A new liquid chromatographic method for routine determination of oxytetracycline marker residue in the edible tissues of farm animals

A simple, rapid, and specific ion-pair liquid chromatographic method for routine determination of the marker residue of oxytetracycline (OTC), namely OTC and 4-epi-oxytetracycline (4-epiOTC), in edible animal tissues (muscle, liver, kidney, and fat) has been developed. Minced tissue samples were acidified at pH 2.7 with 2 mol L(-1) sulfuric acid and extracted with acetonitrile. The extracts were purified by treatment with ammonium sulfate solution and concentrated into 0.1 mol L(-1) phosphoric acid. Baseline separation was carried out isocratically on a Nucleosil 100-5 C(18), 5-microm column using an acetonitrile-0.01 mol L(-1) disodium hydrogen phosphate (20:80, v/v) mobile phase that contained both positively (tetrabutylammonium) and negatively (octanesulfonate) charged pairing ions and EDTA, and was adjusted to pH 3.8. Detection was by UV at 370 nm. The method was fully validated according to Commission Decision 2002/657/EC. Overall recoveries were better than 82.6% and overall relative standard deviation was better than 6% for all the tissues examined. The good analytical characteristics of the method allowed limits of quantification as low as 30 ng g(-1) for muscle and fat and 50 ng g(-1) for liver and kidney, for both OTC and 4-epiOTC, to be realized. The method was successfully used to determine the OTC marker residue in tissues of two sheep intramuscularly administered a commercial OTC formulation.     

An efficient synthesis of the 4'-epimer of 2-fluoronoraristeromycin

The 4'-epimer of 2-fluoronoraristeromycin was synthesized by employing bis-t-butoxycarbonyl (Boc) protected 2-fluoroadenine as a superior substrate for the Mitsunobu reaction with the appropriate cyclopentenol. Unlike the unsubstituted counterpart 2-fluoroadenine, this substrate is completely soluble in THF and resulted in a very good yield in the Mitsunobu coupling reaction as well as subsequent steps.     

An improved asymmetric synthesis of malyngamide U and its 2'-epimer

An accelerated and improved asymmetric synthesis of malyngamide U (1) and its 2'-epimer (2'-epi-1) was accomplished from readily available n-hexanal, ethanolamine and (R)-(-)-carvone. The key steps involved a Johnson-Claisen rearrangement in the synthesis of an unsaturated carboxylic acid 4 and an aldol reaction in the construction of the skeleton of 1 and 2'-epi-1. There are 13 steps in the synthesis, with a 2.7% overall yield for 1 and a 0.4% yield for 2'-epi-1.     

Quantitative analysis of oxytetracycline and its 4-epimer in calf tissues by high-performance liquid chromatography combined with positive electrospray ionization mass spectrometry

Tetracycline antibiotics are commonly used in veterinary medicine because of their broad spectrum activity and cost effectiveness. Oxytetracycline (OTC) is one of the most important members of this antibiotic family. The purpose of this study was to develop and validate a method to determine OTC residues in edible tissues of calf. Extraction of OTC and its 4-epimer (4-epiOTC), in the presence of the internal standard demethylchlortetracycline (DMCTC), was performed using a liquid extraction with sodium succinate solution (pH 4.0), followed by protein removal with trichloroacetic acid and paper filtration. Further solid-phase extraction clean-up on an HLB polymeric reversed phase column was performed to obtain an extract suitable for LC-MS-MS analysis. Chromatographic separation of the internal standard, and especially OTC and its 4-epimer, was achieved on a PLRP-S polymeric reversed phase column, using a mixture of 0.001 M of oxalic acid, 0.5% (v/v) of formic acid and 3% (v/v) of tetrahydrofuran in water (mobile phase A) and tetrahydrofuran (mobile phase B) as the mobile phase, and at a column temperature of 60 degrees C. OTC and its 4-epimer could be identified using the MS-MS detection technique, and were subsequently quantified. The method has been validated according to the requirements of the EC at the MRL (maximum residue limit, 100 ng g(-1) for muscle, 300 ng g(-1) for liver and600 ng g(-1) for kidney), half the MRL and double the MRL levels, as well for OTC as for 4-epiOTC. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested (r > 0.99 and goodness of fit < 10%). Limits of quantification of half the MRLs were obtained for the analysis of OTC and 4-epiOTC in muscle, liver and kidney tissues of calf. Limits of detection ranged for both components between 0.8 and 48.2 ng g(-1). The within-day and between-day precisions, expressed as RSD values, were all below the maximum allowed RSD values calculated according to the Horwitz equation. The results for accuracy fell within the -20% to +10% range. Recoveries were between 47 and 56% for OTC, and between 52 and 62% for 4-epiOTC, depending on the tissue. The method has been successfully used for the quantitative determination of OTC and 4-epiOTC in tissue samples of calves medicated with OTC by intramuscular injection.     

Liquid chromatographic determination of para-toluenesulfonamide in edible fillet tissues from three species of fish.

Chloramine-T (N-sodium-N-chloro-p-toluene-sulfonamide) is a candidate therapeutic drug for treating bacterial gill disease, a predominant disease of a variety of fish species. Research has been initiated to obtain the U.S. Food and Drug Administration's (FDA) approval for the use of chloramine-T on a variety of fish species. An attribute of a therapeutic aquaculture drug that must be characterized before the FDA approves its use is depletion of the drug's marker residue (the drug's parent compound or metabolite of highest concentration in an edible tissue). para-Toluenesulfonamide (p-TSA) is the primary degradation product and marker residue for chloramine-T in rainbow trout. To conduct residue depletion studies for chloramine-T in fish, a robust analytical method sensitive and specific for p-TSA residues in edible fillet tissue from a variety of fish was required. Homogenized fillet tissues from rainbow trout (Oncorhynchus mykiss), walleye (Stizostedion vitreum), and channel catfish (Ictalurus punctatus) were fortified at nominal p-TSA concentrations of 17, 67, 200, 333, and 1000 ng/g. Samples were analyzed by isocratic reversed-phase liquid chromatography (LC) with absorbance detection at 226 nm. Mean recoveries of p-TSA ranged from 77 to 93.17%; relative standard deviations ranged from 1.5 to 14%; method quantitation limits ranged from 13 to 18 ng/g; and method detection limits ranged from 3.8 to 5.2 ng/g. The LC parameters produced p-TSA peaks without coelution of endogenous compounds and excluded chromatographic interference from at least 20 chemicals and drugs of potential use in aquaculture.     

Derivatization and gas chromatographic-mass spectrometric detection of anabolic steroid residues isolated from edible muscle tissues

A method was developed for the detection of anabolic steroid residues in edible muscle tissues. After enzymic digestion of the tissue and purification on disposable C18 solid-phase extraction columns, the extract was injected onto a C18 reversed-phase high-performance liquid chromatographic column. Three fractions or windows were collected, each containing specific analytes. After evaporation to dryness, the residues were subjected to a derivatization procedure which yielded suitable derivatives. After gas chromatographic-mass spectrometric analysis, both gas chromatographic retention data and mass spectral data were used for the detection and identification of nortestosterone, testosterone, estradiol, ethinylestradiol, trenbolone, methyltestosterone, chlormadinone acetate, medroxyprogesterone acetate and megestrol acetate.     

Development of liquid chromatographic methods for determination of quinocetone and its main metabolites in edible tissues of swine and chicken

Quinocetone (QCT), a new antimicrobial growth promotant of quinoxalines, can effectively improve the growth and feed efficiency of food animals with more safety than is provided by olaquindox and carbadox. To clarify its metabolism and residue levels in animals, a liquid chromatographic method with UV-Vis detection was developed for the determination of QCT and its main metabolites, desoxyquinocetone (DQCT) and 3-methylquinoxaline-2-carboxylic acid (MQCA), in muscle, liver, kidney, and fat of swine and chicken. For sample pretreatment, QCT and DQCT were extracted with ethyl acetate and purified with iso-octane; after alkaline hydrolysis of the tissue, MQCA was extracted with ethyl acetate and citric acid buffer (pH 6.0), and the extract was purified over a cation-exchange column (AG MP-50 resin). Detection was at 312 and 320 nm for QCT and DQCT, respectively, and at 320 nm for MQCA. The observed limit of detection for the 3 compounds was 0.025 microg/g in various tissues. The methods were linear over the concentrations range of 0.01-0.64 microg/mL with mean recoveries of approximately 71-86% and relative standard deviations of about 4-12% at the levels of 0.05, 0.10, and 0.20 microg/g. The method is highly selective and can be applied to the determination of QCT and its main metabolites in animal tissues, which would accelerate the pharmacokinetic and residue study of QCT in food animals.     

Determination of ciprofloxacin and enrofloxacin in edible animal tissues by terbium-sensitized luminescence.

A terbium-sensitized luminescence method is described for the determination of the sum of residues of enrofloxacin and its major metabolite ciprofloxacin in edible animal tissues. Several parameters affecting both detection and extraction were studied. Analytes were extracted from spiked samples of chicken and trout tissues with pH 7.4 buffer-dichloromethane. The organic extract was evaporated and the residue dissolved in aqueous nitric acid and defatted with hexane. Determination was carried out directly in the aqueous phase (in a micellar medium). The calibration curves were linear up to 75 micrograms l-1. The detection limit was 3.5 micrograms kg-1 (for a 5 g sample) and the repeatability was 7.0% (n = 7). The sensitivity was similar for both quinolones and therefore calibration can be carried out with either ciprofloxacin or enrofloxacin. In any case, the differences were < 10%.     

Simplified high-performance liquid chromatographic determination of residual amprolium in edible chicken tissues

A simplified determining method for the routine monitoring of residual amprolium in edible chicken tissues (muscle and liver) is developed using a high-performance liquid chromatographic (HPLC) method with a photodiode-array detector after sample cleanup by an Ultrafree-MC/PL centrifugal ultrafiltration unit. For the HPLC determination and identification, a Mightysil RP4 GP column and a mobile phase of an ethanol-5 mM 1-heptanesulfonic acid sodium salt solution (35:65, v/v) using an ion-pairing system with a photodiode-array detector are used. Average recoveries (spiked at 0.3-3.0 microg/g) are > 90%. The inter- and intravariabilities are 1.9-2.4%. The limits of quantitation are 0.22 microg/g for muscle and 0.25 microg/g for liver. The total time and solvent required for the analysis of one sample are < 20 min and < 2 mL of ethanol, respectively. No toxic solvents and regents are used.     

Simplified high-performance liquid chromatographic determination of residual amprolium in edible chicken tissues

A simplified determining method for the routine monitoring of residual amprolium in edible chicken tissues (muscle and liver) is developed using a high-performance liquid chromatographic (HPLC) method with a photodiode-array detector after sample cleanup by an Ultrafree-MC/PL centrifugal ultrafiltration unit. For the HPLC determination and identification, a Mightysil RP-4 GP column and a mobile phase of an ethanol-5 mM 1-heptanesulfonic acid sodium salt solution (35:65, v/v) using an ion-pairing system with a photodiode-array detector are used. Average recoveries (spiked at 0.3-3.0 microg/g) are > 90%. The inter- and intravariabilities are 1.9-2.4%. The limits of quantitation are 0.22 microg/g for muscle and 0.25 microg/g for liver. The total time and solvent required for the analysis of one sample are < 20 min and < 2 mL of ethanol, respectively. No toxic solvents and regents are used.     

Analysis of flumequine and oxolinic acid in edible animal tissues by LC with fluorimetric detection

Summary A simple rapid method has been developed for the determination of flumequine and oxolinic acid in edible animal tissues. Analytes are extracted from the sample matrix using dichloromethane in neutral conditions, then back-extracted in 0.01 M sodium hydroxide and determined by reversed-phase liquid chromatography with fluorimetric detection. The method has been applied to pork, salmon and chicken muscle with recoveries higher than 77% in all matrices. The detection limits achieved (<1 μg kg⁻¹) allow the determination of these drugs at a residue level far below their MRL Moreover, some variables that may affect the spiking process have been studied.     

Selection of sorbent for oxytetracycline preconcentration from solutions

The sorption of oxytetracycline has been studied in the static and dynamic mode on the different types of sorbents, such as hyper-cross-linked polystyrene, copolymer of styrene and divinylbenzene Strata SDB-L, N-vinyl-2-pyrrolidone surface-modified copolymer of styrene and divinylbenzene Strata-X, carbon nanomaterial Taunit, and diethylaminoethylcellulose. The peculiarities of oxytetracycline sorption depending on the solution acidity, oxytetracycline concentration, the nature of the solvent, and the structural characteristics of the sorbents have been explained.     

Determining the procedure for routine residue monitoring of sulfamethazine in edible animal tissues.

A simplified method to determine/identify residual sulfamethazine (SMZ) in edible tissues from cattle, pigs, chickens and sheep by a high-performance liquid chromatography (HPLC) with a photo-diode array detector is presented. The sample preparation was performed by homogenizing with 30% (v/v) ethanol in water followed by an Ultrafree-MC/PL as a centrifugal ultra-filtration unit. For determination/identification of SMZ, a reversed-phase C(4) column and a mobile phase of 15% (v/v) ethanol in water with a photo-diode array detector was used. Average recoveries from spiked SMZ (0.1-1.0 mg kg(-1)) were >or=80% with coefficients of variation between 1.3 and 4.3%. The limits of quantitation were calculated to be 0.057-0.060 mg kg(-1). The total time and solvent required for the analysis of one sample were <40 min and <2 mL of ethanol, respectively.     

Determination of residual pharmaceuticals in edible animal tissues by continuous solid-phase extraction and gas chromatography-mass spectrometry

A sensitive, reliable method using continuous solid-phase extraction and gas chromatography-mass spectrometry was developed for the simultaneous determination of twenty pharmaceuticals including antibacterials, anti-epileptics, antiseptics, β-blockers, lipid regulators, hormones and non-steroidal anti-inflammatories at trace levels in edible animal tissues. The procedure involves deproteination and delipidation of samples by precipitation/centrifugation/filtration, followed by sample enrichment and cleanup by continuous solid-phase extraction. The proposed method was validated with quite good analytical results including low limits of detections (0.4-2.7 ng kg⁻¹ for 2 g of sample) and good linearity (r² > 0.995) throughout the studied concentration ranges. In addition, the method is quite accurate (recoveries ranged from 92 to 101%) and precise (within-day and between-day RSD values were less than 7%), which allows the determination of residual pharmaceuticals in tissues from agricultural farm and fish hatchery animals (pig, veal, lamb and chicken muscle, kidney and liver; and salmon, sea bass and sole flesh). The analytes most frequently found in the studied samples were the hormones estrone and 17β-estradiol, and the antibacterials florfenicol and pyrimethamine.     

New sterols and triterpenoids from four edible mushrooms

Four edible mushrooms, Panellus serotinus, Lepista nuda, Tricholoma matsutake and Naematoloma sublateritium, have been investigated chemically. Two new sterols, 5alpha,9alpha-epidioxy-(22E)-ergosta-7,22-diene-3beta,6alpha-diol (1) and 5alpha,9alpha-epidioxy-(22E)-ergosta-7,22-diene-3beta,6beta-diol (2), have been isolated from Panellus serotinus. Compound 2 was also isolated from Lepista nuda. A new sterol, 3beta,5alpha,9alpha,14beta-tetrahydroxy-(22E)-ergosta-7,22-dien-6-one (3), and compound 2 have been isolated from Tricholoma matsutake. Three new triterpenoids, sublateriols A-C (4-6), have been isolated from Naematoloma sublateritium. The structures of the new compounds were elucidated on the basis of their spectral data.     

Determination of edible bird's nest and its products by gas chromatography

A specific gas chromatographic (GC) detection method for edible bird's nest (EBN) based on identifying the composition of the oligosaccharide chain combined with glycoprotein in EBN is developed. Five monoses (D-mannitose, D-galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, and N-acetyl neuraminate) that constitute the oligosaccharide chain are detected using GC and GC-mass spectrometry techniques; their characteristic GC spectrum can reliably be regarded as EBN's fingerprint. The peak-area ratios in GC spectrum of those five monoses are found to be fixed; therefore, the GC technique developed in this work can conveniently be used to determine various raw EBNs and their products both qualitatively and quantitatively, distinguishing between fake and genuine EBN rapidly.     

Determination of oxytetracycline residues in matrixes from a freshwater recirculating aquaculture system

This paper describes related procedures to determine the amount of oxytetracycline (OTC) present in trout tissue (muscle with skin attached), biofilter sand, sediment, and tank water from a recirculating aquaculture system. OTC was extracted from the matrixes by different techniques, depending on complexity of the matrix and desired OTC detection level in that matrix. Listed in order of increasing complexity, OTC was extracted from tank water by dilution with acidic buffer containing ethylenediaminetetraacetic acid (EDTA); from biofilter sand by shaking with 0.1 N HCl; from sediment by homogenization and shaking with buffer/EDTA; and from ground trout by homogenization and shaking with buffer/EDTA (twice), with further cleanup and concentration of the extract on a polymeric solid-phase extraction cartridge. The 4 procedures all used the same reversed-phase gradient chromatography on a polymeric column with UV detection at 350 nm. The lower limit of detection (estimated) and upper limit of validation for each of these 4 matrixes were 0.04-4.0 microg/g (ppm; trout), 0.03-20 ppm (biofilter sand), 1-6000 ppm (sediment), and 0.003-10 ppm (water). Recoveries ranged from 82 to 108%, with relative standard deviation <20% over the applicable concentration ranges. These procedures were used to monitor OTC residues resulting from medicated feed administered to rainbow trout in a recirculating aquaculture system.     

Application of ultra-performance columns in high-performance liquid chromatography for determination of albendazole and its metabolites in turkeys

Methods for determination of albendazole (ALB), albendazole sulfoxide (SOX) and albendazole sulfone (SON) in turkey blood plasma, using high‐performance liquid chromatography (HPLC) with fluorescence detection, were developed. Moreover, comparison of HPLC columns with ultra‐performance liquid chromatography (UPLC) columns was performed. Albendazol was administered orally in 5‐week‐old birds (n = 18) at a dose of 25 mg/kg b.w. Accuracy and precision of the developed method were satisfactory and stability studies showed acceptable variation (below 15%) in ALB, SOX and SON concentrations when the samples were stored at –75°C for 15 days. UPLC® columns gave higher peaks from typical HPLC columns retaining high quality of analysis. Pharmacokinetic analysis indicated quick elimination of ALB from turkey blood plasma. The mean residence time of SON was at least two times longer than that of SOX and four times longer than that of ALB. The elimination half‐lives for ALB, SOX and SON were 0.7 ± 0.27, 5.37 ± 6.03, 9.17 ± 5.12 h, respectively. The obtained results indicate that the described method allows for precise determination of albendazole and its metabolites in turkey plasma. Moreover, using UPLC columns in HPLC apparatus results in higher sensitivity as compared with the classical HPLC columns. Copyright © 2011 John Wiley & Sons, Ltd.