Jan Zmudzki

Sample preparation for residue determination of gentamicin and neomycin by liquid chromatography

The effect of sample preparation on the determination of gentamicin and neomycin residues in animal tissues was investigated. The extract was mixed with an ion-pair reagent and applied to an octadecyl cartridge. The cartridges were washed with buffer followed by water, and analytes were eluted with ion-pair buffer-acetonitrile mixture. The aminoglycosides were derivatized with 9-fluorenylmethyl chloroformate prior to liquid chromatography using a reversed-phase column and fluorescence detection. Under the conditions applied neomycin was fully separated from all the gentamicin compounds. The highest recoveries of gentamicin and neomycin from spiked tissues were obtained using trichloroacetic acid after initial extraction with phosphate-buffered saline. No interfering peaks from endogenous compounds of matrix were noted at the elution position of the analytes. An intra-laboratory validation of the whole procedure was performed. The calibration graphs were linear from 0.1 to 1.0 mg/kg for gentamicin, and from 0.2 to 1.0 mg/kg for neomycin. Limits of detection were 0.05 mg/kg and 0.10 mg/kg for gentamicin and neomycin, respectively. Limits of quantitation for gentamicin and neomycin were 0.1 and 0.20 mg/kg muscle, liver or kidney tissue, respectively. Recoveries of gentamicin spiked at levels of 0.1 mg/kg porcine tissues ranged from 76 to 86%. Recoveries of neomycin spiked at levels of 0.2 mg/kg porcine tissues ranged from 77 to 83%. The validated procedure was used to determine gentamicin concentrations in porcine tissue after dosing with gentamicin at a level of 5 mg/kg body mass.

Evaluation of sample preparation for control of chloramphenicol residues in porcine tissues by enzyme-linked immunosorbent assay and liquid chromatography

The effects of sample preparation on the determination of chloramphenicol (CAP) residues by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography (LC) were investigated. First, the biological matrices were treated with extraction solutions, which were mixtures of acetate buffer and acetonitrile. The isolation of CAP from previously spiked samples was performed by homogenization or ultrasonication. For clean-up, the suitability of solid phase extraction columns (in reverse phase (RP) or mixed mode) was compared. The level of CAP in tested samples was determined by a commercial ELISA test (R-Biopharm) and LC with UV detection at 270 nm, after separation on a RP column. It was found that the best isolation from interfering, endogenous compounds was obtained after homogenization of sample with a mixture of acetate buffer (pH 5.0) and acetonitrile (2:8, v/v). The clean-up of the sample extracts in a mixed mode rather than reverse phase was more efficient. The recovery of CAP from spiked pig muscle was 82%, and from kidney, 85%. The same analytical protocol is suitable for CAP screening in animal tissues by ELISA as well as by LC.

Screening procedures for clenbuterol residue determination in bovine urine and liver matrices using enzyme-linked immunosorbent assay and liquid chromatography

Abstract The suitability of analytical protocols for simultaneous determination of clenbuterol (CL) by ELISA and liquid chromatography (LC) was investigated. Clenbuterol spiked bovine urine and liver samples were treated with aqueous and organic solutions followed by clean-up by solid phase extraction (SPE). The determination of clenbuterol in prepared solutions was performed by a commercial ELISA test (R-Biofarm) and by LC with UV detection. The optimal conditions for liver were obtained when CL was isolated by a mixture of ethyl acetate–isopropanol (7:3 (v/v)) and purified by a mixed mode SPE column. However, additional purification with perchloric acid should be used to decrease the interference from matrices. For urine samples, the optimal conditions were obtained after dilution of the sample with borate buffer (pH 8.5), and clean-up by mixed mode SPE. The recoveries of CL were >80% from both matrices. The procedures were useful for clenbuterol screening by ELISA as well as by LC.

Determination of malachite green and leucomalachite green residues in water using liquid chromatography with visible and fluorescence detection and confirmation by tandem mass spectrometry.

A liquid chromatography with visible and fluorescence detection (LC-vis/FLD) method for screening and a liquid chromatography with mass spectrometry (LC-MS/MS) method for the confirmation of malachite green (MG) and its major metabolite, leucomalachite green (LMG) residues in water have been described. Water samples were preconcentrated on diol solid-phase extraction columns. Chromatographic separation was achieved by using phenyl-hexyl column with an isocratic mobile phase consisting of acetonitrile and acetate buffer (0.05M, pH 4.5) (70:30, v/v). In screening method liquid chromatography with absorbance detector was used for the detection of MG while LMG was detected by fluorescence detector. Detectors were connected on-line that allowed direct analysis of a sample extract for MG and LMG without the need of any post-column procedure. For the confirmation of MG and LMG in water positive electrospray ionization mass spectrometry in the multiple reaction monitoring mode was used. The developed methods have been validated according to the European Union requirements (Commission Decision 2002/657/EC). For LC-vis/FLD the mean recoveries at three fortification levels (0.4, 1, and 2microgl(-1)) were in the range 95.4-104.7% for MG and 62.2-81.9% for LMG, whereas for LC-MS/MS, recoveries of MG and LMG were in the range 96.9-101.3% and 97.5-104.0%, respectively. Relative standard deviations of recoveries for both methods were less than 3.8 and 8.1% for MG and LMG, respectively. The stability of MG and LMG at 4 degrees C in darkness was observed for at least 10 months. Moreover, photo-oxidative decomposition products of analytes in water samples, observed in stability tests carried out at 20 degrees C, were identified by mass spectrometry as N-demethylated products of MG and LMG. These findings prove that N-demethylated products of MG and LMG, reported as potential carcinogens, may be formed in living fish organisms not only during enzymatic action but also during photo-oxidative degradation in water.

Effects of the matrix and sample preparation on the determination of fluoroquinolone residues in animal tissues

The effects of aqueous-organic solvent extraction and enzymic digestion sonication procedure on the isolation of spiked fluoroquinolones from poultry tissue have been studied. The highest recovery from spiked tissue was obtained using a mixture of trichloroacetic acid-acetonitrile (8:2) as extractant and an SDB1 cartridge for clean-up purposes. Validation data are presented for enrofloxacin, ciprofloxacin, sarafloxacin and difloxacin. The spiking procedure (spike contact time, spike solvent and matrix) had a small influence on the recovery of fluroquinolones from poultry muscle or liver. The effects of a different extraction on the determination of incurred enrofloxacin and its metabolite, ciprofloxacin, residues in poultry tissues have been investigated. The extraction procedures investigated--aqueous-organic solvent extraction, enzymic digestion or sonication--all gave similar results for incurred fluoroquinolone concentration in poultry muscle after correlation for spike recovery. The highest results were obtained in poultry liver when enzymic digestion has been used.

Liquid chromatography-tandem mass spectrometry multiclass method for the determination of antibiotics residues in water samples from water supply systems in food-producing animal farms.

A sensitive liquid-chromatography-electrospray tandem mass spectrometry multiclass method for determination of 45 veterinary compounds belonging to 9 different antibiotic groups, including aminoglicosides (4), β-lactams (13), diaminopyrimidines (1), fluoroquinolones (10), lincosamides (1), macrolides (5), pleuromutilins (1), sulfonamides (6) and tetracyclines (4), in water from breeding animal watering supply system has been developed. Isolation of the analytes was carried out by solid phase extraction with heptafluorobutyric acid as an ion-pair agent on the Strata-X reversed phase cartridges. All analytes were determined simultaneously in one single run on a C18 column with gradient elution and short analysis time (13 min). Method was validated, average relative recoveries were in the range of 84.3-109.3% with satisfactory precision are repeatability for all compounds are in the range of 4.7-12.2%, within-laboratory reproducibility are in the range of 4.4-13.5% for. The limit of quantitation (LOQ) of the method was in the range of 0.02-10 μg L(-1), depending of analyte. The applicability of the method was tested by determining antimicrobial compounds in real water samples collected from water supply systems in breeding animal farms. The average antibiotics concentration in real water samples were, respectively, in the range of 0.14-1670 μg L(-1).

Determination of (fluoro)quinolones in eggs by liquid chromatography with fluorescence detection and confirmation by liquid chromatography–tandem mass spectrometry

A multiresidue analytical procedure for determination of seven fluoroquinolones (marbofloxacin, norfloxacin as internal standard, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin and difloxacin), and three quinolones (oxolinic acid, nalidixic acid and flumequine) in eggs is presented. The procedure is based on dispersive solid-phase extraction technique with acetonitrile as extractant. Norfloxacin and ciprofloxacin - d8 were used as internal standards to quantify the (fluoro)quinolones. Analyses were realised by LC-FLD for screening and LC-MS/MS for confirmatory purposes. The whole procedure was evaluated according to the Commission Decision 2002/657/EC. Specificity, decision limit (CCα), detection capacity (CCβ), recovery (absolute and relative), precision (repeatability and reproducibility) were determined during validation process. Recoveries (relative) for the LC-FLD screening determination ranged from 85% to 93%, repeatability and reproducibility were in the range of 5-9% to 9-16%, respectively. CCα and CCβ were 13-37 and 17-43 μg/kg pending on analite. For the LC-MS/MS confirmatory method, the relative recoveries were satisfactory (92-99%) with repeatability and reproducibility in the range of 4-7% to 6-12%, respectively. CCα and CCβ were 3-7 and 7-11 μg/kg depending on the analite. The results of both prepared methods showed these analytical procedures simple, rapid, sensitive and suitable for routine control of eggs.

Multiresidue method for the determination of nitroimidazoles and their hydroxy-metabolites in poultry muscle, plasma and egg by isotope dilution liquid chromatography–mass spectrometry

A multiresidue analytical procedure for the determination of four nitroimidazoles (metronidazole, dimetridazole, ronidazole, ipronidazole) and their hydroxy-metabolites in poultry muscle, plasma and egg is presented. The procedure is based on ion-exchange solid phase extraction with acetonitrile as an extractant followed by liquid chromatography-mass spectrometry. The separation of analytes was performed on a C18 column using a mobile phase of 0.1% formic acid in acetonitrile and 0.1% formic acid in water with gradient elution. The electrospray ionization was used to obtain the protonated molecules [M+H](+) and two product ions were monitored for each compound. For the quantification stable isotope-labelled analogues of the analytes were used as internal standards. The whole procedure was evaluated according to EU Commission Decision 2002/657/EC requirements. Specificity, decision limit (CCalpha), detection capacity (CCbeta), recovery and precision were determined during validation process. The overall recoveries ranged between 93 and 103% with a good coefficient of variation, less than 14.0% under within-laboratory reproducibility conditions. CCalpha and CCbeta were 0.05-0.44 and 0.08-0.90microgkg(-1) depending on analyte and matrix.

Multi-residue determination of antibiotics in fish by liquid chromatography-tandem mass spectrometry

A multi-residue method for the determination of 34 antibacterial drugs (three aminoglycosides, nine β-lactams, nine fluoroquinolones, three macrolides, five sulfonamides, trimethoprim and four tetracyclines) in fish samples by LC-MS/MS was developed. The procedure enables the isolation of residues based on double extraction of the fish sample with m-phosphoric acid and heptafluorobutyric acid as an ion-pair agent and acetonitrile. All compounds were determined at the same time on a C18 column with gradient elution. The method was validated according to the requirements of European Decision 2002/657/EC. Specificity, recovery, repeatability, within-laboratory reproducibility, decision limit CCα and detection capability CCβ were calculated. CCα ranged from 55.3 to 1085 µg kg−1 and CCβ ranged from 59.5 to 1141 µg kg−1. The overall recoveries were from 96% to 111% with respect to the internal standards.

Rapid method for the determination of tranquilizers and a beta-blocker in porcine and bovine kidney by liquid chromatography with tandem mass spectrometry

A fast and simple liquid chromatography with tandem mass spectrometry method for detection and confirmation of tranquilizers (chlorpromazine, propionylpromazine, acepromazine, triflupromazine, promazine, azaperone and its metabolite, azaperol) and beta-blocker (carazolol) in porcine and bovine kidney has been presented. The method relies on the extraction with acetonitrile followed by centrifugation. After evaporation of acetonitrile, the residue was reconstituted in a mobile phase and filtrated. The separation of analytes was performed on a C18 column using a mobile phase of acetonitrile and ammonium formate buffer (0.05 M, pH 4.5) with gradient elution. The electrospray ionization was used to obtain the protonated molecules [M+H](+) and two product ions were monitored for each compound. For quantification deutered internal standards were used. The whole method has been validated according to the European Union requirements. Specificity, decision limit (CCalpha), detection capability (CCbeta), trueness and precision were determined. The results showed good trueness ranged from 73.2% to 110.6% with a good R.S.D., less than 13.0% under within-laboratory reproducibility conditions. The calculated critical concentrations of CCalpha for phenothiazines were between 5.8 and 6.6 microgkg(-1) while for azaperone CCalpha was 105.5 microgkg(-1) and for azaperol was 121.4 microgkg(-1). CCalpha for carazolol was 16.7 microgkg(-1) in bovine and 21.9 microgkg(-1) in porcine kidney. CCbeta for phenothiazines were between 6.3 and 7.6 microgkg(-1), for azaperone was 119.0 microgkg(-1) and for azaperol was 140.0 microgkg(-1). For carazolol in bovine kidney CCbeta was 18.6 microgkg(-1) whereas in porcine kidney was 24.4 microgkg(-1).