Residue depletion of enrofloxacin and flumequine in feathers of broilers based on quantitative UHPLC-MS/MS detection.

To explore potential factors contributing to high fluoroquinolone resistance levels, it is essential to develop analytical methods capable of detecting residues and trace amounts of antibiotic use in broilers. The aim of the present study was to develop and in-house validate a sensitive UHPLC-MS/MS method capable of determining enrofloxacin (ENR) and flumequine (FLU) residues at slaughter age (day 45) when the animals were treated with these antimicrobials one day after hatching. Residue depletion of ENR and FLU in feathers was also assessed. Two experimental trials were performed, both consisting of 5 different treatment groups. In the first trial animals were treated with ENR and in the second one with FLU. The developed method was successfully validated and was found to be sensitive enough to detect residues of fluoroquinolones in the feathers up until slaughter age in all treatment groups. Average ENR concentration on day 45 was 10 ng g-1 feather after drinking water treatment, with all concentrations above the limit of quantification (LOQ) of 5 ng g-1 feather. For FLU average concentration on day 45 after drinking water administration was 4 ng g-1 feather, with an LOQ of 1 ng g-1 feather. Therefore, the method is suited for application to monitor fluoroquinolone use in broilers.

Prenatal treprostinil improves pulmonary arteriolar hypermuscularization in the rabbit model of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a congenital malformation characterized by pulmonary hypoplasia, pulmonary hypertension, and cardiac dysfunction. Pulmonary hypertension represents the major cause of neonatal mortality and morbidity. Prenatal diagnosis allows assessment of severity and selection of foetal surgery candidates. We have shown that treprostinil, a prostacyclin analogue with an anti-remodelling effect, attenuates the relative hypermuscularization of the pulmonary vasculature in rats with nitrofen-induced CDH. Here we confirm these observations in a large animal model of surgically-created CDH. In the rabbit model, subcutaneous maternal administration of treprostinil at 150 ng/kg/min consistently reached target foetal concentrations without demonstrable detrimental foetal or maternal adverse effects. In pups with CDH, prenatal treprostinil reduced pulmonary arteriolar proportional medial wall thickness and downregulated inflammation and myogenesis pathways. No effect on alveolar morphometry or lung mechanics was observed. These findings provide further support towards clinical translation of prenatal treprostinil for CDH.

Berberine-microbiota interplay: orchestrating gut health through modulation of the gut microbiota and metabolic transformation into bioactive metabolites.

Berberine is an isoquinoline alkaloid found in plants. It presents a wide range of pharmacological activities, including anti-inflammatory and antioxidant properties, despite a low oral bioavailability. Growing evidence suggests that the gut microbiota is the target of berberine, and that the microbiota metabolizes berberine to active metabolites, although little evidence exists in the specific species involved in its therapeutic effects. This study was performed to detail the bidirectional interactions of berberine with the broiler chicken gut microbiota, including the regulation of gut microbiota composition and metabolism by berberine and metabolization of berberine by the gut microbiota, and how they contribute to berberine-mediated effects on gut health. As previous evidence showed that high concentrations of berberine may induce dysbiosis, low (0.1 g/kg feed), middle (0.5 g/kg feed) and high (1 g/kg feed) doses were here investigated. Low and middle doses of in-feed berberine stimulated potent beneficial bacteria from the Lachnospiraceae family in the large intestine of chickens, while middle and high doses tended to increase villus length in the small intestine. Plasma levels of the berberine-derived metabolites berberrubine, thalifendine and demethyleneberberine were positively correlated with the villus length of chickens. Berberrubine and thalifendine were the main metabolites of berberine in the caecum, and they were produced in vitro by the caecal microbiota, confirming their microbial origin. We show that members of the genus Blautia could demethylate berberine into mainly thalifendine, and that this reaction may stimulate the production of short-chain fatty acids (SCFAs) acetate and butyrate, via acetogenesis and cross-feeding respectively. We hypothesize that acetogens such as Blautia spp. are key bacteria in the metabolization of berberine, and that berberrubine, thalifendine and SCFAs play a significant role in the biological effect of berberine.

A High Dose of Dietary Berberine Improves Gut Wall Morphology, Despite an Expansion of Enterobacteriaceae and a Reduction in Beneficial Microbiota in Broiler Chickens.

Phytogenic products are embraced as alternatives to antimicrobials, and some are known to mitigate intestinal inflammation and ensure optimal gut health and performance in broiler chickens. Dietary inclusion of berberine, a benzylisoquinoline alkaloid found in plants, is believed to exert gut health-promoting effects through modulation of the gut microbiota; however, there are only a few studies investigating its effects in chickens. The aim of this study was to investigate the interplay between dietary supplementation of a high concentration of berberine, the gastrointestinal microbiota, and histomorphological parameters in the gut. Berberine was shown to increase villus length and decrease crypt depth and CD3+ T-lymphocyte infiltration in the gut tissue of chickens at different ages. Berberine affected the diversity of the gut microbiota from the jejunum to the colon, both at a compositional and functional level, with larger effects observed in the large intestine. A high concentration of berberine enriched members of the Enterobacteriaceae family and depleted members of the Ruminococcaceae, Lachnospiraceae, and Peptostreptococcaceae families, as well as tended to reduce butyrate production in the cecum. In vivo results were confirmed by in vitro growth experiments, where increasing concentrations of berberine inhibited the growth of several butyrate-producing strains while not affecting that of Enterobacteriaceae strains. Positive correlations were found between berberine levels in plasma and villus length or villus-to-crypt ratio in the jejunum. Our study showed that berberine supplementation at a high concentration improves chicken gut morphology toward decreased inflammation, which is likely not mediated by the induced gut microbiota shifts. IMPORTANCE Dietary additives are widely used to reduce intestinal inflammation and enteritis, a growing problem in the broiler industry. Berberine, with anti-inflammatory, antioxidant, and antimicrobial activity, would be an interesting feed additive in this regard. This study investigates for the first time the impact of berberine supplementation on the chicken gastrointestinal microbiota, as a potential mechanism to improve gut health, together with histological effects in the small intestine. This study identified a dose-effect of berberine on the gut microbiota, indicating the importance of finding an optimal dose to be used as a dietary additive.

Population Pharmacokinetics of Intravenous Amoxicillin Combined With Clavulanic Acid in Healthy and Critically Ill Dogs.

Background: Data regarding antimicrobial pharmacokinetics (PK) in critically ill dogs are lacking and likely differ from those of healthy dogs. The aim of this work is to describe a population PK model for intravenous (IV) amoxicillin-clavulanic acid (AMC) in both healthy and sick dogs and to simulate a range of clinical dosing scenarios to compute PK/PD cutoffs for both populations. Methods: This study used a prospective clinical trial in normal and critically ill dogs. Twelve client-owned dogs hospitalized in the intensive care unit (ICU) received IV AMC 20 mg/kg every 8 h (0.5-h infusion) during at least 48 h. Eight blood samples were collected at predetermined times, including four trough samples before the next administration. Clinical covariates and outcome were recorded, including survival to discharge and bacteriologic clinical failure. Satellite PK data were obtained de novo from a group of 12 healthy research dogs that were dosed with a single AMC 20 mg/kg IV. Non-linear mixed-effects model was used to estimate the PK parameters (and the effect of health upon them) together with variability within and between subjects. Monte Carlo simulations were performed with seven dosage regimens (standard and increased doses). The correlation between model-derived drug exposure and clinical covariates was tested with Spearman's non-parametric correlation analysis. Outcome was recorded including survival to discharge and bacteriologic clinical failure. Results: A total of 218 amoxicillin concentrations in plasma were available for healthy and sick dogs. A tricompartmental model best described the data. Amoxicillin clearance was reduced by 56% in sick dogs (0.147 L/kg/h) compared with healthy dogs (0.336 L/kg/h); intercompartmental clearance was also decreased (p <0.01). None of the clinical data covariates were significantly correlated with individual exposure. Monte Carlo simulations showed that higher PK/PD cutoff values of 8 mg/L could be reached in sick dogs by extending the infusion to 3 h or doubling the dose. Conclusions: The PK of AMC is profoundly different in critically ill dogs compared with normal dogs, with much higher interindividual variability and a lower systemic clearance. Our study allows to generate hypotheses with regard to higher AMC exposure in clinical dogs and provides supporting data to revise current AMC clinical breakpoint for IV administration.

Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

Description of Plasma Penicillin G Concentrations after Intramuscular Injection in Double-Muscled Cows to Optimize the Timing of Antibiotherapy for Caesarean Section.

In order to improve the efficacy of penicillin injection during caesarean section, we aimed to identify the optimal timing of its preoperative administration. A study was conducted in 12 adult, non-pregnant Belgian Blue cows. To evaluate the plasma penicillin concentrations, blood samples were taken from the jugular vein at -5, 15, 30, 45, 60, 120, 240, 480 min relative to the intramuscular (IM) injection of 21,000 IU/kg of body weight of penicillin G. Results showed that plasma concentrations at 15 min after IM injection (668.3 ± 73.7 ng/mL) largely exceeded the minimal inhibitory concentration (MIC) of penicillin-sensitive bacteria (MIC < 125 ng/mL). With increasing time, plasma concentrations continued to rise, attaining an increasing proportion of moderately sensitive bacteria (250 ng/mL > MIC < 2000 ng/mL). The maximal concentration was reached between 1 and 4 h (average: 1.495.1 ± 181.7 ng/mL) after IM injection in the majority of cows, and decreased non-significantly to 1002.1 ± 93.2 ng/mL at 8 h. In conclusion, plasma penicillin concentrations at 15 min after an IM injection inhibit penicillin-sensitive bacteria. However, in order to obtain the maximal protective effect of the antibiotherapy, surgery should be started at 1 to 2 h after IM penicillin injection.

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

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 C(18) reversed-phase column (1.7 µm, 50 mm × 2.1 mm 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 1-2500 ng ml(-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 1 ng ml(-1) and a limit of detection of 0.4 ng ml(-1). The proposed method also shows good characteristics for the analysis of ML-7 in plasma of other animal species and human plasma.

Rapid method for the quantification of amoxicillin and its major metabolites in pig tissues by liquid chromatography-tandem mass spectrometry with emphasis on stability issues.

A fast method for the quantitative determination of amoxicillin (AMO), amoxicilloic acid (AMA) and amoxicillin diketopiperazine-2',5'-dione (DIKETO) in pig edible tissues (kidney, liver, fat and muscle) with liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) is presented. The method uses a simple liquid-liquid extraction of the tissue matrix with a 10 mM potassium dihydrogen phosphate buffer (pH 4.5) as extraction solvent. After deproteinisation by ultrafiltration, the tissue extract was directly injected onto the LC column. Chromatographic separation of the components was performed on a PLRP-S polymeric column using 0.1% of formic acid in water and acetonitrile. The mass spectrometer was operated in the positive electrospray MS/MS mode. The method was fully validated according to EU requirements (linearity, precision, trueness, quantification limit, detection limit and specificity). The stability of the components was evaluated over the pH range from 1.2 to 8.0. Biological samples of pigs medicated with AMO and AMO/clavulanic acid were analyzed using the developed method.

Quantitative determination of dihydrostreptomycin in bovine tissues and milk by liquid chromatography-electrospray ionization-tandem mass spectrometry.

Dihydrostreptomycin (DHS) is an aminoglycoside antibiotic used in veterinary medicine in combination with benzylpenicillin for the treatment of bacterial infections in cattle, pigs and sheep. A method to determine its residues in edible tissues of cattle, as well as in milk, was developed and validated. Extraction of DHS from the tissues was performed using a liquid extraction with a 10 mM phosphate buffer containing 2% (w/v) trichloroacetic acid, while milk samples were treated with a 50% (w/v) trichloroacetic acid solution, followed by a solid-phase clean-up procedure on a carboxypropyl (CBA) weak cation exchange column. Ion-pair chromatography, using a mixture of 20 mM pentafluoropropionic acid in water and acetonitrile as the mobile phase, was used to retain DHS and the internal standard streptomycin (STR) on a Nucleosil (5 microm) reversed-phase C18 column. The components were detected and quantified by electrospray ionization (ESI) tandem mass spectrometry. The method could be validated according to EC (European Community) requirements with respect to linearity, trueness and precision, the latter evaluated at the maximum residue limit (MRL) - 1000 ng g(-1) for kidney, 500 ng g(-1) for muscle, liver and fat, and 200 ng g(-1) for milk -, at one-half of the MRL and at one and a half times the MRL. A limit of quantification of 10 ng g(-1) and 1 ng ml(-1) was obtained for all tissues and for milk, respectively, which is far below one-half of the MRL as requested, while the limit of detection was in the low ppb range, varying between 1.9 and 4.2 ng g(-1) for the different tissues tested, and being 0.6 ng ml(-1) for milk. The method was used for the monitoring of DHS residues in incurred tissue and milk samples coming from cattle medicated with DHS in combination with benzylpenicillin by intramuscular injection, in order to evaluate withdrawal times.

Control of the keto-enol tautomerism of chlortetracycline for its straightforward quantitation in pig tissues by liquid chromatography-electrospray ionization tandem mass spectrometry.

Chlortetracycline (CTC) is a member of the group of the tetracycline antibiotics used in human and veterinary medicine. Its reliable measurement in edible tissues is hampered by the fact that CTC is subjected to keto-enol tautomerism resulting in its keto- and enol-form. Also, it undergoes epimerization to form its 4-epimer. In this paper, we present an LC-electrospray ionization (ESI)-MS/MS method to analyze the CTC content in pig tissues, with the originality that it apports a solution to overcome the possible interference on the measurement of the keto-enol tautomerism equilibrium, which is influenced by several factors. Therefore, the minced tissue samples were incubated at 56 degrees C, forcing the equilibrium in favour of the CTC keto-form. The CTC keto-tautomer, as a measure of the total CTC content, was then quantified by LC-ESI-MS/MS, after further extraction of the tissue samples, consisting of a liquid extraction with sodium succinate solution (pH 4.0), followed by protein removal with trichloroacetic acid and paper filtration, and finally solid-phase clean-up on an hydrophilic-lipophilic balance (HLB) polymeric reversed-phase column. The method could be validated for pig muscle, liver and kidney tissues--for which maximum residue limits (MRLs) have been fixed by the European Union--with respect to linearity, trueness and precision, in accordance with the European guidelines.

Quantitative determination of chlortetracycline content in animal plasma at controlled keto-enol tautomerism by liquid chromatography-electrospray ionization-tandem mass spectrometry.

Chlortetracycline (CTC) is a member of the group of the tetracycline antibiotics used in human and veterinary medicine. Its reliable measurement in plasma - necessary to study its pharmacokinetics - is hampered by the fact that CTC is subjected to keto-enol tautomerism resulting in its keto- and enol-form. Also, it undergoes epimerization to form its 4-epimer. In this paper, we present an LC-ESI-MS/MS method to analyze the CTC content in animal plasma, with the originality that it apports a solution to overcome the possible interference of the keto-enol tautomerism equilibrium, which is influenced by several factors. Therefore, the plasma samples were incubated at 56 degrees C, forcing the equilibrium completely in favour of the CTC keto-form. The CTC keto-tautomer was stable after deproteinization of the samples with concentrated trifluoroacetic acid, and was quantified by LC-ESI-MS/MS, as a measure of the total CTC content. The method could be validated for pig and chicken plasma by a series of parameters - linearity, trueness, precision, limit of quantification, specificity, stability - in accordance with the European guidelines, and was used for pharmacokinetic studies on pigs and chickens medicated with CTC via the feed.

Quantitative determination of dexamethasone in bovine milk by liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

Dexamethasone (DXM) is a synthetic glucocorticoid that is authorized for therapeutic use in veterinary medicine. The European Community (EC) fixed a maximum residue limit (MRL) at 2ng/g for liver, 0.75ng/g for muscle and kidney tissues, and 0.3ng/ml for milk, while its use as growth-promoter is completely banned. The purpose of this study was to develop and validate a simple and reliable method to determine DXM residues in bovine milk. Milk proteins were removed by the addition of concentrated trichloroacetic acid and paper filtration. Solid-phase extraction clean-up on a C18 reversed phase column was performed to obtain an extract suitable for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatographic separation of DXM and the internal standard desoximetasone, was achieved on a PLRP-S polymeric reversed phase column, using a mixture of 0.1% (v/v) acetic acid in water (mobile phase A) and acetonitrile (mobile phase B) as the mobile phases. They were identified using the MS/MS detection technique, and were subsequently quantified. The method has been validated according to the requirements of the EC at 0.15, 0.30 and 0.60ng/ml (being half the MRL, the MRL and double the MRL levels fixed by the EC). Calibration graphs were prepared in the 0.15-5ng/ml range and good linearity was achieved (r>or=0.99 and goodness of fit

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.

Determination of clindamycin in animal plasma by high-performance liquid chromatography combined with electrospray ionization mass spectrometry.

A method for the quantification of clindamycin in animal plasma using high-performance liquid chromatography combined with electrospray ionization mass spectrometry (LC/ESI-MS/MS) is presented. Lincomycin is used as the internal standard. The sample preparation includes a simple deproteinization step with trichloroacetic acid. Chromatographic separation is achieved on an RP-18 Hypersil column using gradient elution with 0.01 M ammonium acetate and acetonitrile as mobile phase. Good linearity was observed in the range 0-10 microg ml(-1). The limit of quantification of the method is 50 ng ml(-1) and the limit of detection is 1.3 ng ml(-1). The method was shown out to be of use for pharmacokinetic studies of clindamycin formulations in dogs.

Quantitative analysis of diclazuril in animal plasma by liquid chromatography/electrospray ionization mass spectrometry.

A novel, sensitive and specific method for the quantitative determination of diclazuril in animal plasma using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with negative ion detection is presented. Extraction of the samples was performed with a rapid deproteinization step using acetonitrile. Chromatography of diclazuril and the internal standard was achieved on a Nucleosil ODS 5-microm column, using a gradient elution with 0.01 M ammonium acetate and acetonitrile. To obtain the highest sensitivity and specificity possible, the mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. The method was validated according to the requirements defined by the European Community. Calibration curves using plasma fortified between 1-100 ng/mL and 100-2000 ng/mL showed good linear correlation (r >or= 0.9991, goodness-of-fit coefficient

Quantitative analysis of amoxycillin and its major metabolites in animal tissues by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

A sensitive and specific method for the quantitative determination of amoxycillin and its major metabolites (amoxycilloic acid, amoxycillinpiperazine-2',5'-dione) in animal tissue samples using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is presented. A liquid extraction using an aqueous 0.01 M potassium dihydrogenphosphate solution as the extraction solvent was performed for a preliminary sample cleanup. The extracts were further purified by a solid-phase extraction using an octadecyl (C18) column. Ampicillin was used as the internal standard. Chromatographic separation of the analytes of interest was achieved on a reversed-phase Hypersil column (100 x 3 mm i.d., dp, 5 microm), using a mixture of 9.6 mM pentafluoropropionic acid in water and acetonitrile as the mobile phase. Gradient elution was performed. To obtain as high sensitivity and selectivity as possible, the mass spectrometer was operated in the multiple reaction monitoring mode. The method was validated for the analysis of amoxycillin and its investigated metabolites in various porcine tissues, kidney, liver, muscle, and 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 range tested (25-500 ng/g, r > or = 0.9974, and goodness of fit < or = 9.6). A limit of quantification of 25 ng/g was obtained for amoxycillin and its metabolites in all tissues, which corresponds to half the maximum residue limit for amoxycillin. Limits of detection ranged from 2.3 to 12.0 ng/g for amoxycillin and from 1.1 to 15.1 ng/g and 0.2 to 2.4 ng/g for amoxycilloic acid and amoxycillinpiperazine-2',5'-dione, respectively. The results for the within-day precision and the trueness fell within the ranges specified. The method has been successfully used for the quantitative determination of amoxycillin and its major metabolites in tissue samples from pigs medicated via the drinking water, proving the usefulness of the developed method for application in the field of residue analysis.