Ultraversatile Fluorescent Sensors Based on Two CoII/NiII Coordination Polymers for Identifying Various Antibiotics via the Turn-On/Off Effect and Detecting pH.

Because the accurate and rapid detection of antibiotics and pH plays an important role in biological systems and environmental fields, developing suitable and efficient sensors that can simultaneously detect antibiotics and pH has become important. In this work, we successfully designed and synthesized two new one-dimensional coordination polymers based on the mixed ligands L [N,N'-bis(4-methylpyridin-4-yl)-2,6-naphthalene dicarboxylamide] and H2CPG [3-(4-chlorophenyl)glutaric acid], [M(L)(HCPG)2(H2O)2] (M = Co for CP 1, and M = Ni for CP 2), which were structurally characterized by single-crystal X-ray diffraction, infrared spectroscopy, powder X-ray diffraction, and thermogravimetric analysis. CP 1 and CP 2 can be used as ultraversatile fluorescent sensors, which can sense erythromycin (ERY) and oxacillin (OXC) by turn-on fluorescent enhancement and detect furaltadone (FTD) via the turn-off fluorescent quenching effect, separately. The concentration ranges of different analytes sensed by CPs 1 and 2 were 0-0.046 and 0-0.069 mM for ERY, 0-0.04 and 0-0.028 mM for OXC, and 0-0.155 and 0-0.019 mM for FTD, respectively. Moreover, CP 2 can effectively sense pH, in both a wide pH range and the fine physiological range. The sensors have a rapid luminescence response, good recyclability, and excellent fluorescence stability. More importantly, they not only represent the first example of detecting ERY or OXC based on fluorescent CPs but also are the very rare ultraversatile fluorescent sensors. The fluorescent sensing mechanism for antibiotics and pH was discussed in detail.

Antibacterial activity of combined aditoprim and sulfamethoxazole against Escherichia coli from swine and a dose regimen based on pharmacokinetic-pharmacodynamic modeling.

The mortality of livestock caused by pathogenic Escherichia coli (E. coli) still accounts for a large proportion of deaths in large-scale production and reproduction, which causes devastating economic losses to the pig breeding industry. The aims of this study were to investigate the antibacterial activity of combined aditoprim (ADP) and sulfamethoxazole (SMZ) against clinical isolates of E. coli from pigs and to develop a pharmacokinetic-pharmacodynamic (PK-PD) model to formulate the optimal dose of ADP/SMZ for the treatment of pig colibacillosis. Blood and ileum fluid samples were collected at different times after single intramuscular injection of ADP/SMZ (5/25 mg/kg b.w.) to healthy pigs and E. coli-infected pigs. Concentrations of ADP and SMZ in plasma and ileum fluid were analyzed by HPLC. The peak concentration (Cmax ) and the area under the concentration-time curve (AUC0-24h ) in ileum fluid of healthy pigs were 1.76 ± 0.27 µg/ml and 18.92 ± 2.87 µg·h/ml for ADP and 19.15 ± 2.63 µg/ml and 125.70 ± 11.86 µg·h/ml for SMZ, respectively. Cmax and AUC0-24h in ileum fluid of infected pigs were 1.88 ± 0.13 µg/ml and 15.12 ± 0.75 µg·h/ml for ADP and 19.71 ± 3.68 µg/ml and 133.92 ± 17.14 µg·h/ml for SMZ, respectively. The minimum inhibitory concentrations (MICs) of combined ADP and SMZ (ADP/SMZ) against 185 strains of E. coli from pigs were determined. The MIC50 and MIC90 of ADP/SMZ were 0.5/2.5 and 4/20 µg/ml, respectively. The MIC of the selected pathogenic E. coli SHC28 was 0.5/2.5 µg/ml in Mueller-Hinton broth and 0.25/1.25 µg/ml in ileum fluid, respectively. In vitro, the mutant prevention concentration, the post-antibiotic effect, growth, and time-killing curves in vitro and ex vivo of ADP/SMZ against the isolate SHC28 were assayed for PD studies. The results showed that ADP/SMZ exhibited strong concentration-dependent antimicrobial activity against E. coli. After integrating the in vivo pharmacokinetic parameters of infected pigs and ex vivo PD data using the sigmoid Emax (Hill) equation, the AUC24h /MIC values in ileum fluid for bacteriostatic, bactericidal, and bacterial eradication were 18.84, 65.39, and 110.68 h, respectively. In conclusion, a dosage of 3.45/17.25 mg/kg ADP/SMZ by intramuscular injection daily for 3 consecutive days may be sufficient to treat swine colibacillosis due to E. coli with a MIC of 0.5/2.5 µg/ml.

Fluorotelomer Alcohols' Toxicology Correlates with Oxidative Stress and Metabolism.

Fluorotelomer alcohols (FTOHs) are widely used as industrial raw materials due to their unique hydrophobic and oleophobic properties. However, because of accidental exposure to products containing FTOHs or with the widespread use of FTOHs, they tend to contaminate the water and the soil. There are reports demonstrating that FTOHs can cause various harmful effects in animals and humans (for example, neurotoxicity, hepatotoxicity, nephrotoxicity, immunotoxicity, endocrine-disrupting activity, and developmental and reproductive toxicities). Oxidative stress is related to a variety of toxic effects induced by FTOHs. To date, few reviews have addressed the relationship between the toxicity of FTOHs and oxidative stress. This article summarises research demonstrating that the toxicity induced by FTOHs correlates with oxidative stress and metabolism. Furthermore, during the metabolic process of FTOHs, a number of cytochrome P450 enzymes (CYP450) are involved and many metabolites are produced by these enzymes, which can induce oxidative stress. This is also reviewed.

Application of physiologically based pharmacokinetic models to promote the development of veterinary drugs with high efficacy and safety.

Physiologically based pharmacokinetic (PBPK) models have become important tools for the development of novel human drugs. Food-producing animals and pets comprise an important part of human life, and the development of veterinary drugs (VDs) has greatly impacted human health. Owing to increased affordability of and demand for drug development, VD manufacturing companies should have more PBPK models required to reduce drug production costs. So far, little attention has been paid on applying PBPK models for the development of VDs. This review begins with the development processes of VDs; then summarizes case studies of PBPK models in human or VD development; and analyzes the application, potential, and advantages of PBPK in VD development, including candidate screening, formulation optimization, food effects, target-species safety, and dosing optimization. Then, the challenges of applying the PBPK model to VD development are discussed. Finally, future opportunities of PBPK models in designing dosing regimens for intracellular pathogenic infections and for efficient oral absorption of VDs are further forecasted. This review will be relevant to readers who are interested in using a PBPK model to develop new VDs.

The dose regimen formulation of tilmicosin against Lawsonia intracellularis in pigs by pharmacokinetic-pharmacodynamic (PK-PD) model.

In this study, the rational dose regimens of tilmicosin against Lawsonia intracellularis (L. intracellularis) were studied using pharmacokinetic-pharmacodynamic (PK-PD) model approach to provide a maximal efficacy. The healthy and infected pigs were orally administrated the tilmicosin premix at a single dose of 10 mg/kg, and then the plasma and ileum content were collected at different time points. The time to peak (Tmax), the peak concentration (Cmax), the area under concentration time curve (AUC0-24h), the apparent volume of distribution by bioavailability (V/F), the body clearance rate by bioavailability (CL/F) and the mean residence time (MRT) of tilmicosin premix for plasma were 2.00 h, 1.08 ± 0.04 µg/mL, 9.61 ± 1.47 µg h/mL, 34.43 ± 1.02 L/kg, 0.71 ± 0.03 L/h/kg and 15.03 ± 0.04 h in healthy pigs, and 2.00 h, 0.99 ± 0.03 µg/mL, 9.30 ± 1.43 µg h/mL, 58.59 ± 1.81 L/kg, 0.44 ± 0.02 L/h/kg and 15.75 ± 0.03 h in infected pigs, respectively. The Tmax, Cmax, AUC0-24h, V/F, CL/F and MRT of tilmicosin premix for ileum content were 2.00 h, 3.78 ± 0.03 µg/mL, 20.41 ± 1.64 µg h/mL, 11.29 ± 0.97 L/kg, 0.44 ± 0.02 L/h/kg and 11.29 ± 0.09 h in healthy pigs, and 2.00 h, 3.41 ± 0.06 µg/mL, 22.65 ± 1.32 µg h/mL, 8.16 ± 1.51 L/kg, 0.41 ± 0.01 L/h/kg and 11.44 ± 0.05 h in infected pigs, respectively. Based on the intracellular minimum inhibitory concentration (MIC) of L. intracellularis isolate was 2 µg/mL, the results of the mutant prevention concentration (MPC), the post-antibiotic effect (PAE) and time-killing curves all showed strong concentration-dependenttendencies. Integrating the in vivo pharmacokinetic data of infected pigs and ex vivo pharmacodynamic data using the sigmoid Emax (Hill) equation to obtain the ileum content AUC0-24h/MIC values of 6.87, 26.80, and 36.02 h to achieve the bacteriostatic activity, bactericidal activity, and virtual eradication of bacteria, respectively. Based on these results, a dosage regimen of daily 14.39 mg/kg for 3 d could be sufficient in the treatment of L. intracellularis. This study will provide a guidance of dosage regimen formulation for drug against animal intracellular bacterial infections.

Antibacterial activity of cyadox against Clostridium perfringens in broilers and a dosage regimen design based on pharmacokinetic-pharmacodynamic modeling.

Necrotic enteritis is an intestinal disease caused by Clostridium perfringens (C. perfringens) that results in high economic losses to the poultry industry. The purpose of this study was to investigate the antibacterial activity of cyadox against C. perfringens and to formulate its dosage regimen based on pharmacokinetics/pharmacodynamics (PK/PD) modeling in broilers. The PK parameters of cyadox in ileum of healthy and infected broilers following oral administration at 30 mg/kg body weight (BW) were investigated and PD study the MIC, MBC, MPC, and PAE were determined. The time-killing curves were established in vitro and ex vivo to evaluate the antibacterial activity of cyadox against C. perfringens. The results revealed that the MIC of cyadox against C. perfringens was 1-16 µg/mL. After oral administration of cyadox, the peak concentration (Cmax), maximum concentration time (Tmax), and area under the concentration-time curve (AUC) in ileum content of broilers were 143.55-161.48 µg/mL, 1.08-1.25 h, and 359.51-405.69 µg h/mL respectively. After Integrating the in vivo PK and ex vivo PD data the AUC24h/MIC values needed for bacteriostatic, bactericidal and bacterial eradication were 27.71 h, 78.93 h, and 165.14 h, respectively. By model validation, the cure rate was 85.71%. In conclusion, a dosage regimen of 14.02 mg/kg repeated after every 12 h for 3-5days was expected to be therapeutically effective in broilers against C. perfringens with MIC ≤2 µg/mL.

Integration of PK/PD for dose optimization of aditoprim against Trueperella pyogenes causing endometritis in bovines.

Trueperella pyogenes is a major pathogenic organism of bovine uterus causing devastating economic losses. Clinical isolates of T. pyogenes demonstrated severe infection with high rate of disease progression than other pathogenic bacteria of uterus. We aimed to investigate the effectiveness of aditoprim, a novel dihydrofolate reductase inhibitor, based upon the ex-vivo pharmacodynamic analysis by using uterine fluid of cattle. In-vivo pharmacokinetic parameters were measured by high performance liquid choromatography and analyzed by winonline software (version 5.2.1). In-vitro minimum inhibitory concentration, mutant prevention concentration and time kill curves were determined with clinical isolates of Trueperell pyogenes. Our data showed that peak concentration (Cmax) and area under the concentration time curve (AUC) were 6551.43 ± 1296.13 and 23585.22 ± 5126.47 µg/mL, respectively. Aditoprim showed potent antimicrobial activity against T. pyogenes (MIC = 0.25 µg/mL) and exhibited the concentration dependent antibacterial effect and produced in-vitro post antibiotic effect which was less than 1 h and increased with concentration. Pharmacodynamics values were modeled with pharmacokinetics parameters (PK/PD modeling) to simulate the efficacy of aditoprim for different dosage regimens. It was concluded that a dose of 2 mg/kg every 12h was expected to reach a bactericidal activity against T. pyogenes in endometritis.

Optimal regimens based on PK/PD cutoff evaluation of ceftiofur against Actinobacillus pleuropneumoniae in swine.

BACKGROUND: Actinobacillus pleuropneumoniae formerly known as Haemophilus pleuropneumoniae, can cause pleuropneumoniae in pigs, which lead to significant mortality. Ceftiofur was the first cephalosporin antibiotic used in animals, which was effective against gram-negative and gram-positive bacterium. This study aimed to formulate a rational dosage strategy and review the preceding recommended dosage based on PK/PD modeling and Establish Clinical breakpoint of ceftiofur against Actinobacillus pleuropneumoniae based on the pharmacodynamic-pharmacokinetic cutoff. RESULTS: The epidemiologic cutoff value was 0.125 µg/mL. The results of the pharmacodynamic study showed that the MICs of BW39 were 0.5 µg/mL and 1 µg/mL in vitro and ex-vivo, respectively. The minimal bactericidal concentrations (MBCs) under in vitro and ex vivo conditions were both 1 µg/mL. The time-killing profiles of ceftiofur against BW39 were time-dependent with a partly concentration-dependent pattern. Based on the inhibitory sigmoid Emax model, the AUC24 h/MIC values for the bacteriostatic, bactericidal, and elimination effects in serum were 45.73, 63.83, and 69.04 h for healthy pigs separately. According to the Monte Carlo simulation, the COPD was calculated as 2 µg/mL, and the optimized dosage regimen of ceftiofur against Actinobacillus pleuropneumoniae to achieve bacteriostatic, bactericidal, and elimination effects over 24 h was 2.13, 2.97, and 3.42 mg/kg for the 50% target attainment rate (TAR) and 2.47, 3.21, and 3.70 mg/kg for the 90% TAR respectively. CONCLUSIONS: In conclusion, we reveal the EOFF and PK/PD cutoff values of ceftiofur against A. pleuropneumoniae in piglets. However, with the paucity of clinical data for ceftiofur to establish a clinical cutoff against A. pleuropneumoniae, the PK/PD cutoff value of 2 µg/mL will be recommended as surrogate. According to the PK/PD data and the MIC distribution in China, the single bactericidal dose was 3.21 mg/kg for the 90% target, which would be more able to cure Actinobacillus pleuropneumoniae and avoid the emergence of resistance for clinical ceftiofur use in piglet.

Disposition of cyadox in domesticated cats following oral, intramuscular, and intravenous administration.

Cyadox (CYX) is a synthetic antibacterial agent of quinoxaline with much lower toxic effects. A safety criterion of CYX for clinical use was established by studying the pharmacokinetics and metabolism of CYX after oral (PO), intramuscular (IM), and intravenous (IV) administration. CYX was administered in six domesticated cats (three males and three females) by PO (40 mg/kg.b.w.), IM (10 mg/kg.b.w.), and IV (10 mg/kg.b.w.) routes in a crossover pattern. Highly sensitive liquid chromatography with ultraviolet detection (HPLC-UV) method was developed for detection of CYX and its metabolites present in plasma, urine, and feces. The bioavailability of CYX after PO and IM routes was 4.37% and 84.4%. The area under curves (AUC), mean resident time (MRT), and clearance (CL) of CYX and its metabolites revealed that CYX quickly metabolized into its metabolites. The total recovery of CYX and its main metabolites was >60% after each route. PO delivery suggesting first pass effect in cats that might make this route suitable for intestinal infection and IM injection could be better choice for systemic infections. Less ability of glucuronidation did not show any impact on CYX metabolism. The findings of present study provide detailed information for evaluation of CYX.

Construction of an Electrochemical Receptor Sensor Based on Graphene/Thionine for the Sensitive Determination of ß-Lactam Antibiotics Content in Milk.

In antibiotics, ß-lactam is one kind of major concern acknowledged as an unavoidable contaminant in milk. Thus, a facile and sensitive method is essential for rapid ß-lactam antibiotics detection. In our work, a specific electrochemical receptor sensor based on the graphene/thionine (GO/TH) composite was established. The mechanism of the electrochemical receptor sensor was a direct competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP-AMP) to the mutant BlaR-CTD protein by free ß-lactam antibiotics. Then, horseradish peroxidase (HRP) catalyzed the hydrolysis of the substrate hydrogen peroxide (H2O2), which produced an electrochemical signal. Under optimal experimental conditions, this method could quantitatively detect cefquinome from 0.1 to 8 µg L-1 and with the limit of detection (LOD) of 0.16 µg L-1, much lower than the maximum residue limit (MRL) of 5 µg L-1 set by the European Union. In addition, the LOD of spiked milk samples with cefalexin, cefquinoxime, cefotafur, penicillin G and ampicillin were 14.88 µg L-1, 2.46 µg L-1, 17.16 µg L-1, 0.06 µg L-1, 0.21 µg L-1 and the limits of quantitation (LOQ) were 36.09 µg L-1, 5.40 µg L-1, 41.45 µg L-1, 0.13 µg L-1, 0.42 µg L-1, respectively. The sensor showed a favorable recovery of 84.89-102.44%. Moreover, the electrochemical receptor sensor was successfully applied to assay ß-lactam antibiotics in milk, which showed good correlation with the results obtained from liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Establishment of pressurized liquid extraction followed by HPLC-MS/MS method for the screening of adrenergic drugs, steroids, sedatives, colorants and antioxidants in swine feed.

A facile and sensitive multi-residue detection approach of pressurized liquid extraction following high-performance liquid chromatography tandem mass spectrometry was established to detect the residues of adrenergic drugs, steroids, sedative, colorant and antioxidant in feed. The conditions employed for pressurized liquid extraction involved acetonitrile/ethyl acetate (1:1, v/v) as the extracting solvent, the temperature 80°C, two cycles and a static time of 10 min. The extraction was followed by a solid-phase extraction clean-up step. The separation of samples was done by C18 column with the mobile phase of 5 mM ammonium acetate solution and acetonitrile with 0.1% formic acid. The limits of quantification ranged from 0.03 to 1 µg/kg, limits of detection were in a range of 0.01-0.5 µg/kg, and average recoveries were 70.4-98.6%. The pressurized liquid extraction procedure was optimized and overall method was validated in terms of sensitivity, linearity, selectivity, matrix effect, accuracy, recovery and stability of the target drugs in the pressurized liquid extraction extracts solution. The screening method was proved to be fast, selective, accurate and sensitive for screening drugs.

Simultaneous determination of multicomponent of acetylkitasamycin and kitasamycin by LC-MS/MS in swine plasma and its application in a pharmacokinetic study.

A simple and reliable LC-MS/MS method was established for simultaneous determination of 12 components from acetylkitasamycin and kitasamycin in swine plasma. The analytes were separated on a Shim-pack VP-ODS column with a 25 min gradient elution using 5 mmol/L ammonium acetate and acetonitrile as the mobile phase at a flow rate of 0.2 mL/min. Identification and quantification were accomplished by electrospray ionization) in positive mode using multiple reaction monitoring. The limits of quantitation of acetylkitasamycin A1 A3 , A13 and kitasamycin A3 , A13 were 3 µg/L, and that of the other eight components was 5 µg/L. The mean recoveries of kitasamycin and acetylkitasamycin ranged from 85.3 to 103.5%. The developed method was successfully applied to a pharmacokinetic study in swine after intravenous (i.v.) and oral (p.o.) administration of acetylkitasamycin. The result showed that the plasma concentrations of acetylkitsamycin components were much higher than that of kitasamycin in swine after i.v. and p.o., in which acetylkitsamycin A4 A5 was the highest component at each time point.

Construction of Electrochemical Immunosensor Based on Gold-Nanoparticles/Carbon Nanotubes/Chitosan for Sensitive Determination of T-2 Toxin in Feed and Swine Meat.

T-2 toxin (T-2) is one of major concern mycotoxins acknowledged as an unavoidable contaminant in human foods, animal feeds and also agriculture products. Thus, a facile and sensitive method is essential for accurate T-2 toxin detection. In our work, a specific electrochemical immunosensor based on gold nanoparticles/carboxylic group-functionalized single-walled carbon nanotubes/chitosan (AuNPs/cSWNTs/CS) composite was established. The mechanism of the electrochemical immunosensor was an indirect competitive binding to a given amount of anti-T-2 between free T-2 and T-2-bovine serum albumin, which was conjugated on covalently functionalized cSWNTs decorated on the glass carbon electrode. Afterwards, the alkaline phosphatase labeled anti-mouse secondary antibody was bound to the electrode surface by reacting with the primary antibody. Lastly, alkaline phosphatase catalyzed the hydrolysis of the substrate α-naphthyl phosphate, which produced an electrochemical signal. Compared with conventional methods, the established immunosensor was more sensitive and simpler. Under optimal conditions, this method could quantitatively detect T-2 from 0.01 to 100 µg·L-1 with a detection limit of 0.13 µg·L-1 and favorable recovery 91.42⁻102.49%. Moreover, the immunosensor was successfully applied to assay T-2 in feed and swine meat, which showed good correlation with the results obtained from liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Magnetic solid-phase extraction based on carbon nanotubes for the determination of polyether antibiotic and s-triazine drug residues in animal food with LC-MS/MS.

Carbon nanotubes-magnetic nanoparticles, comprising ferroferric oxide nanoparticles and carbon nanotubes, were prepared through a simple one-step synthesis method and subsequently applied to magnetic solid-phase extraction for the determination of polyether antibiotic and s-triazine drug residues in animal food coupled with liquid chromatography with tandem mass spectrometry. The nanocomposites were characterized by transmission electron microscopy, X-ray diffraction, and vibrating sample magnetometry. The components within the nanocomposites endowed the material with high extraction performance and manipulative convenience. Compared with carbon nanotubes, the as-prepared carbon nanotubes-magnetic nanoparticles showed better extraction and separation efficiencies for polyether antibiotics and s-triazine drugs thanks to the contribution of the iron-containing magnetic nanoparticles. Various experimental parameters affecting the extraction efficiency had been investigated in detail. Under the optimal conditions, the good linearity ranging from 1 to 200 µg/kg for diclazuril, toltrazuril, toltrazuril sulfone, lasalocid, monensin, salinomycin, narasin, nanchangmycin, and maduramicin, low limits of detection ranging from 1 to 5 µg/kg, and satisfactory spiked recoveries (77.1-91.2%, with the inter relative standard deviation values from 4.0 to 12.2%) were shown. It was confirmed that this novel method was an efficient pretreatment and enrichment procedure and could be successfully applied for extraction and determination of polyether and s-triazine drug residues in complex matrices.

Aqueous two-phase system (ATPS): an overview and advances in its applications.

Aqueous two-phase system (ATPS) is a liquid-liquid fractionation technique and has gained an interest because of great potential for the extraction, separation, purification and enrichment of proteins, membranes, viruses, enzymes, nucleic acids and other biomolecules both in industry and academia. Although, the partition behavior involved in the method is complex and difficult to predict. Current research shows that it has also been successfully used in the detection of veterinary drug residues in food, separation of precious metals, sewage treatment and a variety of other purposes. The ATPS is able to give high recovery yield and is easily to scale up. It is also very economic and environment friendly method. The aim of this review is to overview the basics of ATPS, optimization and its applications.

Synthesis, 3D-QSAR analysis and biological evaluation of quinoxaline 1,4-di-N-oxide derivatives as antituberculosis agents.

A series of quinoxaline 1,4-di-N-oxide derivatives variously substituted at C-2 position were synthesized and evaluated for in vitro antimycobacterial activity. Seventeen compounds exhibited potential activity (MIC ⩽6.25µg/mL) against Mycobacterium tuberculosis (H37Rv), in particular the compounds 3d and 3j having an MIC value of 0.39µg/mL. None of the compounds exhibited cytotoxicity when using an MTT assay in VERO cells. To further investigate the structure-activity relationship, CoMFA (q(2)=0.507, r(2)=0.923) and CoMSIA (q(2)=0.665, r(2)=0.977) models were performed on the basis of antimycobacterial activity data. The 3D-QSAR study of these compounds can provide useful information for further rational design of novel quinoxaline 1,4-di-N-oxides for treatment of tuberculosis.

Targeted analysis and determination of ß-agonists, hormones, glucocorticoid and psychiatric drugs in feed by liquid chromatography with electrospray ionization tandem mass spectrometry.

A comprehensive strategy combining a quantitative method was developed for 30 banned drugs including ß-agonists, hormones, glucocorticoid and psychiatric drugs in swine and chicken feeds. This rapid, simple and effective extraction method was based on matrix solid-phase dispersion and electrospray ionization tandem mass spectrometry. The quantitative method was validated after previous statistical optimization of the main parameters of matrix solid-phase dispersion. The limit of quantification of dopamine hydrochloride, chlormadinone acetate, melengestrol acetate, testosterone propionate, nandrolone and midazolam was 2 µg/kg and that of the other 24 drugs was 1 µg/kg. The recoveries of ß-agonists, hormones, glucocorticoid and psychiatric drugs spiked in swine and chicken feeds at a concentration range of 1-8 µg/kg were above 70.1% with inter-day relative standard deviations less than 15.8%. The analytical strategy was applied to 100 feed samples collected from a local market in Wuhan (China). Clenbuterol, ractopamine and melengestrol acetate were identified and quantified at the level 0.2∼3.5 µg/kg. The rapid and reliable method can be used to efficiently separate, characterize and quantify the residues of 30 banned drugs in swine and chicken feeds with advantages of simple pretreatment and environmental friendly nature.

Multiclass method for the quantification of 92 veterinary antimicrobial drugs in livestock excreta, wastewater, and surface water by liquid chromatography with tandem mass spectrometry.

A simple multiresidue method was developed for detecting and quantifying 92 veterinary antimicrobial drugs from eight classes (ß-lactams, quinolones, sulfonamides, tetracyclines, lincomycins, macrolides, chloramphenicols, and pleuromutilin) in livestock excreta and water by liquid chromatography with tandem mass spectrometry. The feces samples were extracted by ultrasound-assisted extraction with a mixture of acetonitrile/water (80:20, v/v) and edetate disodium, followed by a cleanup using solid-phase extraction with an amino cartridge. Water samples were purified with hydrophilic-lipophilic balance solid-phase extraction column. Urine samples were extracted with acetonitrile and edetate disodium. Detection of veterinary antimicrobial drugs was achieved by liquid chromatography with tandem mass spectrometry using both positive and negative electrospray ionization mode. The recovery values of veterinary antimicrobial drugs in feces, urine, and water samples were 75-99, 85-110, and 85-101% and associated relative standard deviations were less than 15, 10, and 8%, respectively. The limits of quantification in feces, urine, and water samples were 0.5-1, 0.5-1, and 0.01-0.05 µg/L, respectively. This method was applied to determine real samples obtained from local farms and provides reliable quantification and identification results of 92 veterinary antimicrobial drugs in livestock excreta and water.

A high throughput method for rapid screening of chitosanase-producing fungal strain under acidic conditions.

A novel high-throughput method was established for rapid screening of a large numbers of Aspergillus fumigatus (A. fumigatus) mutants with high chitosanase production under acidic culture condition by exploiting the fact that iodine can be used as the indicator to stain chitosan but is ineffective for chitooligosaccharides. The mutant population was generated by irradiating A. fumigatus CICC 2434 with Co(60)-γ rays. Mutants were cultured on acidic plates containing colloidal chitosan and preliminary screened according to diameter of haloes formed around colonies. Then, chitosanase production of the isolates were verified by dinitrosalicylic acid assay. Lastly, molecular masses on enzymolysis products of isolated mutants were rapidly compared by aniline blue plate assay. Using this method, the mutant strain Co-8 was selected, which had chitosanase activity of 24.87 U/mL (increased by 369.2 % as compared to that of its parental strain).Taking together, the method is easy, efficient and particularly suited to rapid screen acidophilic fungal strains with high chitosanase-production.

Development of a novel genetically modified bioluminescent-bacteria-based assay for detection of fluoroquinolones in animal-derived foods.

Fluoroquinolones (FQNs) are broad-spectrum antibacterial agents widely used in animal husbandry and aquaculture. The residues and antimicrobial resistance of such antibiotics are a major public health concern. To realize multianalyte detection of FQN residues, a genetically modified bacterium, Escherichia coli pK12 harboring plasmid pRecAlux3, was constructed in this study to develop a bioluminescent-bacteria-based assay for the detection of FQNs in animal-derived foods. This assay was based on the principle of induction of an SOS response by FQNs via inducing the recA-promoter-fused luciferase reporter gene existing on the plasmid pRecAlux3. E. coli pK12 was able to recognize 11 FQNs: difloxacin, enrofloxacin, ciprofloxacin, sarafloxacin, norfloxacin, danofloxacin, ofloxacin, pefloxacin, lomefloxacin, marbofloxacin, and orbifloxacin. This method could be applied to 11 edible tissues, including milk, fish muscle, and the muscles, livers, and kidneys of cattle, chickens, and pigs, with a very simple and rapid sample extraction procedure using only phosphate-buffered saline. The limits of detection of the FQNs were between 12.5 and 100 µg kg(-1), all of which were lower than the maximum residue limits. Most of the recoveries of the FQNs were in the range from 60 to 120 %, and the interassay coefficients of variation were less than 30 %. This method, confirmed by high-performance liquid chromatography, is reliable and can be used as both a screening test and a semiquantitative assay, when the identity of a single type of FQN is known.