The impact of accuracy and precision of analytical test methods on the determination of withdrawal periods.

Treatment of food-producing animals with veterinary medicinal products (VMPs) can result in residues in foodstuffs (e.g. eggs, meat, milk, or honey) representing a potential consumer health risk. To ensure consumer safety, worldwide regulatory concepts for setting safe limits for residues of VMPs e.g. as tolerances (US) or maximum residue limits (MRLs, EU) are used. Based on these limits so-called withdrawal periods (WP) are determined. A WP represents the minimum period of time required between the last administration of the VMP and the marketing of foodstuff. Usually, WPs are estimated using regression analysis based on residue studies. With high statistical confidence (usually 95% in the EU and 99% in the US) the residues in almost all treated animals (usually 95%) have to be below MRL when edible produce is harvested. Here, uncertainties from both sampling and biological variability are taken into account but uncertainties of measurement associated with the analytical test methods are not systematically considered. This paper describes a simulation experiment to investigate the extent to which relevant sources of measurement uncertainty (accuracy and precision) can impact the length of WPs. A set of real residue depletion data was artificially 'contaminated' with measurement uncertainty related to permitted ranges for accuracy and precision. The results show that both accuracy and precision had a noticeable effect on the overall WP. Due consideration of sources of measurement uncertainty may improve the robustness, quality and reliability of calculations upon which regulatory decisions on consumer safety of residues are based.

Residue depletion profiles and withdrawal interval estimations of meloxicam in eggs and ovarian follicles following intravenous (Meloxicam solution for injection) and oral (Meloxidyl®) administration in domestic chickens (Gallus domesticus).

Meloxicam is a non-steroidal anti-inflammatory drug (NSAID) commonly prescribed in an extralabel manner for treating chickens in urbanized settings. The objectives of this study were to determine meloxicam depletion profiles in eggs and ovarian follicles and to estimate associated withdrawal intervals (WDI) in laying hens following a single intravenous or repeated oral administration. The observed peak concentration of meloxicam in ovarian follicles were consistently higher than in egg yolk and egg white samples. Terminal half-lives were 31-h, 113-h and 12-h in ovarian follicles, egg yolk and egg white samples, respectively, for repeated oral administrations at 1 mg/kg for 20 doses at 12-h intervals. The terminal half-life following a single intravenous administration at 1 mg/kg was 50-h for ovarian follicles. Meloxicam WDI estimations using ovarian follicle and egg yolk concentration data following 20 doses at 12-h intervals were 36 and 12 days, respectively. Meloxicam WDI estimation using egg yolk concentration data following 8 doses at 24-h intervals was 12 days. These results improve our understanding on the residue depletion of meloxicam from chickens' reproductive tracts and egg products and provide WDIs to help ensure food safety for humans consuming eggs from treated laying hens.

Investigations on residual elimination of altrenogest oral solution in pigs and the withdrawal time.

The residual elimination of altrenogest in swine was investigated, preparing for the determination of withdrawal time. The residues of altrenogest in sebum, muscle, liver and kidney were extracted by optimized methods and further analyzed by UPLC-MS/MS. Under experimental conditions, the LOD and LOQ of altrenogest in sebum, muscle, liver and kidney were 0.5 and 1.0 µg/kg, respectively. The recoveries were in the range of 65 and 95% and the inter- and intra-RSD were less than 15%. The established method for the extraction, purification and detection of altrenogest is suitable for the determination of the residue of altrenogest in edible tissues of pigs. It was showed that altrenogest had the highest residual concentration level in liver, followed by kidney, sebum and muscle. Then withdrawal time was set at 9 days. The study provides an effective basis for elimination of altrenogest in swine.

Evaluating the persistence of malachite green residues in tilapia and pacu fish.

Although banned in food-producing animals, residues of malachite green (MG) and its primary metabolite, leucomalachite green (LMG), have been found in fish due to illegal use in aquaculture and the release of industrial wastewater, which represent a serious risk to food and environmental securities. This study aimed to investigate the residue depletion profile of MG and LMG in edible tissues of Nile tilapia (Oreochromis niloticus) and pacu (Piaractus mesopotamicus) cultured simultaneously under the same environmental conditions to support control measures in case of abuse. An analytical method involving QuEChERS sample preparation and liquid chromatography coupled to tandem mass spectrometry was developed, validated, and applied to quantify MG and LMG residues in fish fillets from two depletion experiments after treatment by immersion bath (MG at 0.10 mg L-1 for 60 min). During the experiment, the average water temperature was 30 ºC, while the pH was 6.9. The method is selective, precise (CV = 0.4 - 22%) and accurate (recovery 92 - 114%). The limits of detection and quantification are 0.15 and 0.5 ng g-1, respectively. In both species, the sum of MG and LMG residues were quantified up to the 32nd day post-exposure, and the concentrations were significantly higher in the pacu fillets (up to 3284 ng g-1) than in Nile tilapia (up to 432 ng g-1). The sums of MG and LMG residues were below 2 ng g-1 at 44 days and 342 days for Nile tilapia and pacu, respectively - the Minimum Required Performance Limit (MRPL) for analytical methods intended to monitor forbidden substances in food according to old European Commission guidelines. The persistence of MG residues in pacu may be attributed to its higher lipid content, which favors the accumulation of the non-polar metabolite LMG. These results provide insights into the concern about human, animal, and environmental health risks resulting from unauthorized use or aquatic contamination by industrial wastewater containing MG residues.

Comparison of gamithromycin residue depletion in yellow-feather and white-feather broilers after one single subcutaneous injection.

This study aimed to compare the residue depletion of gamithromycin in yellow-feather and white-feather broilers, using Sanhuang and Arbor Acres chickens as typical examples, respectively. Each breed (54 chickens) received a single subcutaneous dose of gamithromycin at 7.5 mg/kg bodyweight (BW). Tissues, including muscle, skin + fat, liver, kidney, and injection site, were collected at 6 h, 3, 5, 7, 10, 14, 21, 28, and 35 d postdrug administration. Gamithromycin concentrations in these tissues were determined using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The kinetics of gamithromycin were analyzed in different tissues using a noncompartmental method in the Phoenix software. Differences were observed in gamithromycin concentrations and kinetic characteristics in both breeds of chickens, with higher residue concentrations and longer residue times found in yellow-feathered broilers. In Sanhuang broilers, the elimination rates of gamithromycin followed this order: injection site > muscle > liver > kidney > skin + fat. The corresponding elimination half-lives (t1/2λzs) in these samples were 1.22, 1.30, 1.71, 2.04, and 2.52 d, respectively. In contrast, in Arbor Acres broilers, a different order was noted: muscle > injection site > kidney > liver > skin + fat, with corresponding t1/2λzs of 1, 1.23, 1.88, 1.93, and 2.21 d, respectively. These differences may be related to variations in pigments in various tissues of chickens of the 2 breeds. However, further investigations are warranted to discern the underlying reasons.

In vitro antibacterial activity of danofloxacin against Escherichia coli in Gushi chickens and its residue depletion following multiple oral administration.

This study aimed to investigate the in vitro antibacterial activity of danofloxacin against Escherichia coli isolated from Gushi chickens, as well as the tissue distribution and residue depletion of danofloxacin in Gushi chickens following multiple oral administration. A total of 42 clinical E. coli strains were isolated from the cloaca of locally farmed Gushi chickens between August and October 2023. Then the minimum inhibitory concentration (MIC) of danofloxacin against these isolates was determined by broth microdilution method. Additionally, 42 healthy Gushi chickens were randomly divided into 6 groups, and danofloxacin was orally administered at a dose of 5 mg/kg body weight (BW) for 3 consecutive days. Plasma, intestinal content, and tissue samples, including muscle, skin + fat, liver, kidney, lung, and intestine, were collected at 4, 12, 24, 48, 72, and 120 h after the last administration. Danofloxacin concentrations in all samples were determined using a high-performance liquid chromatography (HPLC) method. The average concentration vs. time data were then subjected to noncompartmental analysis using Phoenix software, and withdrawal periods for danofloxacin in Gushi chickens were further determined with WT1.4 software, setting a 95% confidence interval. Results indicated a notable inhibitory effect of danofloxacin on E. coli, with an MIC50 of 0.5 µg/mL. Additionally, danofloxacin exhibited widespread distribution in Gushi chickens, detectable in all collected samples. Among all tissues, the liver exhibited the highest concentration, followed by the intestine. Even on the fifth day postadministration, danofloxacin persisted in skin + fat, liver, and lung. The elimination half-lives (t1/2λzs) of danofloxacin varied across samples: skin + fat (47.87 h), lung (30.61 h), liver (22.07 h), plasma (16.05 h), muscle (12.53 h), intestine (9.83 h), and kidney (6.34 h). Considering residue depletion and the maximum residue limit (MRL) of danofloxacin in poultry set by Chinese regulatory authorities, withdrawal periods for the kidney, muscle, liver, and skin + fat were determined as 1.03, 1.38, 3.34, and 5.85 d, respectively, rounded to a final withdrawal time of 6 d.

Assessing safety, efficacy and residue depletion in golden mahseer, Tor putitora (Hamilton, 1822): biochemical and physiological responses to graded concentrations of oxytetracycline dietary supplementation.

The safety and effectiveness of oxytetracycline can potentially manage bacterial infections in fish. This, in turn, might reduce the concerns related to its use in aquaculture and human consumption, such as toxicity, antimicrobial resistance, and other associated risks. The primary objective of this study was to assess how adding oxytetracycline dihydrate to the diet affects its effectiveness, safety, and the presence of residues in T. putitora. T. putitora fingerlings, subjected to experimental infection with Aeromonas hydrophila at a concentration of 108 CFU mL- 1, received an oral administration of oxytetracycline dihydrate. The oxytetracycline dihydrate was added to the feed (corresponding to 2% of the fish body weight) at concentrations of 44.1, 88.2, 132.3 and 176.4 mg Kg- 1 fish body weight per day. This treatment was carried out for 10 consecutive days. The biochemical and physiological responses of T. putitora and efficacy of oxytetracycline dihydrate were determined through estimation of microbial load (CFU mL- 1), haematogram, serum biomarkers, behavioral characteristics, non-specific immunity and residue depletion. Experimentally infected fish showed disease progression and induced histopathological conditions with highest microbial load (CFU mL- 1) in the muscle of both control and treated fish. The fish haematogram showed increased leucocyte and haemoglobin content, influenced by dietary oxytetracycline dihydrate. The fish demonstrated adaptive physiological response to oxytetracycline dihydrate at 44.1 to 88.2 mg and resulted in increased albumin and globulin content. The serum-enzyme assay showed significant increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT) and plasma alkaline phosphatase (ALP) activities in the test fish (< 0.05). Oxytetracycline dihydrate at 88.2 to 132.3 mg Kg- 1 fish body weight per day recorded higher feed intake (75%), significant survivability (66-68%) and histopathological recovery. The suppressed immune response was manifested with decreased respiratory burst and lysozyme activity. The palatability, treatment of bacterial infection, histopathological changes and survivability by fingerlings of golden mahseer determined the safety and optimized the therapeutic potential of the oxytetracycline dihydrate at 88.2 mg Kg- 1 fish body weight per day for 10 days to contain the infection by A. hydrophila. A withdrawal period of 8-d was recommended as oxytetracycline dihydrate concentration depleted below the legal maximum residue limit (MRL 2.0 mg g- 1) in the edible muscle of the golden mahseer reared at an average water temperature of 20 °C. This is considered safe for human consumption.

Estimation of withdrawal interval recommendations following administration of fenbendazole medicated feed to ring-necked pheasants (Phasianus colchicus).

Introduction: Prescribing fenbendazole medicated feed for pheasants in the USA is considered extra-label drug use under CPG Sec 615.115, and a safe estimated withdrawal interval (WDI) must be applied following administration to this minor food-producing species. This study sought to determine the pharmacokinetic and residue depletion profile for fenbendazole and its major metabolites to estimate a WDI for pheasants following fenbendazole administration as an oral medicated feed. Method: Pheasants (n = 32) were administered fenbendazole as an oral medicated feed (100 ppm) for 7 days. Fenbendazole, fenbendazole sulfoxide, and fenbendazole sulfone (FBZ-SO2) in liver and muscle samples were analyzed using HPLC-UV. Tissue WDIs were estimated using FDA, European Medicines Agency (EMA), and half-life multiplication methods for US poultry tolerances, EMA maximum residue limits, and the analytical limit of detection (LOD; 0.004 ppm). Terminal tissue elimination half-lives (T1/2) were estimated by non-compartmental analysis using a naïve pooled data approach. Results: The tissue T1/2 was 14.4 h for liver, 13.2 h for thigh muscle, and 14.1 h for pectoral muscle. The maximum estimated withdrawal interval was 153 h (7 days) for FBZ-SO2 in pectoral muscle using the FDA tolerance method (95% confidence interval for the 99th percentile of the population), and the LOD as the residue limit. Discussion: The results from this study support the use of FBZ-SO2 as the marker residue in the liver of pheasants and the provision of evidence based WDIs following the extra-label administration of fenbendazole medicated feed (100 ppm) for 7 days.

Pharmacokinetics and residue depletion of erythromycin in rainbow trout Oncorhynchus mykiss (Walbaum).

Erythromycin (ERY) is a drug active against Gram-positive bacteria such as Lactococcus garvieae, a pathogen responsible for an important disease that may cause a substantial decrease in rainbow trout Oncorhynchus mykiss (Walbaum) production, the species of fish most commonly produced in Italy. In the literature, studies on the kinetics behaviour of ERY in fish are limited. Therefore, the aim of the present study was to evaluate the pharmacokinetics of ERY in rainbow trout after a single oral treatment with 75 mg kg⁻¹ body weight (b.w.) of ERY and the residue depletion after multiple oral administration of 75 mg kg⁻¹ b.w. day⁻¹ of ERY for 10 days. Blood concentrations of ERY increased up to 20.24 ± 13.32 µg mL⁻¹ at 6 h, then decreased to 5.97 ± 3.89 µg mL⁻¹ at 24 h. The time during which the antibiotic remains in the bloodstream at concentrations exceeding the MIC (T > MIC) and the area under the serum concentration-time curve (AUC)/MIC are both pharmacokinetic-pharmacodynamic (PK/PD) predictors of ERY efficacy, and the data obtained allowed us to hypothesize that a dosage of 75 mg kg⁻¹ b.w. day⁻¹ of ERY could treat the lactococcosis in trout. Regarding the study of ERY depletion, rapid elimination was observed in tissue (muscle plus adherent skin); in fact the concentrations were below the limit of quantification in all samples (except two) by day 10 post-treatment. ERY is not licensed in Europe for use in aquaculture, and its use is possible only by off-label prescription with a precautionary withdrawal time of 500 degree-days, as established by Directive 2004/28/EC. From the data obtained in this study, a withdrawal time of 8.90 days was calculated, corresponding, in our experimental conditions, to 117.5 degree-days, a value significantly lower than that established by the European directive.

Florfenicol depletion in edible tissue of rainbow trout, Oncorhynchus mykiss (Walbaum), and sea bream, Sparus aurata L.

An increase in fish production has consequently brought an increase in infectious diseases in fish farms. The use of chemotherapic drugs is the most effective instrument against common bacterial agents. The number of registered drugs for use in aquaculture is limited and often veterinary practitioners resort to the off-label use of chemotherapic agents authorized for different food-producing animal species. Florfenicol is well known for its outstanding effect against various pathogenic bacteria affecting fish, and therefore, it may be a useful drug for off-label use in aquaculture. The aim of this study was to evaluate the depletion of florfenicol and its major metabolite, florfenicol amine, from the edible tissue of two fish species, rainbow trout and sea bream, following treatment with medicated feed at a dosage of 10 mg kg(-1) of bw day(-1) , for 10 consecutive days. At prefixed time points after the end of administration (0.25, 1, 2, 3, 4, 6, 7, 10, 14 and 21 days after treatment), edible tissues (muscle plus adherent skin) from 15 individuals in each group were collected and analysed by HPLC, to determine concentration of the drug in the tissue. On the basis of the obtained concentrations, withdrawal times of florfenicol in the two species were calculated. The results indicate that a drug withdrawal time of 500 °C-day, as established by Directive 2004/28/EC, for off-label drug use is more than satisfactory to guarantee the healthiness of fish products against the risk of drug residues.

Stereoselective Pharmacokinetics and Residue Depletion of Praziquantel and Its Metabolites, 4-Hydroxypraziquantel Enantiomers, in Swine.

Praziquantel (PZQ) is administered as a racemic mixture during swine production to treat parasitic diseases. Despite its widespread application, the pharmacokinetics, residue depletion, bioactivity, and toxicity of PZQ enantiomers in swine remain largely unknown. In this study, a systematic investigation of the pharmacokinetics, tissue distribution, and residue depletion of PZQ, its major metabolites (trans- and cis-4-OH-PZQ), and their enantiomers was conducted in swine. The findings indicated that PZQ was absorbed and metabolized rapidly. In swine plasma, the concentrations of S-PZQ, S-trans-4-OH-PZQ, and R-cis-4-OH-PZQ were higher than those of their respective enantiomers. The three analytes exhibited significant tissue distribution and stereoselectivity in 10 swine tissues. Notably, the two enantiomers of PZQ demonstrated comparable tissue concentrations except in the liver and lung. Moreover, the concentrations of S-trans-4-OH-PZQ and R-cis-4-OH-PZQ were higher than those of their respective enantiomers in the 10 tissues. This study has significant implications for the development of rational dosing strategies, reducing drug usage, and minimizing side effects, as well as accurately assessing the risks associated with PZQ administration and, by extension, other chiral drugs. Furthermore, it lays a theoretical foundation for the future use of the active enantiomer, R-PZQ.

Pharmacokinetics, tissue residue depletion, and withdrawal interval estimations of florfenicol in goats following repeated subcutaneous administrations.

Florfenicol is a broad-spectrum antibiotic commonly used in the U.S. to treat respiratory and enteric infections in goats in an extra-label manner, which requires scientifically based withdrawal intervals (WDIs) for edible tissues. This study aimed to determine the depletion profiles for florfenicol and florfenicol amine in plasma and tissues samples and to estimate WDIs for goats following subcutaneous injection of 40 mg/kg florfenicol, twice, 96 h apart. The samples were collected up to 50 days after the second dose. Pharmacokinetic parameters were calculated using non-compartmental analysis. Three different pharmacostatistical methods with different operational tolerances were used to calculate WDIs. The plasma half-life was 101.80 h for florfenicol and 207.69 h for florfenicol amine after the second dose. Using the FDA tolerance limit method, WDIs were 202 and 101 days, while the EMA maximum residue limit method estimated 179 and 96 days for the respective tissue concentrations to fall below limits of detection (0.12 µg/g for liver and 0.05 µg/g for kidney). This study characterizes plasma pharmacokinetics and tissue depletion profiles of florfenicol and florfenicol amine in goats following subcutaneous injections and reports estimated WDIs for food safety assessment of florfenicol in goats.

Biosafety, histological alterations and residue depletion of feed administered anti-parasitic drug emamectin benzoate in golden mahseer, Tor putitora (Hamilton, 1822) as a model candidate fish for sport fishery and conservation in temperate waters.

In the present experiment, the attempt has been made to study the biosafety, toxicity, residue depletion and drug tolerance of graded doses of emamectin benzoate (EB) in juveniles of golden mahseer, Tor putitora as a model candidate fish for sport fishery and conservation in temperate waters through an extended medicated feeding. The graded doses of EB viz., 1× (50 µg/kg fish/day), 2 × (100 µg/kg fish/day), 5 × (250 µg/kg fish/day) and 10 × (500 µg/kg fish/day) were administered to golden mahseer juveniles through medicated diet for 21 days at water temperature of 18.6°C. The higher doses of EB did not cause any mortality during and 30 days after the end of medication period, but considerable variations in feeding and behavior were observed. Severe histological alterations observed after EB-diets (5 × and 10×) were vacuolation, pyknotic nuclei, melanomacrophage centre and necrosis in liver; Bowman's capsule dilation, degenerated renal tubules in kidney; myofibril disintegration, muscle oedema, splitting of muscle fibres, migration of inflammatory cells in muscle; and abundant goblet cells, dilated lamina propria and disarrangement of mucosa in intestine tissues. The residual concentrations of EB metabolites Emamectin B1a and B1b were analyzed using muscle extracts and were found to be peaked during medication period followed by gradual depletion in post-medication period. The outcome of this study showed that the Emamectin B1a residual concentration in fish muscle in 1×, 2×, 5×, and 10× EB treatment groups were 1.41 ± 0.49, 1.2 ± 0.7, 9.7 ± 3.3, and 37.4 ± 8.2 µg/kg at 30 days of post-medication period, respectively, which falls under the maximum residue limits (MRLs) of 100 µg/kg. The results support the biosafety of EB at recommended dose of 50 µg/kg fish/day for 7 days. As residue of EB is recorded falling within the MRL, no withdrawal period is recommended for golden mahseer.

Simultaneous determination of praziquantel and its main metabolites in the tissues of black goats and their residue depletion.

Praziquantel (PZQ) is a pyrazino-isoquinoline compound with broad spectrum of activity against parasitic trematodes and cestodes, and a key veterinary drug in the parasitic disease control field. However, PZQ residues caused by non-conforming or excessive use in food-producing animals may pose a serious threat to human health. Herein, a simple, sensitive and reproducible LC-MS/MS method was developed for the simultaneous determination of praziquantel and trans- and cis-4-hydroxypraziquantel in black goat tissues to guide the reasonable use of PZQ. The mean recoveries for three target analytes were 71.2 ∼ 117.6%, and the limits of quantification were 1.0 µg/kg. Twenty-five healthy black goats were administered a single dose of praziquantel tablets at a dose of 35 mg/kg of body weight for residue elimination study, The results revealed that praziquantel and 4-hydroxypraziquantel were rapidly depleted in goat tissues and the elimination half-lives did not exceed 1 day in all tissues except for muscle and lung. It provides guidance for the establishment of maximum residue limit of praziquantel in goat.

The multicomponent residue depletion of Gelsemium elegans in pig tissues, urine, and plasma.

Introduction: Gelsemium elegans (G. elegans) as a traditional medicinal plant used in livestock production. The use of G. elegans in veterinary clinics may pose safety risks to human health. Objectives: The aim of this study was to investigate tissue residue depletion in pigs fed G. elegans powder. Methods: A precise quantitation method and a simultaneous semi-quantitation method for multiple components independently of standards in pig tissues were developed for the first time. The two methods were validated in terms of specificity, LODs, LOQs, linearity, accuracy, precision, and matrix effects. They were then applied to a tissue residue depletion study after G. elegans powder at a dose of 2% per kg feed were fed to pigs. Results: Compared with precise quantitation, the method validation results indicated that the semi-quantitation method was reliable and acceptable for multicomponent quantification independent of standards. Many G. elegans alkaloids are widely distributed in most tissues of pigs. Tissue residue depletion studies indicated that 14-hydroxygelsenicine, 11-hydroxygelsenicine, and gelsemoxonine could be used as potential residue markers, and pancreas, small intestine, and lung tissues could be considered as potential residue target tissues of G. elegans. In addition, both urine and plasma could be used to predict 14-hydroxygelsenicine and gelsemoxonine residues in the liver, pancreas, and small intestinal tissues of pigs. Conclusion: The developed semi-quantification method can be applied to monitor the application and residue of G. elegans. The results provide scientific evidence for evaluating the safety of animal-derived food from G. elegans for consumers and will be helpful for its application and future development.

Influence of dietary emamectin benzoate on the biological responses of monosex (all-male) Oreochromis niloticus (L.) fries.

The application of antiparasitic drugs plays a crucial role in the removal of infectious parasites in aquaculture. Emamectin benzoate (EB) is predominantly used as a feed premix against ectoparasites on temperate fish. This study evaluated the influence of 14 days of EB-dosing at 0-10 times the recommended dose (1X: 50 µg/kg biomass/day) on the biological responses and accrual/depletion of EB-residues in a tropical fish monosex Oreochromis niloticus fries. A significant dose-dependent reduction in feed intake by 3.50% in 1X and 43.00% in 10X groups, and an increase in mortalities from 2.92% (1X) to 11.25% (10X) during the EB-dosing period was noted. A significant increase in glucose and alkaline phosphatase and reduction in calcium and chloride ions, superoxide dismutase (SOD) and acetylcholinesterase levels in the muscle and/or brain tissue was observed. On day 21 post-EB-dosing, the levels of muscle glucose and SOD reached normalcy in the 1X group, while the levels of other biomarkers failed to recuperate. The EB-residue levels peaked on day 14 EB-dosing (2.77 ng/g) in the 1X group and decreased later with detectable levels (0.03 ng/g) even on day 21 post-EB-dosing. The EB-residue levels were within the permissible limits of the Canadian Food Inspection Agency and the European Commission. The EB-dosing negatively influenced the health of O. niloticus by altering the physiological state in a dose- and time-dependent way. The results suggested that the use of EB might be plausibly risky in tropical aquaculture.

Residue Depletion of Imidocarb in Bovine Tissues by UPLC-MS/MS.

In this study, an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the residue depletion of imidocarb (IMD) in bovine tissues, and the drug withdrawal time of IMD was determined. Twenty-five clinically healthy cattle (body weight 300 kg ± 15 kg) were randomly divided into five groups of five cattle each. The cattle were treated subcutaneously injecting a single dose of a generic IMD formulation, at the recommended dosage of 3.0 mg/kg. The five groups of cattle were slaughtered respectively at 96, 160, 198, 213, and 228 days after IMD administration. Samples from the liver, kidney, muscle, fat, and injection site were collected from each animal. After subtilis proteinase was used to digest the tissue, the content of IMD in the samples was analyzed by UPLC-MS/MS method. In conclusion, the method validation results showed that the method meets the criteria, and the longest withdrawal time of 224 days for the liver can be selected as the conclusive withdrawal time to guarantee consumer safety.

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 (N 1-deoxyolaquindox), O2 (deoxyolaquindox), O3 (2-carboxamide-3-methylquinoxaline-N 4-oxide), O4 (2-carboxymethylaminocarbonyl-3-methylquinoxaline-N 4-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 t 1/2 1.78-2.28 days vs. t 1/2 2.04-2.46 days). The elimination half-lives of deoxyolaquindox residue in broilers' liver and kidney tissues (t 1/2 >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.

Research Note: Study on the residue depletion of febrifugine and isofebrifugine in broiler chicken.

In this study, 105 broiler chickens were fed with dietary feeds containing different contents of Dichroae Radix extract for 10 consecutive days. Then the residue depletions of its main alkaloids (febrifugine and isofebrifugine) in muscle, kidney and liver samples at different withdrawal times were determined by an ultra-performance liquid chromatography method. Results showed that the 2 alkaloids were mainly at tissue-bound formation. At withdrawal period of 0 d, their concentrations in all samples were high but decreased rapidly after 1 day of cessation (35-91%). After 5 to 7 days of cessation, their residues in muscle and kidney were not detectable, and after at least 10 days of cessation they were not detectable in liver. These results indicated that an appropriate withdrawal time for Dichroae Radix preparation was required if it is licensed as a new drug, and the best target tissue for monitoring its residue was liver.

Interaction Between Florfenicol and Doxycycline Involving Cytochrome P450 3A in Goats (Capra hricus).

When two drugs are combined, drug-drug interactions (DDI) often occur. Metabolic DDI usually occur due to inhibition of the metabolism of one drug by the other. This leads to an increase in the plasma concentration of the drug whose metabolism is inhibited. The objective of this research study was to verify the DDI risk of two antibacterial, florfenicol (FF) and doxycycline (DOX) due to metabolism. Because food containing residues of any pharmacologically active substance could potentially constitute a public health hazard, we selected a food producing animal, goat, goat liver microsomes and recombinant metabolic enzymes, for in vivo and in vitro metabolism studies. In vitro experiments showed that CYP3A was the key enzyme subfamily in FF metabolism, DOX slowed down FF metabolism and R440 was possibly the key amino acid in the metabolic interaction between FF and DOX. In vivo studies in the goats showed that DOX inhibited up-regulation of CYP3A24 gene expression produced by FF; in liver and kidney, DOX slightly slowed down FF metabolism. Quantitative prediction of DDI risk suggest that when DOX is used in combination with FF in veterinary medicine, may result in a clinical significant increase of FF plasma and tissue concentrations, resulting a prevalence of harmful tissue residues of medicinal products in the food chain. Through our experimentation, when DOX is used in combination with FF, the withdrawal period of FF in the kidney was extended by 1 day. Otherwise, an appropriate withdrawal period (20 days) of FF was established for FF and DOX combined use to ensure that the animal can be safely slaughtered for food.