Β-lactam Antibiotics In Milk Research Articles

Electromembrane extraction – capillary zone electrophoresis for the quantitative determination of β-lactam antibiotics in milk samples

Three penicillin-based β-lactam antibiotics (benzylpenicillin, amoxicillin, and ampicillin) were extracted by electromembrane extraction (EME) and determined in the resulting extracts by capillary zone electrophoresis (CZE) with UV–Vis detection. The EME was optimized for the simultaneous clean-up of complex samples and preconcentration of the three antibiotics and employed 1-octanol as the organic phase interface (impregnated in the pores of a hollow fiber), acidified donor solution (pH 3), and phosphate buffer (pH 5.6) as the acceptor solution. The EMEs were carried out for 20 min at 300 V and constant stirring (750 rpm) of the donor solution. At the optimized EME-CZE conditions, the sensitivity of the analytical method was sufficient for the determination of the three β-lactam antibiotics in undiluted cow's milk at concentrations below the EU maximum residue limits (4 μg/L) in foodstuffs. The method was simple, rapid, and convenient and offered extraction recoveries of 13.5 – 87.3 %, enrichment factors of 23.6 – 152.8, repeatability (RSD values) better than 7.6 %, linear analytical response in the 1 – 100 μg/L (3 – 100 μg/L for benzylpenicillin) concentration range with correlation coefficients ≥ 0.9997, and limits of detection from 0.2 to 1.2 μg/L. The proposed analytical concept was used for the rapid control of milk quality (i.e. assessment of excessive use of antibiotics in dairy animals), moreover, it was further extended to the trace determination of β-lactam antibiotics in other complex samples, such as in wastewater.

Integration of antibody-antigen and receptor-ligand reactions to establish a gold strip biosensor for detection of 33 β-lactam antibiotics

A novel lateral flow immunoassay biosensor, combining the receptor-ligand reaction and antigen-antibody reaction was developed for the detection of β-lactam antibiotics in milk. The receptor protein, the carboxy-terminal domain of β-lactam sensor-transducer mutant from Bacillus. licheniformis, served as the recognition element. Colloidal gold (CG)-labelled receptor antibody-receptor ternary complex was applied as a signal transducer probe. The presence of β-lactams was identified through a color change in the test zone, where the reaction between the complex of CG/anti-receptor antibody/receptor and β-lactam antigen on the test line could be inhibited by β-lactam residues in the sample, thus leading to a reduction in color signal. Based on the indirect labeling of the receptor with an antibody as a linker, the ability to detect 33 β-lactams was below or near the corresponding maximum residue limit. The proposed lateral flow immunoassay biosensor appears to be an excellent field-based screening tool for the qualitative screening of β-lactams in milk.

Construction of an Electrochemical Receptor Sensor Based on Graphene/Thionine for the Sensitive Determination of β-Lactam Antibiotics Content in Milk.

In antibiotics, β-lactam is one kind of major concern acknowledged as an unavoidable contaminant in milk. Thus, a facile and sensitive method is essential for rapid β-lactam antibiotics detection. In our work, a specific electrochemical receptor sensor based on the graphene/thionine (GO/TH) composite was established. The mechanism of the electrochemical receptor sensor was a direct competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP-AMP) to the mutant BlaR-CTD protein by free β-lactam antibiotics. Then, horseradish peroxidase (HRP) catalyzed the hydrolysis of the substrate hydrogen peroxide (H2O2), which produced an electrochemical signal. Under optimal experimental conditions, this method could quantitatively detect cefquinome from 0.1 to 8 μg L−1 and with the limit of detection (LOD) of 0.16 μg L−1, much lower than the maximum residue limit (MRL) of 5 μg L−1 set by the European Union. In addition, the LOD of spiked milk samples with cefalexin, cefquinoxime, cefotafur, penicillin G and ampicillin were 14.88 μg L−1, 2.46 μg L−1, 17.16 μg L−1, 0.06 μg L−1, 0.21 μg L−1 and the limits of quantitation (LOQ) were 36.09 μg L−1, 5.40 μg L−1, 41.45 μg L−1, 0.13 μg L−1, 0.42 μg L−1, respectively. The sensor showed a favorable recovery of 84.89–102.44%. Moreover, the electrochemical receptor sensor was successfully applied to assay β-lactam antibiotics in milk, which showed good correlation with the results obtained from liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Development of a direct ELISA based on carboxy-terminal of penicillin-binding protein BlaR for the detection of β-lactam antibiotics in foods

β-Lactam antibiotics, including penicillins and cephalosporins, are commonly used in veterinary medicine. Illegal use and abuse of β-lactams could cause allergy and selected bacterial resistance. BlaR-CTD, the carboxy-terminal of penicillin-recognizing protein BlaR from Bacillus licheniformis ATCC 14580, was utilized in this study to develop a receptor-based ELISA for detection and determination of β-lactam antibiotics in milk, beef, and chicken. This assay was based on directly competitive inhibition of binding of horseradish peroxidase-labeled ampicillin to the immobilized BlaR-CTD by β-lactams. The assay was developed as screening test with the option as semiquantitative assay, when the identity of a single type of residual β-lactam was known. The IC50 values of 15 β-lactam antibiotics, including benzylpenicillin, ampicillin, amoxicillin, dicloxacillin, oxacillin, nafcillin, cefapirin, cefoperazone, cefalotin, cefazolin, cefquinome, ceftriaxone, cefotaxime, cefalexin, ceftiofur and its metabolite desfuroylceftiofur were evaluated and ranged from 0.18 to 170.81 μg L(-1). Simple sample extraction method was carried out with only phosphate-buffered saline, and the recoveries of selected β-lactam antibiotics in milk, beef, and chicken were in the range of 53.27 to 128.29 %, most ranging from 60 to 120 %. The inter-assay variability was below 30 %. Limits of detection in milk, beef, and chicken muscles with cefquinome matrix calibration were 2.10, 30.68, and 31.13 μg kg(-1), respectively. This study firstly established a rapid, simple, and accurate method for simultaneous detection of 15 β-lactams in edible tissues, among which 11 β-lactams controlled by European Union could be detected below maximum residue limits.

Penicillin-binding protein 3 of Streptococcus pneumoniae and its application in screening of β-lactams in milk

The soluble form of penicillin-binding protein 3 (sPBP3∗) from Streptococcus pneumoniae was expressed in Escherichia coli as a six-histidine fusion protein. The protein was purified and used to develop a microplate assay in direct competitive format for the detection of penicillins and cephalosporins in milk. The assay was based on competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP–Amp) to the sPBP3∗ by free β-lactam antibiotics in milk. Under optimized conditions, most of the β-lactam antibiotics (11 penicillins and 16 cephalosporins) could be detected at concentrations corresponding to the maximum residue limits (MRLs) set by the European Union. Analysis of spiked milk samples showed that acceptable recoveries ranged from 74.06 to 106.31% in skimmed milk and from 63.97 to 107.26% in whole milk, with coefficients of variation (CVs) less than 16%. With the high sensitivity and wide-range affinities to penicillins and cephalosporins, the developed assay based on sPBP3∗ exhibited the potential to be a screening assay for fast detection of β-lactam antibiotics in milk.

An amperometric affinity penicillin-binding protein magnetosensor for the detection of β-lactam antibiotics in milk

The preparation, characterization and performance evaluation of an amperometric affinity disposable magnetosensor, based on the use of a recombinant penicillin-binding protein (PBP) and screen-printed carbon electrodes (SPCEs), for the specific detection and quantification of β-lactam antibiotic residues in milk are reported. The PBP was immobilized onto His-Tag-Isolation-modified magnetic beads (His-Tag-Isolation-MBs), and a direct competitive assay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling was performed. The amperometric response obtained at -0.20 V vs. the Ag pseudo-reference electrode of the SPCE after the addition of H2O2 in the presence of hydroquinone (HQ) was used as the transduction signal. The developed methodology showed very low detection limits (in the low ppb level) for the 6 antibiotics tested in untreated milk samples, and a good selectivity against other antibiotic residues frequently detected in milk and dairy products. Due to the bioreceptor employed, this methodology was able to detect only the active form of β-lactam antibiotics with high affinities for both penicillins and cephalosporins. Moreover, the analysis took only 30 min.

On-line solid phase extraction using organic–inorganic hybrid monolithic columns for the determination of trace β-lactam antibiotics in milk and water samples

A rapid and simple method with on­line solid phase extraction (SPE) has been developed for the simultaneous determination of beta-lactam antibiotics (BLAs) (amoxicillin, cephradine, and cefazolin sodium) in aquatic environment and milk. The epoxy-based organic–inorganic hybrid monolithic column was used as SPE sorbent to simultaneously monitor three analytes. The morphology of monolithic column and pressure drop across the columns were characterized. Excellent permeability and high selectivity were obtained. The linear range of the standard curve was from 2.0 to 500ng/mL (r2≥0.999). Precision for inter- and intra-day assay showed acceptable results for quantitative assay with relative standard deviation (RSD) less than 11%. The accuracy and recovery were found to be within the range of 93–103% and 83–105%. The results indicated that the prepared monolithic column could provide excellent reproducibility and implied that the prepared monolith was feasible to be used as an on-line SPE sorbent material.

Ultrasound‐assisted matrix solid phase dispersive extraction for the simultaneous analysis of β‐lactams (four penicillins and eight cephalosporins) in milk by high performance liquid chromatography with photodiode array detection

The application of ultrasound-assisted matrix solid phase dispersive extraction for the confirmatory analysis of 12 β-lactam antibiotics in milk by high performance liquid chromatography with photodiode array detection has been proposed herein. Four penicillins (cloxacillin, dicloxacillin, oxacillin, and amoxicillin) and eight cephalosporins (cefaclor, cefadroxil, ceftiofur, cefuroxime, cefoperazone, cefazolin, cephalexin, and cefotaxime) are effectively extracted using a mixed sorbent of Quick Easy Cheap Effective Rugged Safe technique and OASIS HLB providing a matrix free from any endogenous interference. Examined analytes were well resolved on an Inertsil ODS-3 analytical column with a mobile phase of CH(3)COONH(4) (0.05 M) and acetonitrile delivered under a gradient program. 1,7-Dimethyl-xanthine was used as internal standard. The method was validated meeting the European Legislation determining linearity, selectivity, stability, decision limit, detection capability, accuracy, precision, and ruggedness according to the Youden approach. Recoveries of all antibiotics rated from 85.0 to 115.7%, while RSD values were <12.7%. Finally, the method was successfully applied to milk samples purchased from local market.

Effect of heat treatments on stability of β-lactams in milk

The presence of residues of antimicrobial substances in milk may have serious toxicological and technical consequences. To date, few studies have been done to evaluate the effect of heat treatments on β-lactam residues in milk. However, the few studies that have been conducted estimate losses of antimicrobial activity under different combinations of temperature and time using microbiological methods. The aims of this study were to calculate the kinetic parameters for the degradation of β-lactam antibiotics in milk and to develop prediction models to estimate the concentration losses of these compounds in conventional dairy heat treatments. To do so, we employed a quantitative HPLC method to calculate losses in concentrations of 10 β-lactam antibiotics in milk with different combinations of temperature and time. Increasing the temperature from 60°C to 100°C decreased the half-life of amoxicillin (372 to 50min), ampicillin (741 to 26min), cloxacillin (367 to 46min), and penicillin G (382 to 43min). These increases in temperature caused further degradation in cephalosporins, which was accompanied by a decrease in half-life times to reach very low values; for instance, 4, 5, and 6min for cefoperazone, cephurexime, and cephapirin, respectively. Kinetic equations were applied to different heat treatments used in dairy processing. Heat treatments at high temperatures and long times (e.g., 120°C for 20min) led to a further degradation of β-lactam antibiotics with percentages close to 100% for cefoperazone and cefuroxime. In contrast, when milk was subjected to heat treatments at lower temperatures and times (e.g., 72°C for 15s), the degradation of β-lactam in milk did not exceed 1% for the 10 antibiotics tested.

Determination of β-lactam antibiotics in milk based on magnetic molecularly imprinted polymer extraction coupled with liquid chromatography–tandem mass spectrometry

In this work, a rapid and selective method was successfully developed using the magnetic molecularly imprinted polymer (MMIP) as sorbent for the extraction of β-lactam antibiotics (BLAs) from milk samples. The MMIP has been prepared using penicillin V potassium (PENV) as template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinking agent and Fe 3O 4 magnetite as magnetic component. The experimental results showed that the MMIP had high affinity and selectivity toward PENV and other structurally related BLAs. The extraction process was carried out in a single step by mixing the extraction solvent, MMIPs and milk samples under ultrasonic action. When the extraction was completed, the MMIPs adsorbing the analytes were separated from the sample matrix by an external magnet. The analytes eluted from the MMIP were analyzed by liquid chromatography–tandem mass spectrometry. For achieving optimal preconcentration and reducing non-specific interactions, various parameters affecting the extraction efficiency such as extraction mode, extraction solvent, the amount of MMIPs, extraction time, washing solution and eluting solution were comprehensively evaluated. Under the optimal conditions, the detection limits of BLAs are in the range of 1.6–2.8 ng mL −1. The relative standard deviations of intra- and inter-day ranging from 3.2% to 8.3% and from 3.6% to 9.8% are obtained, respectively. The method was applied to determine BLAs including PENV, amoxicillin and oxacillin in five milk samples from different provenances. The recoveries of BLAs in these samples from 71.6% to 90.7% are obtained.

Validation of the βeta-s.t.a.r. 1 + 1 for rapid screening of residues of β-lactam antibiotics in milk

The 2-min protocol (1 + 1) for the βeta-s.t.a.r. (manufactured by Neogen Corporation, Lansing, MI, USA) was validated at the Technology and Food Science Unit of the Institute for Agricultural and Fisheries Research according to Commission Decision 2002/657/EC. The test was very selective for the group of β-lactam compounds: the only interference found was by clavulanic acid at 2500 µg kg−1 and above. The modified protocol (βeta-s.t.a.r. 1 + 1) detected all β-lactams with a maximum residue limit (MRL) in milk, but not all these compounds were detected at their respective MRL. The detection of cefalexin (detection capability = 6000 µg kg−1; MRL = 100 µg kg−1) and penethamate (detection capability = 80 µg kg−1; MRL = 4 µg kg−1) was especially poor, and also ceftiofur was only detected from 500 µg kg−1 (MRL = 100 µg kg−1). The repeatability of the reader and of the test was very good. The test was very robust: test results were not significantly influenced by small changes in the test protocol, by the milk composition or by the type of milk. The test was also suitable to test the milk of animal species other than cow. Favourable results were obtained in testing monitoring samples, in two national ring trials, and in an international proficiency test. The βeta-s.t.a.r. 1 + 1 is a very fast, simple, and reliable test that could be used at the farm level to prevent tanker milk contamination by β-lactams.

Analytical methodologies for the detection of β-lactam antibiotics in milk and feed samples

Antibiotic residues in milk are of great concern to dairy farmers, milk processors, regulatory agencies and consumers. In addition, the effectiveness of antibiotics is threatened by extensive, inappropriate use. In veterinary practice, antibiotics are utilized at therapeutic levels primarily to treat diseases and to prevent infections. They are also used at sub-therapeutic levels to increase feed efficiency and to promote growth in food-producing animals. The frequent, sometimes illegal, use of antibiotics may result in residues being found at different concentration levels in products of animal origin, such as milk. β-lactams comprise some of the antibiotics most frequently used for the treatment of sick animals in Europe. This review presents analytical methods that have been developed during the past decade for penicillin and cephalosporin residues in milk and feed samples. It includes microbiological approaches, biosensors, immunochemical techniques and chromatographic methods.

Determination of β-lactam antibiotics in milk using micro-flow chemiluminescence system with on-line solid phase extraction

In this paper, a chemiluminescence (CL) micro-flow system combined with on-line solid phase extraction (SPE) is presented for determination of β-lactam antibiotics (penicillin, cefradine, cefadroxil, cefalexin) in milk. It is based on the enhancement effect of β-lactam antibiotics on the luminol-K 3Fe(CN) 6 CL system. The micro-flow system was fabricated from two polymethyl methacrylate (PMMA) plates (50 mm × 40 mm × 5 mm) with the microchannels of 200 μm wide and 150 μm deep. C 18-modified silica gel was packed into the microchannel (length: 10 mm; width: 1 mm; depth: 500 μm) to serve as SPE device. Extraction and preconcentration of the analytes were carried out using on-line SPE micro-flow system and the selectivity of CL detection was improved. The detection limits were 0.5 μg mL −1 of penicillin, 0.04 μg mL −1 of cefradine, 0.08 μg mL −1 of cefadroxil and 0.1 μg mL −1 of cefalexin. The proposed method was also applied to analyze the β-lactam antibiotics in milk. Experimental results were in good agreement with those obtained by high performance liquid chromatography (HPLC) method with UV detection.

Biosensor Analysis of β-Lactams in Milk Using the Carboxypeptidase Activity of a Bacterial Penicillin Binding Protein

The applicability of a beta-lactam receptor protein for detection of beta-lactam antibiotics in milk using surface plasmon resonance (SPR) biosensor technology was investigated. The advantage of using a receptor protein instead of antibodies for detection of beta-lactams is that a generic assay, specific for the active form of the beta-lactam structure, is obtained. Two assays based on the enzymatic activity of the DD-carboxypeptidase from Actinomadura R39 were developed, using a Biacore SPR biosensor. The carboxypeptidase converts a tri-peptide into a di-peptide, a reaction which is inhibited in the presence of beta-lactams. Polyclonal antibodies against the 2 peptides were developed and used to measure the amount of enzymatic product formed (di-peptide assay) or the amount of remaining enzymatic substrate (tri-peptide assay), respectively. The 2 assays showed similar performances with respect to detection limits (1.2 and 1.5 microg/kg, respectively) and precision (coefficient of variation <5%) for penicillin G in milk. Several other beta-lactams were detected at or near their respective maximum residue limit. Furthermore, the 2 peptide assays were evaluated against 5 commercial kit tests in the screening of 195 producer milk samples. The biosensor assays showed 0% false-negative and 27% false-positive results, whereas the figures were 0% false-negative and 27-53% false-positive results for other screening tests investigated.

Development of an optical biosensor assay for detection of β-lactam antibiotics in milk using the penicillin-binding protein 2x*

An assay was developed for the detection of residues of penicillins and cephalosporins in milk using a surface plasmon resonance (SPR) biosensor. The assay was based on the inhibition of the binding of digoxigenin-labelled ampicillin (DIG-AMPI) to a soluble penicillin-binding protein 2x derivative (PBP 2x*) of Streptococcus pneumoniae. Samples were incubated with PBP 2x* in a first step, whereby β-lactams in positive samples would bind to the PBP 2x*. Non-complexed PBP 2x* was then allowed to form a complex with DIG-AMPI in a second incubation step. The formed DIG-AMPI/PBP 2x*-complexes were detected in a SPR-based biospecific interaction assay (BIA) for digoxigenin with an antibody against digoxigenin immobilised on the sensor chip. Although binding of matrix components to the sensor chip (non-specific binding) occurred, benzylpenicillin, ampicillin, amoxicillin, cloxacillin, cephalexin and cefoperazone could be detected in defatted bulk raw milk samples at concentrations corresponding to the maximum residue limits (MRL) set by the European Union. The influence of matrix components on the performance of the assay was examined in more detail by analysing individual raw milk samples from 19 cows. Compared to bulk raw milk samples, individual samples showed a higher level and variation of matrix interferences. Non-specific binding could be reduced to a lower and more constant level by a heat-treatment step, a centrifugation step and the addition of carboxymethylated dextran to the samples. With this sample preparation, benzylpenicillin could be detected at MRL (4 μg kg −1) in individual raw milk samples. Thus, the assay could be the basis for a screening test for routine use.

Biosensor analysis of penicillin G in milk based on the inhibition of carboxypeptidase activity

The β-lactam antibiotics, including penicillins, are the most important antimicrobial substances used for mastitis treatment. Consequently, this is also the most frequently occurring type of antibiotic residues in milk. Today, in addition to the traditional microbial inhibitor tests, rapid and sensitive receptor and immunoassays are used in residue control. Due to the limitations in throughput capacity of these tests, recent applications of automated biosensor technology in food analysis are of great interest. A surface plasmon resonance (SPR)-based biosensor (Biacore) was used to design an inhibition assay to detect β-lactam antibiotics in milk. A microbial receptor protein with carboxypeptidase activity was used as detection molecule. One advantage of using this receptor protein over antibodies that are more commonly used is that only the active, intact β-lactam structure is recognized, whereas most antibodies detect both active and inactive forms. In the presence of β-lactam antibiotics the formation of a stable complex between receptor protein and antibiotic inhibits the enzymatic activity of the protein. The decrease in enzymatic activity was measured using an antibody against the degraded substrate and penicillin G in milk samples was quantitatively determined. The limit of detection of the assay for penicillin G was determined to 2.6 μg kg −1 for antibiotic-free producer milk, which is below the European maximum residue limit (MRL) of 4 μg kg −1. The coefficient of variation at 4 μg kg −1 penicillin G, ranged between 7.3 and 16% on three different days.

Analysis of β-Lactam Antibiotics Using a Microbial Receptor Protein-based Biosensor Assay

An optical biosensor assay for detection of β-lactam antibiotics in milk based on a microbial receptor protein was developed. The assay uses a general sensor surface previously described with a small organic molecule (H1) immobilized. A conjugate between a β-lactam (cephalosporin C) and a monoclonal H1 antibody is injected across the sensor surface before injection of the sample mixed with receptor protein. Receptor inhibited by β-lactam residues in the milk sample will not bind to the sensor surface and the reduction in response is inversely related to the β-lactam concentration of the sample. The detection limit for a number of commonly used β-lactams was below or near the respective maximum residue limit and the relative standard deviation (CV) for penicillin G in milk was 6-12% in the interval 2.0-12.5 μ g kg-1. For application in the field further optimization is needed to solve problems related to non-specific binding to the sensor surface.

Multiresidue determination of β-lactam antibiotics in milk and tissues with the aid of high-performance liquid chromatographic fractionation for clean up

Screening of milk shipments for β-lactam antibiotic residues is mandatory in the USA and is widely used in other countries. Interpretation of positive screening test results has been difficult. Only six β-lactam antibiotics are approved for use in food-producing animals in the USA but many others are used in other countries. A multiresidue procedure was developed for identification and quantitation of unknown β-lactam antibiotics. The residues were extracted with acetonitrile and tetraethylammonium chloride. The extract was concentrated by evaporation and filtered. The concentrated extract was then loaded onto an HPLC column in 100% 0.01 M KH 2PO 4 and eluted with an acetonitrile gradient. Fractions corresponding to analytes of interest were collected and tested for antibiotics using rapid milk screening tests. Fractions testing positive were analyzed by HPLC. The identity of β-lactams was confirmed by treating a replicate with β-lactamase.

Simultaneous determination of β-lactam antibiotics in milk by ion-pair liquid chromatography

A simple and rapid ion-pairing liquid chromatographic method was developed for the simultaneous determination of five penicillins (PCs), ampicillin (AB-PC), benzylpenicillin (PC-G), cloxacillin (MCI-PC), dicloxacillin (MDI-PC) and nafcillin (NF-PC) in milk. These PCs are most frequently used for the treatment of mastitis of cows. These antibiotics were extracted with acetonitrile from milk and cleaned up by solid-phase extraction with a C 18 cartridge. PCs were separated on a Kaseisorb LC ODS-300-5 column with a mobile phase (1 ml/min) of acetonitrile–methanol–0.05 M potassium dihydrogenphosphate (20:10:80, v/v/v) mixture containing 5 m M of sodium 1-decanesulfonate adjusted to pH 3.5 and UV detection at 210 nm. The average recoveries of five PCs from milk fortified at 0.5 and 1.0 μg/ml ( n=5) were 79.8–89.4% with relative standard deviations ranging from 2.7 to 7.2%. The detection limit of PCs in milk were 0.03–0.05 μg/ml.

Determination of Penicillin G in Beef and Pork Tissues Using an Automated LC Cleanup

A procedure previously described for determination of β-lactam antibiotics in milk was modified for use with tissues. Tissues were extracted directly with acetonitrile (procedure I) to avoid degradation of penicillin G in water homogenates. Recovery of penicillin G in acetonitrile extracts of tissues was improved by addition of tetraethylammonium chloride and, in some cases, pH adjustment. The acetonitrile extracts were concentrated by evaporation and filtered. For cleanup, an automated high-performance liquid chromatography (HPLC) fractionation was used. The fraction corresponding to penicillin G was tested by a rapid screening test and, if positive, was analyzed by HPLC. For analysis, an Inertsil ODS-2 column was used with a mobile phase of 0.0067 M KH2PO4, 0.0033 M H3PO4−acetonitrile (68 + 32) with UV detection at 215 nm. For confirmation by treatment with β-lactamase, tissues were homogenized in water. An aliquot of the water homogenate was treated with β-lactamase prior to extraction with acetonitril...