Marc Cherlet

Determination of gentamicin in swine and calf tissues by high-performance liquid chromatography combined with electrospray ionization mass spectrometry.

Gentamicin is a broad-spectrum aminoglycoside antibiotic widely used in veterinary medicine for the treatment of serious infections. The purpose of this study was to develop and validate a method to determine gentamicin residues in edible tissues of swine and calf. Extraction of gentamicin was performed using a liquid extraction with phosphate buffer containing trichloroacetic acid, followed by a solid-phase clean-up procedure on a CBA weak cation-exchange column. Tobramycin was used as the internal standard. After drying of the eluate, the residue was redissolved and further analyzed by reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (MS/MS). Chromatographic separation of the internal standard tobramycin and the gentamicin components was achieved on a Nucleosil (5 microm) column using a mixture of 10 mM pentafluoropropionic acid in water and acetonitrile as the mobile phase. The gentamicin components C1a, C2 + C2a and C1 could be identified with the MS/MS detection, and subsequently quantified. The method was validated according to the requirements of the EC at the maximum residue limit (MRL) (100 ng g(-1) for muscle and fat, 200 ng g(-1) for liver and 1000 ng g(-1) for kidney), half the MRL and double the MRL levels. 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 25.0 ng g(-1) were obtained for the determination of gentamicin in muscle, fat, liver and kidney tissues of swine and calf, which correspond in all cases to at least half the MRLs. Limits of detection ranged between 0.5 and 2.5 ng g(-1) for the tissues. The within-day and between-day precisions (RSD) and the results for accuracy fell within the ranges specified. The method was successfully used for the determination of gentamicin in tissue samples of swines and calves medicated with gentamicin by intramuscular injection.

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.

Evaluation and establishing the performance of different screening tests for tetracycline residues in animal tissues

Four methods intended for screening muscle tissue for residues belonging to the tetracycline group were compared using artificially contaminated as well as incurred samples. Two agar diffusion methods were studied: one with Bacillus subtilis as a test strain, the second with Bacillus cereus. Two variants of each method were compared: thin plates for analysis of intact or minced meat, and thick plates for analysis of meat fluid. The thin plate variants could not be evaluated with artificially contaminated samples because it was impossible to prepare homogeneously spiked, undiluted meat. The thick plates were suited for doxycycline and chlortetracycline, but they did not detect oxytetracycline or tetracycline in spiked meat fluid. The results of these tests done on incurred meat were very good for doxycycline and satisfying or just failing for oxytetracycline, while the best detection capability was obtained when intact frozen meat was examined on thin plates seeded with B. cereus. Two commercially available screening tests were also evaluated. The Premi® test, an inhibitor test with Bacillus stearothermophilus as a test strain and an indicator for growth, was not suited for detection of tetracyclines up to the maximum residue limit. Tetrasensor®, a receptor test specific for tetracyclines, proved a quick and simple test able to detect meat samples artificially contaminated with tetracycline, oxytetracycline, doxycycline or chlortetracycline, as well as meat incurred with oxytetracycline or doxycycline.

Antipyretic effect of oral sodium salicylate after an intravenous e. coli LPS injection in broiler chickens

1. A study was set up to investigate the influence of sodium salicylate on fever and acute phase reaction after lipopolysaccharide (LPS) injection in broiler chickens. 2. An acute phase reaction was provoked through the intravenous injection of Escherichia coli LPS. Four oral doses of sodium salicylate were tested. Apart from body temperature, other inflammation indices, such as plasma corticosterone and ceruloplasmin, serum thromboxane B2 and zinc concentrations were monitored. 3. Intravenous LPS induced a fever of about 1°C. A dose-dependent attenuation of the fever response of the chickens in the salicylate treated groups was observed. LPS-injected chickens also showed elevated plasma corticosterone and ceruloplasmin, while serum thromboxane and zinc concentrations decreased. Except for thromboxane B2, no linear relationship with increasing salicylate dose could be shown for the other blood variables. 4. These data confirm that sodium salicylate is an effective antipyretic agent after injection of LPS in chickens, if used at an appropriate dosage. No dose-related change could be found for the other inflammation indices.

Liquid chromatographic determination of levamisole in animal plasma: ultraviolet versus tandem mass spectrometric detection

This work presents the optimization and validation of a liquid chromatography (LC)-ultraviolet (UV) detection method and a tandem mass spectrometry (LC-MS/MS) method with positive electrospray ionization for the determination of levamisole, an anthelmintic for veterinary use, in animal plasma. A liquid-liquid extraction procedure in alkaline medium, using hexane-isoamyl alcohol (95:5, v/v) as extraction solvent, was performed to clean-up the plasma samples prior to LC-UV analysis. The sample preparation prior to LC-MS/MS analysis consisted of a protein precipitation step, using acetonitrile. Methyllevamisole was used as the internal standard. Chromatographic separation was achieved on a Lichrospher® 60 RP-select B (5 μm) column using a mixture of 0.1 M ammonia acetate in water and acetonitrile as the mobile phase. A gradient (flow rate 1 ml min -1 ) or isocratic (flow rate 0.2 ml min -1 ) elution was performed in case of the LC-UV and LC-MS/MS methods, respectively. UV detection was at 235 nm. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. The methods were validated (linearity, precision, trueness, limit of quantification, limit of detection, specificity) according to the requirements defined by the European Community. Good linearity was achieved over the concentration ranges tested (0-0.5 μg ml -1 and 0-4.0 μg ml -1 , r > 0.99, goodness-of-fit < 10%). Limits of quantification of 0.025 μg ml -1 and 0.1 μg ml -1 were achieved for the LC-MS/MS and LC-UV methods, respectively. The limits of detection were 0.009 and 0.077 μg ml -1 , respectively. The results for precision (within-day and between-day) fell within the ranges specified. The methods have been successfully used for the determination of levamisole in plasma samples from two pigs medicated via drinking water. A good correlation was observed between the results of both methods (r 2 = 0.9831, slope = 1.13, intercept = 0.005 μg ml -1 ), proving their usefulness for the application in the field of pharmacokinetic and residue analysis.

Quantitative analysis of levamisole in porcine tissues by high-performance liquid chromatography combined with atmospheric pressure chemical ionization mass spectrometry

This work presents the development and the validation of an LC-MS-MS method with atmospheric pressure chemical ionization for the quantitative determination of levamisole, an anthelmintic for veterinary use, in porcine tissue samples. A liquid-liquid back extraction procedure using hexane-isoamylalcohol (95:5, v/v) as extraction solvent was followed by a solid-phase extraction procedure using an SCX column to clean up the tissue samples. Methyllevamisole was used as the internal standard. Chromatographic separation was achieved on a LiChrospher 60 RP-select B (5 microm) column using a mixture of 0.1 M ammonium acetate in water and acetonitrile as the mobile phase. The mass spectrometer was operated in MS-MS full scanning mode. The method was validated for the analysis of various porcine tissues: muscle, kidney, liver, fat and skin plus fat, according to the requirements defined by the European Community. 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 5.0 ng/g were obtained for the analysis of levamisole in muscle, kidney, fat and skin plus fat tissues, and of 50.0 ng/g for liver analysis, which correspond in all cases to half the MRLs (maximum residue limits). Limits of detection ranged between 2 and 4 ng/g tissue. The within-day and between-day precisions (RSD, %) and the results for accuracy fell within the ranges specified. The method has been successfully used for the quantitative determination of levamisole in tissue samples from pigs medicated via drinking water. Moreover the product ion spectra of the levamisole peak in spiked and incurred tissue samples were in close agreement (based on ion ratio measurements) with those of standard solutions, indicating the worthiness of the described method for pure qualitative purposes.

Quantitative analysis of an anti-viral immune escape compound ML-7 in feline plasma using ultra performance liquid chromatography/electrospray ionization mass spectrometry.

Abstract An analytical method for the quantitative measurement of ML-7, a product with possible anti-immune escape activity for feline infectious peritonitis virus (FIPV), in feline plasma was developed and validated. The sample preparation consists of a solid-phase extraction step on an MCX cartridge. ML-7 and ML-9, used as the internal standard for the analysis, were separated on an ACQUITY UPLC™ BEH C18 reversed-phase column (1.7μm, 50mm×2.1mm I.D.), using isocratic elution with acetonitrile and 0.1% formic acid in water as the mobile phase. Both compounds were subsequently quantified in MRM mode on a Micromass® Quattro Premier™ XE triple quadrupole mass spectrometer. The use of a Thermo Scientific® Exactive™ orbitrap mass spectrometer made it possible to confirm the proposed fragmentation pattern of both ML-7 and ML-9. A validation study according to EC requirements was carried out, in which the method showed good performance. Linear behaviour was observed in the 1–2500ngml−1 range, which is relevant for real sample analysis. Accuracy and precision were within the criteria requested by the EC requirements throughout this concentration range. Extraction recovery of ML-7 was 72%. Matrix effect for ML-7 was not higher than 8%. The method was successfully used for the monitoring of ML-7 in feline plasma after intravenous, subcutaneous or oral administration of an ML-7 formulation, for the determination of pharmacokinetic parameters, with a limit of quantification of 1ngml−1 and a limit of detection of 0.4ngml−1. The proposed method also shows good characteristics for the analysis of ML-7 in plasma of other animal species and human plasma.