C until analysis. Analytical Method. The determination of oxytetracycline and 4epi-oxytetracycline in tissues samples was adopted from previously described LC−MS/MS method for muscle samples.18 The method was validated for the liver and kidney analysis. For the measurement of oxytetracycline and 4-epi-oxytetracycline concentrations in oral fluid of pigs, a high-performance liquid chromatography with tandem mass spectrometry (LC−MS/MS) method was developed. Analytical Reagents and Solvents. Analytical reference standard of oxytetracycline, demeclocycline 3 (Figure 1), and trichloracetic acid were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). 4epi-Oxytetracycline analytical standard was purchased from LGC Standards (Teddington, Middlesex, U.K.). Acetonitrile was from J.T. Baker (Deventer, The Netherlands). Formic acid was from Fluka (St. Louis, MO). All organic solvents were HPLC grade and all chemicals were analytical grade. Syringe 0.22 μm Hydrophilic Polyvinylidene Fluoride (PVDF) Membrane Filters were from Restek, (College, PA). Extraction and Cleanup. Before extraction, the oral fluid samples were centrifuged at 9447 × rcf. Then, 1 g of oral fluid was weighed into a polypropylene centrifuge tube, and the internal standard (demeclocycline) was added. For the oxytetracycline extraction, 1 mL of 10% trichloracetic acid was used. Next, the sample was mixed and centrifuged for 10 min at 9447 × rcf. Then, the extract was filtered through a 0.22 mm PVDF filter into an amber vial. LC−MS/MS Analysis. For the analysis of oxytetracycline and 4-epioxytetracycline, an Agilent series 1200 HPLC system (Agilent Technologies, Waldbrom, Germany) connected with an API 4000 triple quadrupole mass analyzer with a TurboIonSpray source (Applied Biosystems, Toronto, ON, Canada) was used. The MS detector was configured for electrospray ionization (ESI). The ESI was operated in the positive ion mode, and MS data acquisition was performed in the multiple reaction monitoring (MRM) mode. The precursor → product ion pairs for oxytetracycline and 4-epioxytetracycline were 461 → 426, 461 → 443 and for demeclocycline 465 → 448. The mass spectrometry optimization was conducted both with direct infusion of working standard solution from a syringe pump and with LC injection. Analyst 1.5 software controlled the LC−MS/ MS system and processed the data. Nitrogen was used as the nebulizer gas, curtain gas, and collision gas. Collision energy (CE) was optimized to maximize the relative abundance for ion transition. The following mass spectrometer parameters were used: resolution Q1 and Q3, 1; nebulizer gas, 40 psi; auxiliary gas, 50 psi; curtain gas, 20 psi; collision gas, 3 psi; ion spray voltage, 5500 V; temperature, 500 °C. The fragmentation reactions used for monitoring were selected on the basis of their significance in product ion spectra. The declustering potential (DP), cell exit potential (CXP), and entrance potential (EP) were set as 65, 15, and 10 V, respectively. Dwell time was 200 ms for the both transitions. The chromatographic column used was a 50 mm × 2.0 mm i.d., 3 μm, Luna C18, with a 2 mm × 4 mm i.d. guard column of the same material (Phenomenex). The column was held at 35 °C with a flow rate of 0.4 mL/min, and the injection volume was 20 μL. The resulting total run time was 10 min. The mobile phase consisted of solvent A, 0.1% formic acid in water (v/v); and solvent B, 0.1% formic acid in acetonitrile. The elution was performed in a gradient mode. The mobile phase starting conditions were 95% of eluent A and then decreased to 40% within 5 min. This composition was maintained up to 6.30 min and then increased to 95% of eluent A. Validation. The validation study was evaluated in terms of linearity, recovery, specificity, and precision (repeatability and withinlaboratory reproducibility). The detection limit (LOD) and the limit of quantitation (LOQ) of the method were evaluated. The LOD was calculated on the basis of the signal-to-noise ratio (S/N = 3) on the chromatograms of 20 oral fluid blank samples. The LOQ was calculated as the lowest oxytetracycline concentration on the matrixmatched calibration curve that could be quantitated with a precision level not exceeding 20% and with accuracy within 20% of nominal. Linearity was tested by preparing in duplicate matrix-matched

calibration curves on eight levels corresponding to 5, 10, 20, 50, 100, 200, 500, and 1000 μg/kg by plotting the response of the analyte/ internal standard peak area ratio versus the analyte concentration. The correlation coefficient was evaluated. The specificity of this method was evaluated by analyzing 20 different oral fluid blank samples. Repeatability and within-laboratory reproducibility were determined by the repeated analysis (n = 6) of oral fluid samples spiked with oxytetracycline and 4-epi-oxytetracycline before extraction at three concentrations, corresponding to 5, 50, and 200 μg/kg, from run-torun during 1 day for repeatability and 3 day for within-laboratory reproducibility, calculated in reference to internal standard using a matrix-matched calibration curve. Precision was evaluated by calculating the relative standard deviation (RSD) of the results obtained for each level of the target compound. The overall mean concentrations obtained in the reproducibility study were used to calculate the accuracy expressed as percent. To assess the matrix effect, 6 blank samples of oral fluid were spiked after the sample preparation step at the level of 50 μg/kg, by addition of the appropriate amount of standard solution. Simultaneously, a standard solution at the same concentration level was also prepared. Spiked extracts and standard solution were injected and analyzed by LC−MS/MS. The following equation was used for matrix effect (ME) calculation: ME (%) = B/A × 100 where B is the average analyte peak area in the extract of the blank sample spiked after the extraction, and A is the average analyte peak area in the standard solution. Signal suppression is observed when the ME value is less than 100%. The LC−MS/MS method previously described and validated for muscle was validated for the determination of oxytetracycline and 4-epi-oxytetracycline in kidney and liver samples.18

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RESULTS AND DISCUSSION The quantitative analysis of oxytetracycline and 4-epi-oxytetracycline in oral fluid was performed by the liquid chromatography−tandem mass spectrometry method. The optimal isolation was achieved with trichloracetic acid. For the cleanup, PVDF filters were found most suitable. The optimal separation of oxytetracycline and 4-epi-oxytetracycline was made using octadecyl chromatographic column with mobile phase containing acetonitrile and formic acid. For the detection of oxytetracycline and 4-epi-oxytetracycline, the best product ions were obtained choosing collision energy at the level of 30 V for the first transition 461 → 426 (detection) and the collision energy at the level of 17 V for the second transition 461 → 443 (confirmation). The developed procedure was sensitive with satisfactory precision and linearity. A good linear response was observed with correlation coefficient r2 > 0.99. The validation results obtained for presented method were repeatable and reproducible with the repeatability RSDs lower than 10% at all fortification levels (at the range of 4.2−7.0% for oxytetracycline and 4-epi-oxytetracycline) and within-laboratory reproducibility lower than 15% (at the range of 11.1−15.6 for both compounds). The accuracy was calculated in the range of 96.5−102.6% and 98.7− 100.6% for oxytetracycline and 4-epi-oxytetracycline, respectively. The LOD and LOQ were established as 2 μg/kg and 5 μg/kg, for both compounds. The matrix effect calculated for oxytetracycline in oral fluid was 88.4%. To completely eliminate this effect, for the quantitative analysis of oxytetracycline, matrix-matched calibration curves were used. The validation results obtained for kidney and liver matrixes showed good accuracy ranging from 98.2% for kidney and 97.7% for liver tissue with a good RSD, less than 10.0% of within-laboratory reproducibility. During the validation process, a good linear response for kidney and liver was observed with correlation coefficients over r2 > 0.98. The procedure was satisfactory C

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Figure 2. LC−MS/MS chromatograms of (A) blank (control) oral fluid sample, (B) oral fluid sample spiked with 4-epi-oxytetracycline and oxytetracycline at a concentration level of 100 μg/kg, and (C) oral fluid sample with oxytetracycline at the level of 68.6 ± 29 μg/kg determined at the 336 h after medication.

sensitive with LOD established at 2 μg/kg and LOQ as 5 μg/ kg, both in kidney and liver samples. Before oxytetracycline administration, at zero time, all pigs were negative for oxytetracycline and 4-epi-oxytetracycline. After experimental injection of one dose of oxytetracycline, all oral fluid samples showed positive results for oxytetracycline presence by LC−MS/MS. However, the 4-epi- metabolite, was not found. Oral fluid was absorbed on a rope during chewing by pigs. After 30 min, about 30−40 mL of oral fluid was collected from each pen at each time point. Oxytetracycline was detectable from 2 h up to 21 days after administration. The highest concentrations in oral fluid at the level of 10653 ± 1421 μg/kg in group 1 and 7456 ± 1145 μg/kg in group 2, 2 h after

medication were observed. At 6 h, oxytetracycline concentration was reduced by almost 50% in each group. The content of antibiotic decreased gradually over time to 30.8 ± 9.4 μg/kg in group 1 and 11.6 ± 5.6 μg/kg in group 2 at the 21st day of sampling. After 21 days, the oxytetracycline concentrations were still much higher than the detection limit of the developed method. Figure 2A−C show the LC−MS/MS chromatograms of blank (control) and spiked with oxytetracycline and 4-epioxytetracycline at the concentration of 100 μg/kg oral fluid samples as well as oral fluid sample collected from pigs (group 1) at 336 h after oxytetracycline injection. In group 2, where half of the pen was left untreated, the obtained concentrations were found to be lower than in group D

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Figure 3. Mean (±SD) values of oxytetracycline concentrations in oral fluid determined until 504 h after medication.

As a diagnostic antemortem fluid, oral fluid shows advantages over serum, because it can be collected noninvasively. The pigs’ natural behavior of chewing and biting the rope is used in the sampling, so from the perspective of animal welfare, oral fluid sampling offers an “animal friendly” system. During the fluid collection, no needles or snout loop to immobilize pigs are necessary, so the stress of animals is minimized. The technique requires minimal staff training and the time of oral fluid sampling is much reduced, compared to the time of blood collection. Additionally, it offers a cost-effective approach for the screening of large populations of swine. The material is collected from many of the animals at the same time and provides an opportunity to detect the presence of antibiotics in a large number of animals by taking one bulk sample, as one rope should be sufficient for 100 pigs. The analysis cost of bulk sample is greatly reduced compared to testing hundreds of individual samples, as in plasma analysis. Moreover, the implementation of a new technique of oral fluid sampling to antemortem identification of antibiotics on farms would allow for a rapid evaluation of antibiotics administration on farms with pigs intended for slaughter and subsequently for sale. The antemortem control of antibiotics at the farm level could significantly reduce the losses caused by utilization of meat in the case of detecting the presence of drugs. The route of medicine administration is an important factor in oral fluid testing. In the present study, to evaluate and prove the possibility of antibiotics detection, administration of the drug by intramuscular injection was chosen. In pigs treated by medicated feed or drinking water, the presence of drugs in saliva is rather evident, although some data report that β-lactam antibiotics are neutralized by different enzymes present in saliva.25 The pilot study conducted in our laboratory at a few pig farms, where the treatment was not confirmed, showed the presence of doxycycline in oral fluid at concentrations range of 10−50 μg/kg (unpublished data). These levels of antibiotics can indicate the illegal administration or lack of water or feed supplying systems cleaning after previous treatment. It proves the adequacy of oral fluid analysis as an important “guard tool” for rapid verification of farms, where no antimicrobial treatment is declared. Additionally, such control enables checking whether the water supply system and feed dispensers are properly cleaned after antibiotics treatment. The presented control

1. The differences between the oxytetracycline concentrations in group 1 and group 2 depended on the time point of oral fluid collection. At the beginning of oral fluid sampling, the concentrations in pens with all animals treated were 1.4−1.5 times higher than in pen, where 50% of the pigs received the oxytetracycline. Increase in concentration differences over time between two groups was observed. At 21 days, the level of oxytetracycline in group 1 was 2.8 times greater than in group 2. All oral fluid samples from the untreated pen (control group) remain negative in all experiments. Results of oral fluid samples for oxytetracycline presence, expressed as mean values (±SD), are reported in Figure 3. The concentrations of oxytetracycline and 4-epi-oxytetracycline in tissue samples collected at withdrawal period (21st day), determined as a mean values (±SD), were as follows: 34.5 ± 8.2 μg/kg in muscle, 76.8 ± 22 μg/kg in liver, and 204 ± 49 μg/kg in kidney. Human medicine has shown the utility of oral fluid as a diagnostic specimen for a long time.19 Over the past few years, an increase in the application for detection of a wide range of infectious and noninfectious diseases in veterinary medicine, like porcine respiratory and reproductive syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), swine influenza virus (SIV), Mycoplasma hyopneumoniae, Haemophilus parasuis, or Mycoplasma hyorhinis, has been observed.13,20−22 Oral fluid is a mixture of saliva and serum transudate that crosses the oral mucosa (oral mucosal transudate) and gingiva (gingival crevicular fluid) from capillaries located in the oral mucosa and the gingival tissues.23 Oral fluid can include antibodies and antigen-specific pathogens. Oral fluid harbors a wide spectrum of proteins, nucleic acid, electrolytes, and hormones that originate from multiple local and systemic sources. Therefore, this unique fluid is a good diagnostic medium both in humans and animals, which can reflect bodily health and well-being. The process of passive transudation was first demonstrated by intravenous injection of fluorescein dye into the hind leg of dogs and recording fluorescence on filter paper strips collected within and at the gingival crevice.24 In those experiments, the dye appeared at the gingival crevice within 30 s, demonstrating the connection between the circulatory system and the oral cavity. E

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were comparable with concentrations determined in pig muscle. At the 21st day, levels in oral fluid, in group 1, were 30.8 ± 9.4 μg/kg, while in muscle the determined concentrations were 34.5 ± 8.2 μg/kg. The concentrations determined in liver and kidney were higher, but it should be noted that MRL values in these tissues are much higher than in muscle. The withdrawal time and MRL values are crucial factors of antibiotic residues control in animal products. The oxytetracycline levels in oral fluid of group 1 remained comparable to MRL values in kidney (600 μg/kg) until 72 h. Regarding the liver MRL (300 μg/kg), much higher concentrations of oxytetracycline (445 ± 111 μg/kg in group 1) at 192 h were observed. The concentrations of oxytetracycline in oral fluid fell below the lowest MRL value (100 μg/kg) on day 14, indicating a long oxytetracycline persistence in oral fluid at the level of MRL established for muscle. Oral fluid appears to be a stable and noninvasive way for testing the oxytetracycline presence in pigs. This study proved that oxytetracycline is retained in oral fluid for a long time after intramuscular injection. The presence of 4-epi-oxytetracycline was not found in collected oral fluid samples. The developed LC−MS/MS method allows for rapid detection of the tested compound. Additionally, the method of oral fluid collection, presented in this study, allows for fast oral fluid sampling at pig farms. Analysis of oral fluid on farms seems to be the an effective tool for monitoring the reasonable treatment and residue avoidance. It offers a cost-effective approach for the screening of large populations of animals. The comparable oxytetracycline concentrations in oral fluid and muscle at the withdrawal period confirmed the utility of this fluid as a biological material for noninvasive oxytetracycline detection at the farm level. In the case of positive oral fluid analysis results, the slaughtering of animals is delayed, reducing the costs associated with destruction of noncompliant meat.

procedure can motivate farmers and feed industry to a more prudent use of veterinary drugs in piggeries. The analytical sensitivity and specificity required for the analysis of oral fluid as a medium for drugs determination are necessary.26 The availability of suitable methods for the quantitative determination of antibiotics at low concentrations are essential analytical tools. The developed LC−MS/MS method provided satisfactory determination of oxytetracycline and 4-epi-oxytetracycline in the oral fluid of pigs. It is also checked for the usefulness of the method for the analysis of other tetracycline antibiotics used in veterinary practice (tetracycline, 4-epi-tetracycline, chlortetracycline, 4-epi-chlortetracycline, doxycycline) with satisfactory results.28 However, because of the experiment with oxytetracycline only, this study is focused on the isolation and validation of tetracycline with its 4-epi form. The method validation results indicate that the presented method can successfully extract oxytetracycline and 4-epi-oxytetracycline from fortified oral fluid samples as well as from naturally incurred samples. Additionally, the sample preparation step in this method is very simple and laborefficient. For the effective isolation of oxytetracycline from different biological matrixes, mainly EDTA−McIlvaine buffer, oxalic acid buffer, citrate buffer have been described.2,27−31 For the effective cleanup, many SPE cartridges like Oasis HLB, C18, Strata X, have been reported.2,30−32 A matrix solid-phase dispersion with heated water for determining oxytetracycline antibiotic was also reported.33 In the present method, the extraction “dilute and shoot” with 10% trichloracetic acid and cleanup with PVDF filters only enables analysis of many samples in a short time. The use of Luna C18 analytical column with mobile phase consisting of formic acid in water and formic acid in acetonitrile provides a good separation of oxytetracycline and 4-epi-oxytetracycline. The validation protocol proves the selectivity, sensitivity, reliability, and accuracy of the presented method. The results of this study demonstrate the utility of oral fluid as a medium for oxytetracycline detection after intramuscular administration. Oxytetracycline appeared in oral fluid in a short time after administration. The high concentrations both in group 1 (10653 ± 1421 μg/kg) and in group 2 (7456 ± 1145 μg/kg), 2 h after injection obtained, indicate the rapid oxytetracycline transition to oral fluid. The long period of samples collection allowed determination of the time of oxytetracycline persistence in analyzed fluid. The presence of antibiotic throughout the withdrawal time (up to 21 day) was confirmed. Additionally, it was demonstrated, that with the number of treated animals reduced by half housed in one pen, the presence of antibiotic can be detected. Both in group 1, where all pigs were medicated and group 2 with reduced by half the number of treated animals, oxytetracycline was detected for the same period of time. However, at the beginning of samples collection, the differences of oxytetracycline between the groups were smaller than at the end of sampling, where concentrations in group 2 were halved. In this study, the samples of edible tissues (muscle, liver and kidney) at withdrawal period of the administered medicinal product containing oxytetracycline (at the 21st day) were also collected. Withdrawal time for antibiotics is defined as the interval of time required following administration for the levels of antibiotic in the whole organism to fall below the maximum permitted concentration. The strong correlation between the oxytetracycline residues in oral fluid and muscle tissues was observed. The concentrations of oxytetracycline in oral fluid

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AUTHOR INFORMATION

Corresponding Author

*Phone: +48-81-889-32-40. E-mail: [email protected]. pl. ORCID

Anna Gajda: 0000-0003-2895-4482 Funding

This work was funded by KNOW (Leading National Research Centre) Scientific Consortium “Healthy Animal-Safe Food”, decision of the Ministry of Science and Higher Education No. 05-1/KNOW2/2015. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Nisha, A. R. Antibiotic residues − a global health hazard. Vet. World 2009, 1, 375−377. (2) Oka, H.; Ito, Y.; Matsumoto, H. Chromatographic analysis of tetracycline antibiotics in foods. J. Chromatogr. A 2000, 882, 109−133. (3) Katz, S. E.; Brady, M. Antibiotic residues in food and their significance. Food Biotechnol. 2000, 14, 147−171. (4) Report from 2012 on the results from the monitoring of veterinary medicinal product residues and other substances in live animals and animal products. EFSA Supporting Publications, 2014, 11, 1−65 http://ec.europa.eu/food/food/chemicalsafety/residues/docs/ workdoc_2012_en.pdf (Accessed: June 13, 2014).

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DOI: 10.1021/acs.jafc.6b05205 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.6b05205 J. Agric. Food Chem. XXXX, XXX, XXX−XXX