Marker Residue Research Articles

Residue, biotransformation, risk assessment and withdrawal time of enrofloxacin in red swamp crayfish (Procambarus clarkii)

Crayfish is a very popular aquatic food in many countries, and enrofloxacin (ENR) and ciprofloxacin (CIP) was the most frequently detected in aquatic products. However, limited information is available on the residue characterization, biotransformation rate and withdrawal period (WT) of ENR and CIP in crayfish and health risk via consumption of ENR and CIP remained crayfish. Herein, a comprehensive investigation was conducted to study residue depletion, biotransformation, ingestion risk, and WT of ENR and its predominate metabolite CIP in crayfish following different routes with repeated doses. The results showed that the elimination half-life (T1/2) of target compounds in crayfish were all in order of hepatopancreas > muscle > gill, and the order of T1/2 in different crayfish tissues were intramuscular (IM) route > oral (PO) treatment > immersion (IMMR) administration. The biotransformation rates from ENR to CIP varied from 0.75% to 3.45% in crayfish tissues following different exposure routes. The high dietary risk (RQ > 1) consuming muscle and hepatopancreas of ENR and CIP remained crayfish occurred at early after different administrations. WT is the key to control the drug residue risk, and the longest WT of marker residue of ENR in crayfish was calculated to be 51 d (1275 °C-day).

Feasibility of establishing a veterinary marker to total residue in edible tissues with non-radiolabeled study using high-resolution mass spectrometry

Traditionally, in vivo metabolism and total residue studies in veterinary drug research were conducted using radiolabeled drug where information on metabolite profiles and marker residue to total residue ratio is obtained. The Veterinary International Conference on Harmonisation (VICH) guideline GL46 indicates that the metabolism and residue kinetics in food-producing animals may be documented by an alternative approach, one other than the traditional radiolabeled study. High-resolution mass spectrometry (HRMS) has been widely used in human pharmaceutical R&D from metabolite profiling and identification in early drug discovery to first-in-human (FIH) studies in development. Recent advances in data mining tools have greatly improved the metabolite profiling capability with HRMS. It is now routine to study metabolism using non-radiolabeled samples without missing any major metabolites. In the current paper, we explored the feasibility of conducting non-radiolabeled marker residue studies to obtain metabolism information using HRMS. Metabolite profiles of gamithromycin in edible tissues of sheep treated with 6 mg/kg body weight subcutaneous injections were obtained with HRMS. The semi-quantitative relationship between the level of gamithromycin and the total treatment-related residues was established by determining the percentages of extracted ion chromatograms for metabolites and parent compound residues in each tissue. Major components (gamithromycin and its metabolite, declad) were measured quantitatively using a validated liquid chromatography/tandem mass spectrometry (LC-MS/MS) method. Metabolite profiles in excreta were also obtained and the major components measured quantitatively with a LC-MS/MS method to ensure no major metabolite was missing. Combining previous knowledge of marker residue studies in cattle and swine, as well as an in vitro comparative metabolism study with metabolite data across various species, gamithromycin was designated as the marker residue in sheep edible tissues. The marker to total residue ratios were established using a combination of the semi-quantitative HRMS results and quantitative results with the major components: the marker residue and declad. The pros and cons of the HRMS method as well as the appropriate use of the method for marker residue studies are discussed.

Verification of Bound Aminoguanidine as the Marker Residue for the Banned Antibiotic, Nitrovin, in Pig Tissues.

Nitrovin (NTV) belongs to a class of antibiotics called nitrofurans, which are classified as nonallowed pharmacologically active substances that do not have a maximum residue limit listed in EU legislation. The objectives of this study were to confirm aminoguanidine (AGN) as a suitable marker residue to monitor NTV abuse and to investigate its persistence in porcine tissues. In this work, pigs were fed with NTV-medicated feed (50 mg/kg), and tissues (kidney, muscle, and liver) and plasma were collected on different withdrawal days. All samples were analyzed for bound AGN, total AGN, and the parent drug NTV itself. The highest concentrations of AGN residues were found in the liver, while the lowest were in muscle. Parent NTV was only detected in the kidney at low levels on day 0 of withdrawal. The findings are in support of using AGN as the marker residue for monitoring the illegal use of NTV in animal-derived products.

MRM3-based UHPLC-MS/MS method for quantitation of total florfenicol residue content in milk and withdrawal study profile of milk from treated cows

Florfenicol is a broad spectrum antibacterial, licensed globally for treatment of animal and aquaculture diseases. In the EU, Canada and US it is not permitted for use in animals producing milk or eggs. There are no published methods for analysis of total florfenicol content in milk/milk products as these lack a hydrolysis step, failing to meet the marker residue definition. A method for determining total florfenicol content in milk that meets this definition is reported for the first time. Use of a UHPLC-MS/MS multiple reaction monitoring-cubed method improved the selective detection and quantitation of lower levels of florfenicol amine in milk compared to MRM only. Single laboratory validation data and withdrawal profile in bovine milk are presented. A withdrawal period of over 50 days is indicated in case of off-label use. Requirement for hydrolysis is demonstrated.

Efficacy study of non-lanthanide small luminescent molecules as gunshot residue indicators

The efficiency of using small, non-lanthanide and readily available molecules was tested as luminescent markers for gunshot residue (GSR) analysis. Three luminophores, namely pyrene (Py), fluorescein (Fl) and a Pt-CNN complex (Pt-C) were used in the present study by using their 5 wt% additives to gunpowder filled in a cartridge followed by firing with a 9 mm pistol. The easy visualization of GSR location, collection of GSR samples followed by their identification through various characterization techniques and the possibility of strategically using these markers as a cost-effective alternative compared to any lanthanide material were evaluated through this work. The comparison of physical mixture of marker and gunpowder and surface soaked gunpowder with the same marker (Py) was also evaluated. Spectroscopic (Optical, Luminescence and Raman), Microscopic, Spectrometric (Mass) and thermal analysis (Differential Scanning Calorimetry, DSC) of the marker, gunpowder and GSR residues implies that both the dyes and gunpowder retains their individual properties and those are merely a physical mixture. Overall, the present study clearly demonstrates that these small organic/metal complex based luminophores are cost effective luminescent marker as compared to lanthanide materials for GSR detection / collection and can be strategically use to track the illegal / unauthorized use of gunpowder.

Depletion study and estimation of the withdrawal period for albendazole in tambaqui (Colossoma macropomum) parasitised by acanthocephalan (Neoechinorhynchus buttnerae) treated with albendazole-containing feed

ABSTRACT This study provides the first data related to albendazole (ABZ) and its main metabolites [albendazole sulphoxide (ABZSO), albendazole sulphone (ABZSO2), and albendazole-2-amino sulphone (ABZ-2-NH2-SO2)] residue depletion in tambaqui (Colossoma macropomum) parasitised by acanthocephalan (Neoechinorhynchus buttnerae). The ABZ withdrawal period was also calculated. The fish received a daily dose of 10 mg ABZ kg−1 body weight (b.w.) via medicated feed for 34 days. Samples of target tissue (muscle plus skin in natural proportions) were collected 24, 48, 72, 120, 168, 240, and 336 h after the end of ABZ administration. The quantitation of ABZ residues and its metabolites in the target tissue was performed using a validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analytical method. After treatment, ABZ in the target tissue was rapidly metabolised over time, and ABZSO was the most persistent metabolite and was shown to be at the highest levels in the target tissue. Considering the maximum residue limit (MRL) established by Codex Alimentarius in the muscle (100 μg kg−1, species not specified), a withdrawal period of 4 days (112 °C-day) was estimated for the total residue (sum of ABZ and its metabolite residues). Considering data reported in the literature and data obtained in this study, it is suggested that the total residue be considered as marker residue to be adopted for fish in the legislative framework.

Design, Synthesis, and Characterization of Tracers and Development of a Fluorescence Polarization Immunoassay for Rapid Screening of 4,4'-Dinitrocarbanilide in Chicken Muscle.

The compound, 4,4′-dinitrocarbanilide (DNC), is the marker residue of concern in edible tissues of broilers fed with diets containing anticoccidial nicarbazin (NIC). In this study, 25 fluorescein-labeled DNC derivatives (tracers) are synthesized and characterized to develop a rapid fluorescence polarization immunoassay (FPIA) for the detection of DNC in chickens using DNC monoclonal antibodies (mAbs). The effect of the tracer structure on the sensitivity of the FPIA is investigated. Our results show that after optimization, the half maximal inhibitory concentrations (IC50) and limit of detection (LOD) of the FPIA in the buffer are 28.3 and 5.7 ng mL−1, respectively. No significant cross-reactivity (CR < 0.89%) with 15 DNC analogues is observed. The developed FPIA is validated for DNC detection in spiked chicken homogenates, and recoveries ranged from 74.2 to 85.8%, with coefficients of variation <8.6%. Moreover, the total time needed for the detection procedure of the FPIA, including sample pretreatment, is <40 min, which has not been achieved in any other immunoassays for DNC from literature. Our results demonstrate that the FPIA developed here is a simple, sensitive, specific, and reproducible screening method for DNC residues in chickens.

Marker residue types at the structural regions of transmembrane alpha-helical and beta-barrel interfaces.

Membrane proteins play a variety of biological functions to the survival of organisms and functionalities of these proteins are often due to their homo- or hetero-complexation. Encoded by ~30% of the genome in most organisms, they represent the target of over half of nowadays drugs. Spanning the entirety of the cell membrane, transmembrane proteins are the most common type of membrane proteins and can be classified by secondary structures: alpha-helical and beta-barrel structures. Protein-protein interaction (PPI) have been widely studied for globular proteins and many computational tools are available for predicting PPI sites and construct models of complexes. Here, the structural regions of a non-redundant set of 232 alpha-helical and 37 beta-barrel transmembrane complexes and their interfaces are analyzed. Using the residue composition, frequency and propensity, this study brings the light on the marker residue types located at the structural regions of alpha-helical and beta-barrel transmembrane homomeric protein complexes and of their interfaces. This study also shows the necessity to relate the frequency to the composition into a ratio for immediately figuring out residue types presenting high frequencies at the interface and/or at one of its structural regions despite being a minor contributor compared to other residue types to that location's residue composition.

Tissue Depletion of Olaquindox and Its Six Metabolites in Pigs and Broilers: Identification of a Suitable Marker Residue.

The depletion profiles of olaquindox and its six major metabolites, including O1 (N1-deoxyolaquindox), O2 (deoxyolaquindox), O3 (2-carboxamide-3-methylquinoxaline-N4-oxide), O4 (2-carboxymethylaminocarbonyl-3-methylquinoxaline-N4-oxide), O5 (2-carboxymethylaminocarbonyl-3-methylquinoxaline), and O6 [3-methyl-quinoxaline-2-carboxylic acid (MQCA)] were studied with a sensitive and accurate HPLC-UV method in pigs and broilers after oral administration of olaquindox at the rate of 50 mg kg−1 feed for 14 consecutive days. Five medicated pigs and six medicated broilers and one control animal for each time point were anesthetized and killed at different time points (6 h and 1, 3, 7, and 14 days for pigs and 6 h and 1, 3, 5, and 7 days for broilers) after ingestion of the medicated feed ceased and samples of muscle, liver, kidney, and fat were collected. The samples were assayed using a liquid chromatographic method. Mean concentrations of O2 (deoxyolaquindox) metabolite residues in all tissues of pigs were higher than other metabolite residues at each time point. MQCA was detected at lower concentrations and eliminated more rapidly than deoxyolaquindox (calculated t1/2 1.78–2.28 days vs. t1/2 2.04–2.46 days). The elimination half-lives of deoxyolaquindox residue in broilers' liver and kidney tissues (t1/2 >4 days) were much longer than those in pigs. Thus, the use of olaquindox in poultry is clearly inappropriate, as significant drug residues will occur without a withdrawal time. The results that deoxyolaquindox occurs at higher concentrations in kidney tissue and is more persistent than other residues in edible tissues of pigs which indicate that deoxyolaquindox is the most relevant marker residue and should be monitored in the routine surveillance of olaquindox-related residues in foods of animal origin.

Oral and topical extra-label administration of fipronil to laying hens: Assessment of the egg residue patterns.

This experimental work reproduces the fipronil extra-label administration performed by producers in laying hens. The scientific goal was to characterize the residual concentrations in eggs from treated hens and suggest the withdrawal periods that should be respected to avoid risk for consumers. Thirty-four laying hens were allocated into two groups: Group A was treated with fipronil in feed, two single doses of 1mgkg-1 day-1 ; Group B was administered a single dose of 1mgkg-1 by the topical route. Fipronil egg residues were quantified by HPLC-MS/MS. Fipronil and its sulphone metabolite (fipronil-SO2 ) were measured in egg after both treatments. The highest egg residual profile was always for fipronil-SO2 . Mean maximum egg concentrations (Cmax ) of 228.5±79.8ng/g (fipronil) and 1,849±867ng/g (fipronil-SO2 ) were found after fipronil administration in feed. The lowest residual levels were quantified after the topical treatment with Cmax of 27.1±4.9 and 163±26ng/g for fipronil and fipronil-SO2 . Mean fipronil marker residues and established MRLs allowed calculating the withdrawal periods, the shortest being 74days after topical administration. Such a long withdrawal period is difficult to meet in egg production systems. Thus, the extra-label use of fipronil in laying hens should not be recommended under any circumstances.

Determination of 8α-hydroxymutilin as a Marker Residue for Tiamulin in Swine Tissue by Liquid Chromatography-Tandem Mass Spectrometry

Tiamulin is a semi-synthetic derivative of the natural antibiotic pleuromutilin and is widely used as a veterinary drug for swine. Herein, we report the development of a sensitive and reliable method for determining 8α-hydroxymutilin as a marker residue for tiamulin in swine tissue using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method consists of sample extraction with acetone, defatting by acetonitrile/hexane partitioning, hydrolysis of the tiamulin metabolites to 8α-hydroxymutilin under alkaline conditions, liquid-liquid extraction with ethyl acetate, cleanup using a primary secondary amine cartridge, and LC-MS/MS analysis. The developed method was validated for 8α-hydroxymutilin in swine muscle, fat, and liver at two levels, namely 0.01 mg/kg and the maximum residue limits established in Japan (i.e., 0.1 mg/kg for swine muscle and fat, and 0.6 mg/kg for liver). The trueness ranged from 82 to 89%, and the relative standard deviations ranged from 1 to 3%. No chromatographic interference was observed near the retention time of 8α-hydroxymutilin, and matrix effects were negligible for all matrices, suggesting that the cleanup protocol was effective. The calibration curve was linear in the 0.005–0.5 μg/mL range, with a coefficient of determination greater than 0.997. The developed method enabled accurate quantification using solvent-based calibration without compensating for matrix effects and losses during sample preparation. The limit of detection of the method was 0.0005 mg/kg for each matrix. The developed method is suitable for regulatory-purpose analysis of 8α-hydroxymutilin as a marker residue for tiamulin as defined by the European Union and several other countries.

Evaluation of ELISA kits for the screening of four nitrofuran metabolites in aquaculture products according to the European guideline for the validation of screening methods

ABSTRACT The administration of nitrofurans to livestock to treat or prevent animal diseases has been banned in the EU for the production of food of animal origin. The corresponding marker residues are tissue-related metabolites AMOZ, AHD, SEM, and AOZ. The MRPL (minimum required performance limit)/RPA (Reference point for action) was set at 1 µg kg−1 in the EU. Thus, all the laboratories involved in the control of nitrofuran metabolites must detect at least at this analytical limit of performance. The objectives of the work reported here were to evaluate the performance of ELISA kits from two different manufacturers (R-Biopharm, Germany; Europroxima, the Netherlands) for the individual screening of the four nitrofuran metabolites (AOZ, AMOZ; AHD; and SEM) in aquaculture products (fish, shrimps), and then to validate the kits according to the European Decision EC/2002/657 and to the European guideline for the validation of screening methods. The false positive rates were below 9 % for the kits from both manufacturers. The detection capabilities CCβ determined were all below the current RPA (1 µg/kg). However, regarding the updated RPA at 0.5 µg/kg that shall apply in 2022, the AMOZ and SEM kits from R-Biopharm and the SEM kit from Europroxima will not be able to reach it.

Review of the existing maximum residue levels for Streptomyces lydicus strain WYEC 108 according to Article 12 of Regulation (EC) No 396/2005.

According to Article 12 of Regulation (EC) No 396/2005, EFSA has reviewed the maximum residue levels (MRLs) currently established at European level for the pesticide active substance Streptomyces lydicus strain WYEC 108. Considering the information provided by Member States, neither EU uses nor import tolerances are currently authorised for Streptomyces lydicus strain WYEC 108 within the EU. Furthermore, no MRLs are established by the Codex Alimentarius Commission (codex maximum residue limits) for this active substance. Therefore, residues of Streptomyces lydicus strain WYEC 108 are not expected to occur in any plant or animal commodity and therefore a consumer risk assessment is not required. Nevertheless, the available information allowed EFSA to propose a marker residue definition noting that methods for identification and quantification of Streptomyces lydicus strain WYEC 108 are available; however, a method for enforcement fully validated at a specific limit of quantification (LOQ) was not provided. An inclusion of Streptomyces lydicus strain WYEC 108 in Annex IV of (EC) No 396/2005 is not recommended.

Sex-related differences in disposition of sulfamethoxazole, N-acetyl-sulfamethoxazole and trimethoprim in yellow catfish (Pelteobagrus fulvidraco) following a single oral administration

The aim of this present study was to explore the sex-related differences in disposition regularities of sulfamethoxazole (SMZ), N-acetyl-sulfamethoxazole (N-ac-SMZ) and trimethoprim (TMP) in male and female yellow catfish. Concentrations of target compounds were determined using a reliable HPLC-MS/MS approach, which was validated according to the guidelines defined by FDA. The results showed that there are significant sex-associated differences in SMZ concentrations of muscle with skin and liver, in N-ac-SMZ levels of muscle with skin and kidney, and in TMP concentrations of muscle with skin, liver and kidney. Whereas, no significant sex differences were also observed in SMZ, N-ac-SMZ and TMP levels of plasma, in SMZ concentrations of kidney and in N-ac-SMZ concentrations of liver. Longer residue time of N-ac-SMZ than SMZ was observed in male and female yellow catfish. Withdrawal time (WT) of SMZ (N-ac-SMZ as the marker residue) calculated by the WT 1.4 software, was 11 days (male) and 9 days (female), respectively. By contrast, WT of SMZ (SMZ as the marker residue) was 9 days (male) and 7 days (female), respectively. These results could serve as reference data for establishing disparate use regimen and drug withdrawal time of SMZ and TMP in male and female yellow catfish.

Review of the existing maximum residue levels for propineb according to Article 12 of Regulation (EC) No 396/2005.

According to Article 12 of Regulation (EC) No 396/2005, EFSA has reviewed the maximum residue levels (MRLs) currently established at European level for the pesticide active substance propineb. Although this active substance is no longer authorised within the EU, due to insufficient information in order to conclude on the consumer risk assessment for the active substance, the toxicity of the metabolite propane‐1,2‐diamine (PDA) the impact on non‐target organisms and the risk to honeybees, MRLs based on the use of propineb were established by the Codex Alimentarius Commission (codex maximum residue limits; CXLs) and are still in place. Additionally, propineb has potential endocrine‐disrupting properties related to the hazards of its major metabolite 4‐methylimidazolidine‐2‐thione (PTU). Lacking a full toxicological characterisation for the compound PDA, exposure data for metabolites propineb‐DIDT in plants and PDA in processed commodities and considering that propineb‐MRLs correlated to CXLs were based on EU uses that were withdrawn following the non‐renewal and are no longer in place, it was not possible for EFSA to perform an assessment of these MRLs and their incorporation in European legislation cannot be recommended. Nevertheless, available data allowed EFSA to propose a marker residue and a limit of quantification (LOQ) for enforcement against potential illegal uses.

Determination of 5-nitro-2-furaldehyde as marker residue for nitrofurazone treatment in farmed shrimps and with addressing the use of a novel internal standard

We developed a significantly improved ultra-high performance liquid chromatography-tandem mass spectrometry method for determination of 5-nitro-2-furaldehyde (NF) as a surrogate using a novel internal standard for the detection of nitrofurazone. We used 2,4-dinitrophenylhydrazine derivatization and furfural as the internal standard. Derivatization was easily performed in HCl using ultrasonic manipulation for 5 min followed by liquid extraction using ethyl acetate. The samples were concentrated and purified using reverse phase and alumina cartridges in tandem. The derivatives were separated using a linear gradient elution on a C18 column with methanol and water as the mobile phase in negative ionization mode and multiple reaction monitoring. Under the optimized conditions, the calibration curves were linear from 0.2 to 20 μg/L with correlation coefficients >0.999. Mean recoveries were 80.8 to 104.4% with the intra- and inter-day relative standard deviations <15% at spiking levels of 0.1 to 10 μg/kg. The limits of detection and quantification were 0.05 and 0.1 μg/kg, respectively. This method is a robust tool for the identification and quantitative determination of NF in shrimp samples.

Simultaneously Quantification of Ethanamizuril Marker Residue in Edible Tissues of Chicken by Ultra-high Performance Liquid Chromatography–Mass Spectrometry

Ethanamizuril (EZL) is a novel anti-coccidiosis triazine compound synthesized in our laboratory. The marker residue of EZL in chicken tissues was EZL and its metabolite deacetylethanamizuril (deacetylEZL). In order to quantitatively analyze the EZL and deacetylEZL in chicken tissues, a rapid, simple, and specific ultra-high-performance liquid chromatography–mass spectrometry (UPLC-MS/MS) method was developed. Validation was performed according to the European Commission Decision 2002/657/EC guidelines. The detection limit was 5 μg kg−1, 10 μg kg−1, and 5 μg kg−1 in muscle, liver, and skin+fat for EZL. And the detection limit of deacetylEZL was 2 μg kg−1, 10 μg kg−1, 2 μg kg−1, and 10 μg kg−1 in muscle, liver, skin+fat, and kidney, respectively. The mean recoveries from fortified samples ranged from 80.15 to 99.50%, with inter assay coefficients of variation ranging between 5.03 and 15.84%. Based on this method, the depletion profile of EZL was studied in 60 healthy chickens after administration of 10 mg L−1 via drinking water for 3 days. Six medicated and one control chicken were sacrificed at 4, 8, 12, 24, 48, 60, 72, 96, and 120 h after the cessation of treatment. The highest residue concentrations of marker residue were attained at the first day, and those of all samples were below the MRL at 68.74 h post-treatment. The concentration of marker residue in kidney tissue was significantly higher than those in liver, skin+fat, and muscle tissues. The statistically estimated withdrawal period of EZL with oral administration was 4 days.

Depletion study and withdrawal period calculation of florfenicol in the crucian carp (Carassius auratus) following multiple intramuscular injections.

The previously adopted marker residue for florfenicol (FF) in China was only florfenicol amine (FFA); however, the marker residue has been changed to FF plus FFA since the end of 2017. The previous official withdrawal period determined based on the only concentration of FFA may no longer be suitable. Therefore, the present study aimed to determine the depletion profiles of FF and FFA and further calculate the withdrawal period in the crucian carp (Carassius auratus) based on the new marker residues. Florfenicol was intramuscularly administered at 10mg/kg bodyweight daily for five consecutive days to crucian carps reared in freshwater at 10°C. After the last dose, plasma and tissue samples were randomly collected from 10 fish at different time points. The FF and FFA concentrations were simultaneously determined by high-performance liquid chromatography (HPLC) with a fluorescence detector and further subjected to noncompartmental analysis. The elimination half-life (h) of FF in different tissues decreased as follows: liver (39.1)>kidney (36.3)>skin plus muscle (34.6)>plasma (31.7), whereas that of FFA decreased as follows: kidney (41.4)>skin plus muscle (39.4)>liver (39.3)>plasma (35.7). Considering a maximum residue limit of 1μg/g for the total concentration of FF and FFA in the skin plus muscle, a withdrawal period of 6days was calculated based on the upper limit of the one-sided 95% confidence interval.

Tissue residue depletion and estimation of extralabel meat withdrawal intervals for tulathromycin in calves after pneumatic dart administration.

The objectives of this study were to evaluate the injection site pathology and determine tissue residue depletion of tulathromycin in calves following pneumatic dart administration and to calculate the associated extralabel withdrawal interval (WDI). Castrated male Holstein calves were injected with ~2.6 mg/kg tulathromycin via pneumatic dart administration. At 1 (n = 2), 6, 12, 18, and 24 d after drug injection (n = 3/time point), calves were euthanized, and muscle, liver, kidney, fat, and injection site samples were harvested and analyzed for tulathromycin concentrations using a LC-MS/MS method. Gross pathology and histopathology evaluations on the injection site samples were also performed. Pneumatic dart administration of tulathromycin caused severe localized lesions of hemorrhage and edema on days 1 and 6, as well as severe pathological reactions in the subcutaneous muscle on days 1, 6, and 12. Slight to moderate reactions were still observed in the majority of the skin or subcutaneous/muscle samples on day 24. Measured tulathromycin concentrations were converted to calculate the concentrations of the marker residue CP-60,300 by dividing a conversion factor of 1.4. The data were used to calculate extralabel WDIs based on the guidelines from U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). The results showed that tulathromycin concentrations were the highest in the liver (4,877.84 ± 65.33 µg/kg), kidney (5,819.52 ± 1,087.00 µg/kg), muscle (1,717.04 ± 140.35 µg/kg), injection site (51,884.05 ± 7,529.34 µg/kg), and fat (161.69 ± 36.48 µg/kg) at 6, 1, 1, 1, and 1 d, respectively, after treatment. Tulathromycin concentrations remained above the limit of quantification of 5 µg/kg in all tissues at 24 d. The calculated WDIs based on kidney data were 26 d using EMA method, 36 d using FDA method based on CP-60,300 data, and 45 d using FDA method based on tulathromycin data. These results suggest that pneumatic dart administration of tulathromycin causes injection site reactions in calves and an extended WDI is needed. One limitation of this study was the small sample size of 3 that did not meet FDA guideline requirement. Therefore, the calculated WDIs should be considered as preliminary and additional studies that use a larger number of animals and directly measure the concentrations of the marker residue CP-60,300 are needed to make a more conclusive recommendation on the extralabel WDI.

Discovery of the Marker Residue of Olaquindox in Pigs, Broilers, and Carp.

The excretion, metabolism, distribution, and residue depletion of olaquindox (OLA), an antibacterial and growth-promoting agent used in food-producing animals for decades without a clear understanding of metabolic fate, was completely studied in pigs, broilers, carp, and rats using a radio-tracing approach combined with liquid chromatography-ion trap/time-of-flight mass spectroscopy to define the scientific marker residue (MR). After a single gavage of [3H]OLA, over 92% of the dose was excreted via urine. OLA was transformed into eight metabolites (O1-O8) in pigs and broilers, four metabolites (O1, O2, O4, and O7) in carp, and nine metabolites (O1-O9) in rats. O2 was the major residue in edible tissues of four species and persisted for the longest time in the kidneys with the longest half-life of 3.52-4.6 d. Bisdesoxyolaquindox (O2) is designated to be the MR, and the kidneys are considered to be the target tissue for OLA in food producing animals. Monitoring for this metabolite would improve the food safety evaluation and residue control of this drug.