Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection

A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively.     

Determination of chloramphenicol,thiamphenicol, florfenicol,and florfenicol amine in poultry and porcine muscle and liver by gas chromatography-negative chemical ionization mass spectrometry

A sensitive and reliable method using gas chromatography-negative chemical ionization mass spectrometry (GC-NCI/MS) was developed for the simultaneous determination of chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) at trace levels in muscle and liver. Before extraction with ethyl acetate, CAP-d₅ was added to tissue samples as internal standard. The organic extracts were frozen to remove lipid and further purified by liquid–liquid extraction (LLE) with hexane and solid-phase extraction (SPE) using Oasis HLB cartridges. The target compounds were derivatized with BSTFA + 1% TMCS prior to GC-NCI/MS determination in selected ion monitoring mode (SIM). The recovery values ranged from 78.5 to 105.5%, with relative standard deviations (RSD) <17%. The limits of detections (LODs) of 0.1 μg/kg for CAP and 0.5 μg/kg for TAP, FF, and FFA were obtain. Incurred sample and samples from local market were successfully analyzed using this method.     

Simultaneous determination of thiamphenicol,florfenicol and florfenicol amine in swine muscle by liquid chromatography–tandem mass spectrometry with immunoaffinity chromatography clean-up

A rapid and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method to quantify thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in swine muscle is described. An immunoaffinity chromatography (IAC) column based on polyclonal antibodies and protein A-sepharose CL 4B was used to clean-up extracted samples. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of TAP, FF, and FFA. The dynamic column capacity was more than 512 ng/mL of gel after being used for 15 cycles. From fortified swine muscle samples at levels of 0.4–50 ng/g, the average recoveries were 85.2–98.9% with intra- and inter-day variations less than 9.8% and 12.4%, respectively. The limit of quantitation ranged from 0.4 to 4.0 μg/kg.     

分散固相萃取剂-液相色谱串联质谱法测定鸡肉和鸡蛋中氟苯尼考和氟苯尼考胺残留

建立分散固相萃取剂-液相色谱串联质谱法(HPLC-MS/MS)同时检测鸡肉及鸡蛋中氟苯尼考和氟苯尼考胺的方法.样品用乙腈提取,C18分散固相萃取填料净化,乙腈饱和的正己烷脱脂,电喷雾离子源正负模式切换,HPLC-MS/MS多反应监测(MRM),同位素内标法定量.氟苯尼考和氟苯尼考胺线性范围分别为0.1 ng/mL～2....     

不同水温下氟甲砜霉素在斑点叉尾鮰体内的药代动力学研究

研究不同水温(18℃和28℃)条件下,单剂量(10mg/kgb·w)强饲氟甲砜霉素,在斑点叉尾鮰(Ictaluruspunc-tatus)体内药代动力学特征.采用高效液相色谱紫外检测法可以同时检测血浆中氟甲砜霉素及其代谢物氟甲砜霉素的浓度.用3p97药代动力学软件处理药时数据.结果表明:在不同水温条件下氟甲砜霉素在斑点叉尾鮰体内的药时数据均符合一室开放式模型.药时规律符合理论方程C血浆=71921(e-0.036t-e-0.18t)和C血浆=91061(e-0.081t-e-0.301t).18℃和28℃的条件下,主要药代动力学参数:吸收半衰期T1/2ka分别为31845h和21301h,消除半衰期T1/2ke分别为191118h和81519h,达峰时间Tpeak分别为111136h和51953h,最大血药浓度Cmax分别为41074μg/mL和41226μg/mL,曲线下面积AUC分别为1741547(μg/mL)/h和811279(μg/mL)/h,平均驻留时间MRT分别为271581h和121290h,相对表观分布容积V/F(c)分别为11580L/kg和115121L/kg.采用氟甲砜霉素防治斑点叉尾鮰细菌性疾病,建议在18℃左右口服10mg/kg体重剂量的氟甲砜霉素,2d给药1次;在28℃左右口服10mg/kg体重剂量的氟甲砜霉素,1d给药1次.试验过程中在斑点叉尾鮰血浆样品中未检测到氟甲砜霉素的主要代谢物氟甲砜霉素胺.       

Pharmacokinetics of florfenicol in cod Gadus morhua and in vitro antibacterial activity against Vibrio anguillarum

The pharmacokinetic profile of the antibacterial agent florfenicol was studied in plasma after intravenous (i.v.) injection and in plasma, muscle and liver following oral (p.o.) administration to cod Gadus morhua, held in seawater at 8 degrees C and weighing 100 to 200 g. Following i.v. injection, the plasma drug concentration-time profile showed 2 distinct phases. The plasma distribution half-life (t1/2alpha) was estimated to be 1.6 h, the elimination half-life (t1/2beta) to be 43 h, the total body clearance (ClT) to be 0.015 1 kg(-1) h(-1) and mean residence time (MRT) to be 74 h. The volume of distribution at steady state, Vd(ss), was calculated to be 1.1 l kg(-1). Following p.o. administration, the bioavailability was estimated to be 91%, the peak plasma concentrations (Cmax) to be 10.8 microg ml(-1) and the time to peak plasma concentrations (Tmax) to be 7 h. Corresponding Cmax and Tmax values were 13.0 microg g(-1) and 9 h, respectively, in muscle and 12.1 microg g(-1) and 9 h, respectively, in liver. The in vitro minimum inhibitory concentration (MIC) values of florfenicol against 3 Vibrio anguillarum strains isolated from diseased cod (A-21, HI-610, HI-618) were 0.5 microg ml(-1) for all 3 strains.     

Simultaneous determination of seven nitroimidazole residues in meat by using HPLC-UV detection with solid-phase extraction

A method was developed for the determination of the seven nitroimidazoles including metronidazole (MNZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole (TNZ), ornidazole (ONZ), secnidazole (SNZ) and the common metabolite of RNZ and hydroxydimetridazole (DMOHZ) in poultry and pork muscles by high-performance liquid chromatography (HPLC) with ultraviolet detection (UV). After extraction with ethyl acetate and evaporation, the nitroimidazoles were redissolved in ethyl acetate and purified using strong cation exchange (SCX) solid-phase extraction (SPE) column. The HPLC separation was carried through on a C(18) bonded silica column with a deionized water-methanol-acetonitrile mobile phase using a gradient elution procedure. The limit of detection of all the seven nitroimidazoles was 0.2 microg/kg. The recoveries of the seven nitroimidazoles for chicken, pork and bacon samples spiked with 1-20 microg/kg were in the range of 71.4-99.5%. The linearity is satisfactory with a correlation coefficient of >0.998 at concentrations ranging from 0.7 to 60 microg/kg. The relative standard deviations of 10 measurements for spiked chicken, pork and bacon samples at the concentration of 1 and 20 microg/kg were in the range of 6.2-13.9% and 4.0-8.7%, respectively. The intra-day precision (n=5) for nitroimidazoles residues in chicken spiked at 20 microg/kg is 6.9%, and the inter-day precision for 5 days (n=25) is 11%. The method is capable of identifying nitroimidazole residues at > or =0.7 microg/kg levels and was applied in the determination of nitroimidazole residues in meat sample.     

High-performance liquid chromatographic method with fluorescence detection for the screening and quantification of oxolinic acid,flumequine and sarafloxacin in fish

A previously published liquid chromatographic method for determining residues of nine quinolones in chicken, porcine, bovine and ovine muscle was adapted and applied to fish tissue for simultaneous determination of three quinolones (flumequine, oxolinic acid and sarafloxacin). The analytes were extracted from homogenised muscle using an acetonitrile basic solution. After centrifugation, partial evaporation and cleaning with hexane, direct injection was possible. Separation was achieved on PLRP-S column and detection was performed with a programmable fluorescence detector. Chromatographic conditions were optimised to be compatible with the determination of the three quinolones in a single run. The linearity, recovery, accuracy and precision of the method were evaluated from fortified tissue samples at concentration levels ranging from 15 to 120 microg kg(-1) for sarafloxacin and 75 to 600 microg kg(-1) for oxolinic acid and flumequine according to the EU maximum residue limit of each quinolone. The limits of detection were estimated to be 2, 5 and 7 microg kg(-1), respectively, for sarafloxacin, oxolinic acid and flumequine. The limits of quantification were validated at 15 microg kg(-1) for sarafloxacin and 75 microg kg(-1) for oxolinic acid and flumequine. Mean extraction recoveries of quinolones in fish ranged from 56.9 to 71.0%. This simple and rapid method is suitable for residue control.     

Enzyme-linked immunosorbent assay of chlorampenicol in foodstuff

A test-system based on enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of chloramphenicol (CAP) in foodstuff has been developed. The detection limit of the method was 0.05 μg/l. The procedures for milk samples preparation of various fat content and chicken muscles were optimized. Before the analysis milk was diluted 5-fold with a buffer. The detection limit for milk was 0.3 μg/l; recoveries varied from 74 to 118%. Two protocols for chicken muscles preparation were elaborated; extraction with buffer (the express method) and extraction with acetonitrile. The detection limits of CAP in chicken muscles were 0.5 and 0.3 μg/kg, respectively; recovery values were 71–107% and 95–115%, respectively. The results of residual amounts of CAP detection in foodstuff by ELISA and HPLC-MS were in good correlation.     

High-performance liquid chromatographic determination of gossypol and gossypolone enantiomers in fish tissues using simultaneous electrochemical and ultraviolet detectors

This paper describes a method for residue analysis of difloxacin and sarafloxacin in chicken muscle. Clean-up and preconcentration of the samples are effected by solid-phase extraction (C18) and the determination is carried out by capillary electrophoresis using a photodiode array detection system. The method was validated with satisfying results. The calibration graphs are linear for difloxacin and sarafloxacin from 50 to 300 microg/kg. The limit of detection obtained for difloxacin and sarafloxacin are 10 and 25 microg/kg, respectively, which allows the detection of positive muscle samples at the required maximum residue limits of European Union.     

Liquid chromatographic determination of florfenicol in the plasma of multiple species of fish

A simple method was developed for determining florfenicol concentration in a small volume (250 micro l) of plasma from five phylogenetically diverse species of freshwater fish. Florfenicol was isolated from the plasma matrix through C(18) solid-phase extraction and quantified by reversed-phase high-performance liquid chromatography with UV detection. The accuracy (84-104%), precision (%RSD相似文献   

Validation of an HPLC-UV method according to the European Union Decision 2002/657/EC for the simultaneous determination of 10 quinolones in chicken muscle and egg yolk

Herein two different methods are proposed for the determination of 10 quinolones (enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, oxolinic acid, nalidixic acid and flumequine) in chicken muscle and egg yolk. Two different HPLC systems were used comparatively and the respective methods were fully validated. The analytes were initially extracted from chicken muscle and egg yolk and purified by a solid phase extraction using LiChrolut RP-18 cartridges. Recoveries varied between 96.6 and 102.8% for chicken muscle and 96.4-102.8% for egg yolk. HPLC separation was performed at 25 degrees C using an ODS-3 PerfectSilTarget (250 mmx4 mm) 5 microm analytical column (MZ-Analysentechnik, Germany). The mobile phase consisted of a mixture of 0.1% trifluoroacetic acid (TFA)-ACN-CH3OH, delivered by a gradient program, different for each method. In both cases caffeine was used as internal standard at the concentration of 7.5 ng/microL. Column effluent was monitored using a photodiode array detector, set at 275 and 255 nm. The developed methods were validated according to the criteria of Commission Decision 2002/657/EC. The LODs for chicken muscle varied between 5.0 and 12.0 microg/kg and for egg yolk was 8.0 microg/kg for all examined analytes.     

Use of florfenicol in non-human primates

Studies were undertaken to determine if florfenicol, an antimicrobial agent structurally similar to chloramphenicol, could be used as an effective broad spectrum antibiotic for the treatment of bacterial infections in primates. Florfenicol was developed as an injectable antibiotic for use in cattle on an every other day dosing schedule. Its broad spectrum activity, long duration of action following i.m. administration, and its safety as compared with chloramphenicol made it an attractive antibiotic for use in non-human primates. Previous studies had shown that florfenicol is effective against common primate pathogens such as Salmonella, Klebsiella, Escherichia coli, Bordetella bronchiseptica, Streptococcus pneumoniae, Staphylococcus spp., and Yersinia pseudotuberculosis. We performed experiments on a total of 15 macaques. The animals were given florfenicol at 50 mg/kg i.m. and blood samples taken at various time points. Serum was evaluated for florfenicol absorption. Necropsies were also performed to determine if major organs were affected and to determine the effects of i.m. injection of florfenicol. We determined that florfenicol given every 48 hours in rhesus macaques results in blood levels that were acceptable for therapeutic use. The effect on muscle tissue of i.m. injection was similar to ketamine and normal saline. There were no gross lesions observed and no changes with tissues submitted for histology. Our work shows that with further studies, florfenicol may be useful when injectable antibiotic therapy is required in non-human primates.     

环丙沙星与新氟康杀灭鳗弧菌及其影响因素研究

对环丙沙星与新氟康杀灭鳗弧菌及其影响因素进行了研究,结果表明：环丙沙星（Ciproflozacin）和新氟康（Florfenicol）杀灭鳗弧菌的最高稀释度分别为10^-5和10^-4。在20℃～50℃的范围里,环丙沙星与新氟康杀灭鳗弧菌的效果随着温度升高而下降;杀菌最佳pH值分别为3和5;两种药品杀灭鳗弧菌的效果均随着可溶性淀粉浓度的增加而减弱。综合各最佳因素,环丙沙星在20℃和pH3时,杀灭鳗弧菌效果提高了19．13％;新氟康在20℃和pH5时,杀灭鳗弧菌效果提高了59．47％。     

Liquid chromatography analysis of enrofloxacin and ciprofloxacin in chicken blood spotted on filter-paper disks

A simple, low-cost, sensitive and selective LC method was developed for the determination of enrofloxacin and ciprofloxacin in chicken blood. The method was applied to whole blood from a chicken using dried blood spots on filter paper disks. The detection limits of enrofloxacin and ciprofloxacin (100 microl of whole blood on a disk) were 0.005 and 0.01 microg/ml, respectively. The whole procedure was verified in intra-laboratory studies (recoveries of both compounds were above 90%), and its applicability was tested with blood from the chicken receiving enrofloxacin in a single oral dose at a level of 10 mg/kg body mass. The method permits the use of a small volume of blood from a chicken and should be useful for pharmacokinetic studies.     

Effects of high fat provision on muscle PDH activation and malonyl-CoA content in moderate exercise.

This study examined the effects of elevated free fatty acid (FFA) provision on the regulation of pyruvate dehydrogenase (PDH) activity and malonyl-CoA (M-CoA) content in human skeletal muscle during moderate-intensity exercise. Seven men rested for 30 min and cycled for 10 min at 40% and 10 min at 65% of maximal O(2) uptake while being infused with either Intralipid and heparin (Int) or saline (control). Muscle biopsies were taken at 0, 1 (rest-to-exercise transition), 10, and 20 min. Exercise plasma FFA were elevated (0.99 +/- 0.11 vs. 0.33 +/- 0.03 mM), and the respiratory exchange ratio was reduced during Int (0.87 +/- 0.02) vs. control (0.91 +/- 0.01). PDH activation was lower during Int at 1 min (1.33 +/- 0.19 vs. 2.07 +/- 0.14 mmol. min(-1). kg(-1) wet muscle) and throughout exercise. Muscle pyruvate was reduced during Int at rest [0.17 +/- 0.03 vs. 0.25 +/- 0.03 mmol/kg dry muscle (dm)] but increased above control during exercise. NADH was higher during Int vs. control at rest and 1 min of exercise (0.122 +/- 0.016 vs. 0.102 +/- 0.005 and 0.182 +/- 0.016 vs. 0.150 +/- 0.016 mmol/kg dm), but not at 10 and 20 min. M-CoA was lower during Int vs. control at rest and 20 min of exercise (1.12 +/- 0.22 vs. 1.43 +/- 0.17 and 1.33 +/- 0.16 vs. 1.84 +/- 0.17 micromol/kg dm). The reduced PDH activation with elevated FFA during the rest-to-exercise transition was related to higher mitochondrial NADH at rest and 1 min of exercise and lower muscle pyruvate at rest. The decreased M-CoA may have increased fat oxidation during exercise with elevated FFA by reducing carnitine palmitoyltransferase I inhibition and increasing mitochondrial FFA transport.     

Development and validation of a method for the confirmation of halofuginone in chicken liver and eggs using electrospray tandem mass spectrometry

A method is described for the quantitative confirmation of halofuginone (HFG) residues in chicken liver and eggs. This method is based on LC coupled to positive ion electrospray MS-MS of the tissue extracts, prepared by trypsin digestion of the tissues followed by liquid-liquid extraction and final clean-up using Solid Phase Extraction (SPE). The [M+H](+) ion at m/z 416 is monitored along with four transitions at m/z 398, 138, 120 and 100. The method has been validated according to the draft EU criteria for the analysis of veterinary drug residues at 15, 30 and 45 microg kg(-1) in liver and 5, 15 and 50 microg kg(-1) in eggs. The new analytical limits, CCalpha and CCbeta were calculated for liver and were 35.4 and 43.6 microg kg(-1), respectively.     

Simultaneous determination of fluoroquinolones and tetracyclines in chicken muscle using HPLC with fluorescence detection

A multiresidue method has been developed which allows for the simultaneous determination of both fluoroquinolones and tetracyclines in chicken muscle. Samples were extracted with a mix of acetonitrile and 0.1 M citrate, 150 mM MgCl(2), pH 5.0. After centrifugation and evaporation, the extracts could be analyzed by liquid chromatography with fluorescence detection. Good recoveries (63-95%) were obtained from samples fortified with a mix of five fluoroquinolones and three tetracyclines, with satisfactory relative standard deviations. Limits of detection were 0.5 ng/g (danofloxacin), 1 ng/g (oxytetracycline, ciprofloxacin, enrofloxacin), 1.5 ng/g (tetracycline), 2 ng/g (difloxacin) and 5 ng/g (sarafloxacin, chlortetracycline). Enrofloxacin and its metabolite ciprofloxacin, as well as oxytetracycline were determined in enrofloxacin and oxytetracycline incurred chicken muscle using this method.     

Determination of sodium nifurstyrenate and nitrovin residues in edible food by liquid chromatography-tandem mass spectrometry after ultrasound-assisted extraction

A specific and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of nitrovin and sodium nifurstyrenate residues in muscle and liver of swine and chicken and in muscle of fish. Sample preparation procedure includes ultrasound-assisted extraction with acetonitrile, defatting with n-hexane and final clean-up with solid phase extraction (SPE) on Oasis HLB cartridges. The analytes were detected in multiple reaction monitoring (MRM) under negative scan mode acquiring two diagnostic product ions for sodium nifurstyrenate and under positive mode for nitrovin. The averaged decision limits (CCα; α 1%) ranged 0.09-0.26 μg/kg while the detection capability (CCβ; β 5%) was 0.33-0.97 μg/kg in the tissues. Reasonable recoveries (71-110%) spiked in muscle and liver showed excellent relative standard deviation (RSD). The validated method was simple, rapid, sensitive, and complied with the regulations for the determination of nitrovin and sodium nifurstyrenate residues in food matrices.     

Effects of chloramphenicol on brain energy metabolism using 31P spectroscopy: influences on sleep-wake states in rat

Effects of chloramphenicol (antibiotic inhibiting complex-1 of respiratory chain) and thioamphenicol (TAP, a structural analog of CAP inactive on complex-1) were examined on cerebral energy metabolites and sleep-wake cycle architecture in rat. In the first group, animals were chronically equipped with a cranial surface resonator and ³¹P spectroscopic measurements were performed using a 2 T magnetic resonance spectrometer (operating frequency 34.46 MHz). CAP administration (400 mg/kg, tail vein, light period) induced deficits in phosphocreatine (−30%, p  < 0.01) and ATP (−40%, p  < 0.01), whereas TAP (400 mg/kg) had no effect. In the second group, animals were chronically implanted with polygraphic electrodes for EEG and electromyogram recordings. CAP administered intraperitoneally at light-onset reduced rapid-eye movement (REM) sleep (−60% in the first 6 h of light period, p  < 0.01), increased waking state (+65% in the first 6 h of light period, p  < 0.01), and slightly affected slow-wave sleep (SWS). During waking state, θ and σ power bands of the EEG were, respectively, increased and decreased ( p  < 0.05). During SWS, delta power band was reinforced ( p  < 0.05), while θ, α, and σ bands were decreased ( p  < 0.05). No changes occurred during REM sleep. TAP had no effect on sleep-wake states and spectral components of the EEG. Overall, these data indicate that REM sleep occurrence is linked to an aerobic production of ATP.