Development and validation of a multi-residue analytical method for veterinarian and human pharmaceuticals in livestock urine and blood using UHPLC-QTOF.

The aim of this work is to contribute to the assessment of multi-residue analysis of veterinarian and human pharmaceuticals using UHPLC-QTOF in livestock urine and blood (cattle, chicken, sheep and pig). Firstly, an in-house database including compound name, monoisotopic mass, chemical formula, retention time, chemical structure, and three CID MS-MS spectra of the 234 selected drugs were built for qualitative detection. Secondly, the method validation result showed that all the 234 drugs exhibited good linearity with determination coefficients (R2) higher than 0.999. Then, the distribution of the drugs recoveries, intra-day RSD and inter-day RSD results for all seven matrices were tested. Finally, after a carefully cross check, 150 veterinarian and human pharmaceuticals could meet the methodological requirements (recovery, 50-120%; intra-day RSD ≤ 15%, inter-day RSD ≤ 20%) in all seven matrices. Our results suggested that although the main applications of UHPLC-QTOF are directed towards detection and identification of the compounds, this method should be also applied for quantitative purposes.

Novel Eco-friendly HPLC Methods Using Refractive Index Detector for Analysis of Three Veterinary Antibiotics in Pharmaceutical Formulations and Rat Plasma.

Antibiotic resistance increases the human mortality rate nowadays. The main purpose of the present study was to develop green reversed-phase high-performance liquid chromatography (RP-HPLC) methods with a refractive index detector for the assay of the three veterinary antibiotics (VAs), i.e., maduramicin ammonium (MA), apramycin sulfate (AS) and clarithromycin (CLA) in pharmaceutical dosage forms and spiked rat plasma. The method utilized isocratic elution using an ODP-40 C18 column, the flow rate was set at 1.0 mL/min and negative polar signals. The linearity ranges were 3.0-18.0 µg/mL for MA, 1.5-4.0 µg/mL for AS and 0.5 to 3.0 µg/mL for CLA, respectively. Liquid-liquid extraction (LLE) procedure was optimized in plasma samples. The recoveries percentages were 85.4, 81.2 and 88.8 correspondingly, in rat plasma. However, the drugs extraction by protein precipitation method yields very poor recoveries (around 50%). The new HPLC- refractive index (RI) methods are better than the previously reported HPLC-ultra violet methods in terms of greenness and simplicity of procedures. Moreover, the previously reported LC-MS methods lack the simplicity and availability of such expensive techniques in Quality control (QC) labs. The novelty of this research is the use of refractive index detector for the first time for VAs analysis.

Rapid simultaneous determination of 160 drugs in urine and blood of livestock and poultry by ultra-high-performance liquid chromatography-tandem mass spectrometry.

In order to address the specific question of food safety in livestock and poultry, it is imperative to monitor veterinary drugs at every moment in the process of livestock and poultry breeding. Thus, multi-residue analysis of a wide variety of drugs using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has become a tool of critical significance, especially for veterinary drug monitoring programs. A total of 160 compounds, belonging to 17 different families of veterinary drugs, were investigated in the urine and blood of livestock and poultry. Drug samples were extracted using a slightly acidic acetonitrile solution. The QuEChERS (quick, easy, cheap, effective, rugged, and safe) preparation method, combined with dispersive solid phase extraction (d-SPE) was compared with the approach of solid phase extraction (SPE). In the end, the QuEChERS extraction procedure was selected to reduce matrix effects and efficiently extract target veterinary drugs, and d-SPE was applied as a cleanup step. Electrospray ionization coupled with positive dynamic multiple reaction monitoring (dMRM) was utilized for the analysis of 160 different drugs in a single chromatographic run of 24 min. The efficiency of this method was evaluated using 7 matrices (pig blood, cattle blood, sheep blood, chicken blood, pig urine, cattle urine, and sheep urine). Good linearity was obtained for the analytes in a concentration range of 1-100 ng/mL, with correlation coefficients higher than 0.990. Most of the 160 drugs studied gave estimated limits of detection (LOQs) of 1 ng/mL, with some LOQs reaching as much as 5 ng/mL. The mean recoveries at four spike-in levels of 1, 5, 10, and 50 ng/mL, ranged from 60% to 120%. The intra-day precision measurements had coefficients of variation (n = 6) <15%, and the inter-day precision measurements were below 25%. Our method was applied in real samples and proved to be adequate for routine analysis. The proposed method proved to be simple, rapid and reliable for monitoring 160 drugs in the urine and blood of livestock and poultry, and can also be used for food safety monitoring.

Rapid Detection of the Antibiotic Sulfamethazine in Pig Body Fluids by Paper Spray Mass Spectrometry.

We report herein a practical method for nonlethal detection of the antibiotic sulfamethazine in pig body fluids via the combination of simple extraction and paper spray mass spectrometry (PS-MS). This method requires minimal sample preparation while still providing high sensitivities and accuracies in complex matrices including pig whole blood (LOD = 7.9 µg/L; recovery = 95.4-103.7%), pig serum (LOD = 11.5 µg/L; recovery = 103.2-106.2%), and synthetic urine (LOD = 11.2 µg/L; recovery = 99.1-103.2%). Given a known correlation between the level of sulfamethazine in body fluids and edible tissues, this method shows great promise as a practical and nonlethal solution for rapid testing of the drug, which can substantially aid managerial decision in the livestock industry.

Percentage of faecal excretion of meloxicam in the Cape vultures (Gyps corprotheres).

Asian Gyps vulture species are gradually recovering from the devastating effect of diclofenac being present in contaminated carcasses. This drug was responsible for the death of over 10 million vultures in India, Nepal and Pakistan. To prevent the extinction of vultures, meloxicam was introduced after the ban of veterinary diclofenac. Meloxicam's safety in vultures was attributed to its short elimination half-life in contrast with diclofenac. The reason for the rapid elimination of meloxicam is yet to be explained. The aim of this study was to evaluate the role of biotransformation in the elimination of meloxicam. Six Cape griffon vultures (Gyps coprotheres) were treated with 2 mg/kg meloxicam intramuscularly for faecal and plasma quantification of meloxicam concentration over time. In the plasma meloxicam was characterised by a half-life, mean residence time, clearance and volume of distribution at steady state of 0.37 ±â€¯0.10 h, 0.90 ±â€¯0.12 h, 0.02 ±â€¯0.00 l/h kg and 0.02 ±â€¯0.00 l/kg respectively (presented as geometric mean). Over the 24 h monitoring period, the total non-metabolised meloxicam in the faeces was 1.35 ±â€¯0.71% of the total concentration in the plasma. Based on the short meloxicam elimination half-life and low cumulative concentration of total faecal meloxicam over a period in excess of 10 half-lives, this study indicates that Cape griffon vultures are efficient metaboliser of meloxicam, which is suggestive of different set of cytochrome enzymes being involved in the metabolism to that for diclofenac in this species. Identification of orthologous human CYP2C9 and CYP3A4 enzyme families in vultures will be an important further step in explaining the differences in the metabolic pathway(s) of meloxicam and diclofenac for the species.

Translating Pharmacokinetic and Pharmacodynamic Data into Practice.

Pharmacokinetic (PK) and pharmacodynamic (PD) publications provide scientific evidence for incorporation in evidence-based veterinary medicine, aiding the clinician in selecting doses and dosing intervals. PK and PD studies have reported wide variations within exotic species, due to physiologic differences in absorption, distribution, metabolism, and excretion. PK studies offer species-specific data to help tailor doses and dosing routes to individual patients, minimize toxicity, and provide a cornerstone for PD studies to determine drug efficacy. This article reviews the application of PK parameters and the challenges in determining the PD activity of drugs, with a particular emphasis on exotic species.

Investigation of the role of environmental contamination in the occurrence of residues of the veterinary drug phenylbutazone in cattle.

Phenylbutazone is a non-steroidal anti-inflammatory drug licensed for use in horses to treat musculoskeletal disorders. It is not permitted in the European Union for use in animals destined for the food chain. Official statistics provided by the European Food Safety Authority (EFSA) show that 0.18% of bovines tested in the European Union between 2008 and 2014 for non-steroidal anti-inflammatory drugs were non-compliant, with phenylbutazone representing over 28% of these. Anecdotal evidence suggests animals that have not been treated with the drug may have produced non-compliant samples, possibly through some form of contamination. In this study, ultra-high-performance liquid chromatography coupled with mass-spectrometric detection was applied to bovine plasma samples to determine if detectable residues (CCα = 0.28 ng ml-1) may occur in untreated animals as a result of environmental contamination through normal farming practice. The study demonstrates that waste from animals treated with phenylbutazone, and spread on an area of pasture, can contaminate untreated bovines grazing the pasture many weeks later. It was determined that this contamination, which can persist over a significant period, may be due to the ingestion of as little as 30 µg phenylbutazone by a 500 kg bullock.

Distribution and Depletion of Ractopamine in Goat Plasma, Urine and Various Muscle Tissues.

This study investigated the ractopamine (RAC) distribution and depletion process in goat plasma, urine and various muscle tissues which were associated with a potential risk for consumer health. The experiment was carried out in 21 goats (18 treated and 3 controls). Treated animals were administered orally a dose of 1 mg/kg body mass per day for 28 consecutive days and randomly sacrificed on Days 0.25, 1, 3, 7, 14 and 21 of the withdrawal period. RAC in goat samples was analyzed by using ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry. RAC was below the limits of detection (LOD = 0.15 ng/mL) in plasma while which was higher than the LOD in urine on withdrawal day 21. The residues in goat longissimus dorsi muscle, biceps femoris muscle and triceps surae muscle were differed significantly. These findings demonstrated that urine can be used as the target matrix for monitoring RAC abuse in goat.

Food safety in scavenger conservation: Diet-associated exposure to livestock pharmaceuticals and opportunist mycoses in threatened Cinereous and Egyptian vultures.

Pharmaceuticals from veterinary treatments may enter terrestrial food webs when medicated livestock are available to wildlife in supplementary feeding stations aimed at the conservation of endangered scavengers. Here, we hypothesized that the exposure risk to livestock fluoroquinolones, as indicators of pharmaceutical burden in food, is related to the variable reliance of scavengers on domestic versus wild animal carcasses. Since the misuse of broad-spectrum antibiotics is a major predisposing factor for opportunistic mycoses, we evaluated disease signs potentially associated with diet-dependent drug exposure in nestlings of two threatened vultures. A greater occurrence (100%, n=14) and concentration of fluoroquinolones (mean±SD=73.0±27.5µgL-1, range=33.2-132.7), mostly enrofloxacin, were found in Cinereous vultures, Aegypius monachus, due to their greater dependence on livestock carcasses than Egyptian vultures, Neophron percnopterus (fluoroquinolones occurrence: 44%, n=16, concentration: 37.9±16.6µgL-1, range=11.5-55.9), which rely much more on carcasses of wild animals (42% of remains vs. 23% in the cinereous vulture). The chaotic, chronic and pulsed ingestion of these drugs throughout nestling development is proposed as one of the most plausible explanations for the high occurrence and intensity of oral Candida-like lesions in nestling vultures. The high occurrence of fluoroquinolone residues and disease hindered the probing of a cause-effect relationship between both factors in individual vultures. This relationship could be evaluated through a population-based approach by sampling vultures not exposed to these drugs. The high dependence of vultures on domestic animals today compared to past decades and the growing intensification of livestock farming, imply an expected increase in the impact of pharmaceuticals on scavenger populations. This requires further evaluation due to potential consequences in biodiversity conservation and environmental health. We encourage the prioritization of efforts to promote the use of less medicated free-ranging livestock carcasses left in the countryside, rather than stabled stocks made available in vulture restaurants. Additionally, attention should be paid to the population recovery of wild species that dominated scavenger diets in the past.

Pharmacokinetic and pharmacodynamics of xylazine administered to exercised thoroughbred horses.

There is limited data describing xylazine serum concentrations in the horse and no reports of concentrations beyond 24 hours. The primary goal of the study reported here was to update the pharmacokinetics of xylazine following intravenous (IV) administration in order to assess the applicability of current regulatory recommendations. Pharmacodynamic parameters were determined using PK-PD modeling. Sixteen exercised adult Thoroughbred horses received a single IV dose of 200 mg of xylazine. Blood and urine samples were collected at time 0 and at various times for up to 96 hours and analyzed using liquid chromatography tandem mass spectrometry. Xylazine serum concentrations were best fit by a 3-compartment model. Mean ± SEM systemic clearance, volume of distribution at steady state, beta half-life and gamma half-life were 12.7 ± 0.735 mL/min/kg, 0.660 ± 0.053 L/kg, 2.79 ± 0.105 hours and 26.0 ± 1.9, respectively. Immediately following administration, horses appeared sedate as noted by a decrease in chin-to-ground distance, decreased locomotion and decreased heart rate (HR). Sedation lasted approximately 45 minutes. Glucose concentrations were elevated for 1-hour post administration. The EC50 (IC50) was 636.1, 702.2, 314.1 and 325.7 ng/mL for HR, atrioventricular block, chin-to-ground distance and glucose concentrations, respectively. The Emax (Imax) was 27.3 beats per minute, 47.5%, 42.4 cm and 0.28 mg/dL for HR, atrioventricular block, chin-to-ground distance and glucose concentrations, respectively. Pharmacokinetic parameters differ from previous reports and a prolonged detection time suggests that an extended withdrawal time, beyond current regulatory recommendations, is warranted to avoid inadvertent positive regulatory findings in performance horses. Copyright © 2016 John Wiley & Sons, Ltd.

Modelling concentrations of antimicrobial drugs: comparative pharmacokinetics of cephalosporin antimicrobials and accuracy of allometric scaling in food-producing and companion animals.

BACKGROUND: To optimize antimicrobial dosing in different animal species, pharmacokinetic information is necessary. Due to the plethora of cephalosporin antimicrobials and animal species in which they are used, assessment of pharmacokinetics in all species is unfeasible. In this study we aimed to describe pharmacokinetic data of cephalosporins by reviewing the available literature for food producing and companion animal species. We assessed the accuracy of interspecies extrapolation using allometric scaling techniques to determine pharmacokinetic characteristics of cephalosporins in animal species for which literature data is unavailable. We assessed the accuracy of allometric scaling by comparing the predicted and the published pharmacokinetic value in an animal species/humans not included in the allometric modelling. RESULTS: In general, excretion of cephalosporins takes place mainly through renal mechanisms in the unchanged form and volume of distribution is limited in all animal species. Differences in plasma protein binding capacity and elimination half-life are observed but available information was limited. Using allometric scaling, correlations between body weight (BW) and volume of distribution (Vd) and clearance (Cl) were R (2) > 0.97 and R (2) > 0.95 respectively for ceftazidime, ceftiofur, cefquinome and cefepime but not ceftriaxone. The allometric exponent ranged from 0.80 to 1.31 for Vd and 0.83 to 1.24 for Cl. Correlations on half-life ranged from R(2) 0.07-0.655 (literature) and R(2) 0.102-0.876 (calculated). CONCLUSIONS: Allometric scaling can be applied for interspecies extrapolation of cephalosporin pharmacokinetic parameters Vd and Cl, but not elimination half-life. We hypothesize that the accuracy could be improved by using more refined scaling techniques.

Dispersive liquid-liquid microextraction of quinolones in porcine blood: Optimization of extraction procedure and CE separation using experimental design.

A dispersive liquid-liquid microextraction procedure was developed to extract nine fluoroquinolones in porcine blood, six of which were quantified using a univariate calibration method. Extraction parameters including type and volume of extraction and dispersive solvent and pH, were optimized using a full factorial and a central composite designs. The optimum extraction parameters were a mixture of 250 µL dichloromethane (extract solvent) and 1250 µL ACN (dispersive solvent) in 500 µL of porcine blood reached to pH 6.80. After shaking and centrifugation, the upper phase was transferred in a glass tube and evaporated under N2 steam. The residue was resuspended into 50 µL of water-ACN (70:30, v/v) and determined by CE method with DAD, under optimum separation conditions. Consequently, a tenfold enrichment factor can potentially be reached with the pretreatment, taking into account the relationship between initial sample volume and final extract volume. Optimum separation conditions were as follows: BGE solution containing equal amounts of sodium borate (Na2 B4 O7 ) and di-sodium hydrogen phosphate (Na2 HPO4 ) with a final concentration of 23 mmol/L containing 0.2% of poly (diallyldimethylammonium chloride) and adjusted to pH 7.80. Separation was performed applying a negative potential of 25 kV, the cartridge was maintained at 25.0°C and the electropherograms were recorded at 275 nm during 4 min. The hydrodynamic injection was performed in the cathode by applying a pressure of 50 mbar for 10 s.

Comparison of Active Drug Concentrations in the Pulmonary Epithelial Lining Fluid and Interstitial Fluid of Calves Injected with Enrofloxacin, Florfenicol, Ceftiofur, or Tulathromycin.

Bacterial pneumonia is the most common reason for parenteral antimicrobial administration to beef cattle in the United States. Yet there is little information describing the antimicrobial concentrations at the site of action. The objective of this study was to compare the active drug concentrations in the pulmonary epithelial lining fluid and interstitial fluid of four antimicrobials commonly used in cattle. After injection, plasma, interstitial fluid, and pulmonary epithelial lining fluid concentrations and protein binding were measured to determine the plasma pharmacokinetics of each drug. A cross-over design with six calves per drug was used. Following sample collection and drug analysis, pharmacokinetic calculations were performed. For enrofloxacin and metabolite ciprofloxacin, the interstitial fluid concentration was 52% and 78% of the plasma concentration, while pulmonary fluid concentrations was 24% and 40% of the plasma concentration, respectively. The pulmonary concentrations (enrofloxacin + ciprofloxacin combined) exceeded the MIC90 of 0.06 µg/mL at 48 hours after administration. For florfenicol, the interstitial fluid concentration was almost 98% of the plasma concentration, and the pulmonary concentrations were over 200% of the plasma concentrations, exceeding the breakpoint (≤ 2 µg/mL), and the MIC90 for Mannheimia haemolytica (1.0 µg/mL) for the duration of the study. For ceftiofur, penetration to the interstitial fluid was only 5% of the plasma concentration. Pulmonary epithelial lining fluid concentration represented 40% of the plasma concentration. Airway concentrations exceeded the MIC breakpoint for susceptible respiratory pathogens (≤ 2 µg/mL) for a short time at 48 hours after administration. The plasma and interstitial fluid concentrations of tulathromcyin were lower than the concentrations in pulmonary fluid throughout the study. The bronchial concentrations were higher than the plasma or interstitial concentrations, with over 900% penetration to the airways. Despite high diffusion into the bronchi, the tulathromycin concentrations achieved were lower than the MIC of susceptible bacteria at most time points.

Acute acepromazine overdose: clinical effects and toxicokinetic evaluation.

INTRODUCTION: Acepromazine is a phenothiazine that is used exclusively in veterinary medicine for multiple purposes. Human overdoses are rarely reported and toxicokinetic data has never been reported. We present a case of intentional acepromazine overdose resulting in central nervous system and cardiovascular toxicity with confirmatory toxicokinetic data. CASE REPORT: A 54-year-old woman intentionally ingested 950 mg of her dog's acepromazine. Within 3 h of ingestion, she developed central nervous system and respiratory depression along with hypotension requiring non-invasive ventilation and vasopressors. Clinical toxicity resolved over the following 8 h. Serial plasma acepromazine levels were determined using gas chromatography/mass spectrometry. The initial acepromazine level (1-h post-ingestion) was 63 ng/ml. Follow-up levels at 8-, 10.5-, and 13.5-h post-ingestion were 8.9 ng/ml, 7.6 ng/ml, and 6.3 ng/ml, respectively. DISCUSSION: Human acepromazine toxicity is rarely reported but results in clinical toxicity (central nervous system depression, respiratory depression, hypotension) are similar to other phenothiazines. Compared to other phenothiazines, it appears to have a short elimination half-life that may account for the brief duration of clinical toxicity with relatively rapid improvement. No significant human cardiac toxicity has been reported. Treatment is supportive. CONCLUSION: This case highlights the unique toxicity of acepromazine in demonstrating rapid improvement of severe toxicity within 8 h consistent with a short elimination half-life.

Identification and elucidation of the structure of in vivo metabolites of diaveridine in chicken.

Diaveridine (DVD) is a popular antibacterial synergist that is widely used in combination with sulfonamide. It has been reported to be genotoxic to mammalian cells, but more studies are required to clarify this. Moreover, there is very little information on its pharmacokinetics, metabolic elimination and mechanism of toxicity. Therefore, in order to gain a better understanding of the metabolism of DVD, we performed high-performance liquid chromatography linear ion trapped orbitrap mass spectrometer (LC-LTQ-Orbitrap). With this approach, we identified 15 metabolites of DVD in chicken after a single oral administration of DVD; 10 of these metabolites have been identified in vivo for the first time. Nine phase I and five phase II metabolites were detected in the plasma, and eight phase I and six phase II metabolites were found in feces. The major phase I metabolites were formed via the O-demethylation and N-oxidation pathways, and the major phase II metabolites were glucuronide conjugates. These results are essential for understanding this compound more clearly and lay the basis for further studies about the metabolism of DVD. Therefore, using this approach, we were able to identify and characterize metabolites of DVD with high sensitivity and resolution. We were able to detect a broad range of metabolites, even some trace ones and some so far unknown metabolites.

Determination of anxiolytic veterinary drugs from biological fertilizer blood meal using liquid chromatography high-resolution mass spectrometry.

A liquid environment-friendly agricultural material originating from animal blood, blood meal, was employed to detect anxiolytic veterinary drugs using a combination of liquid-liquid extraction (LLE) and positive electrospray ionization Orbitrap mass spectrometry. Every positive ion of the analytes was consistent with [M+H](+) , and the accurate mass analysis and mass spectral filtration with a 2-ppm mass tolerance window were applied to identify and quantitate the analytes and metabolites. The developed LLE method was validated with the lowest calibrated level, linearity (r(2) ), recovery, repeatability and the within-laboratory reproducibility, which were in the ranges of 0.3-1 µg/L, 0.9963-0.9995, 48.3-117.5%, 1.1-12.6% and 2.3-15.7%, respectively. The LLE method was compared with a solid-phase extraction (SPE) method; however, its recoveries were <70% for most of the analytes despite good repeatability of 1.2-7.4%. The analytes and the ascertained acepromazine, azaperone and xylazine metabolites were monitored in four actual liquid blood meal samples, and none of the targeted compounds were observed.

Comparison of ELISA and LC-MS/MS for the measurement of flunixin plasma concentrations in beef cattle after intravenous and subcutaneous administration.

Eight cattle (288 ± 22 kg) were treated with 2.2 mg/kg of body weight of flunixin free acid in a crossover design by subcutaneous (SC) and intravenous (IV) administration. After a minimum 1:10 dilution with 50 mM phosphate buffer, a commercial immunoassay was adapted to determine plasma concentrations of flunixin. The limit of detection was 0.42 ng/mL and the working range was 0.76-66.4 ng/mL when adjusted with the dilution factor. Plasma samples were extracted using mixed-mode cation exchange solid phase extraction prior to the LC-MS/MS analyses. The linear calibration curve for LC-MS/MS was 0.5-2000 ng/mL with a limit of detection of 0.1 ng/mL for flunixin and 0.3 ng/mL for 5-hydroxy flunixin. Flunixin concentrations determined using the ELISAs were compared to concentrations derived from the same samples using LC-MS/MS analyses. Pharmacokinetic parameters of time versus concentration data from each analysis were estimated and compared. Differences (P < 0.05) in estimates of area under the curve, volume of distribution, and clearance were apparent between ELISA and LC-MS/MS analyses after IV dosing; after SC dosing, however, there were no differences among the estimated parameters between the two methods. Quantitative immunoassay was a satisfactory method of flunixin analysis and that it would be difficult to differentiate routes of administration in healthy beef cattle based on the plasma elimination profile of flunixin after IV or SC administration.

Residue depletion of nifuroxazide in broiler chicken.

BACKGROUND: Several nitrofuran drugs have been prohibited for use in food producing animals due to their carcinogenic and mutagenic effects. However, one of the nitrofurans, nifuroxazide, is still used as a veterinary drug in some countries. This study was conducted to investigate the residue depletion of nifuroxazide in broiler chicken. Chickens were fed with dietary feeds containing 50 mg kg⁻¹ of nifuroxazide for seven consecutive days. Liver, kidney, muscle and plasma samples were collected at different withdrawal periods, and the residues of parent nifuroxazide and its acid-hydrolysable side chain, 4-hydroxybenzhydrazide (HBH), in these samples were determined. RESULTS: Nifuroxazide was metabolised in vivo and its metabolite HBH was formed. Parent nifuroxazide was not detectable in these samples after 14 days of cessation. HBH was detectable in these samples even after 28 days of cessation and the total HBH residues were higher than 1.0 ng g⁻¹. Furthermore, the residue level of tissue bound HBH was much higher than that of free HBH. CONCLUSION: The tissue-bound HBH could be used as a marker to monitor the residue of nifuroxazide in chicken and the best target tissue should be liver. This is the first paper reporting the residue depletion of nifuroxazide in chicken.