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Rationale: Cloxacillin and flucloxacillin are prone to degradation and polymerization in humid and hot environments, and their polymers have long been recognized to trigger allergic manifestations. A series of the degradation and polymerized impurities in cloxacillin and flucloxacillin were separated and characterized to ensure safe use of these drugs by the public., Methods: By studying the chromatographic behavior of the degradation impurities and polymerized impurities in reversed-phase high-performance liquid chromatography (RP-HPLC) gradient elution, the impurities in cloxacillin and flucloxacillin were effectively separated and eluted. RP-HPLC tandem ion trap/time-of-flight mass spectrometry (MS) was applied to characterize the structures of unknown impurities eluted from the RP-HPLC methods for cloxacillin and flucloxacillin. The mechanisms of formation of the impurities in cloxacillin and flucloxacillin were also investigated., Results: The structures of 10 unknown impurities in cloxacillin and 8 unknown impurities in flucloxacillin were elucidated based on the high-resolution MS n data at positive and negative modes, respectively. Six polymerized impurities were found and characterized, of which three were from the polymerization of cloxacillin and three were from the polymerization of flucloxacillin., Conclusions: The study on the impurity profiles of cloxacillin and flucloxacillin provided a scientific basis for improving their production processes and quality control., (© 2024 John Wiley & Sons Ltd.)

Cloxacillin and oxacillin are group M penicillins. The therapeutic monitoring of plasma concentrations of these antibiotics and those of their hydroxymethylated metabolites is of great clinical interest, especially in the choice of an adequate dosage allowing an effective treatment while limiting the occurrence of undesirable effects and the development of bacterial resistance. In this context, we conducted this work aiming at developing and validating a method allowing the determination of cloxacillin and oxacillin as well as the identification of their active metabolites in different biological matrices (CSF and plasma) using turbulent flow liquid chromatography coupled to high-resolution mass spectrometry. To do this, we carried out several optimisation tests. Subsequently, we validated our method according to the latest bioanalytical validation recommendations of the European Medicines Agency. The validation results showed that our method is specific and sensitive. We obtained good linearity in the range 0.5 to 100 µg/mL with correlation coefficients above 0.995. The lower limit of quantification was 0.5 µg/mL for each analyte. The method was found to be accurate with repeatability and reproducibility coefficients of variation below 15 %. Our method is also accurate with bias values below 15 %. Recovery values ranged from 87 % to 95 %. Finally, we were able to apply our method to the therapeutic monitoring of the analysed molecules and to identify their active metabolites. Our results suggest that LC-MS shows superiority in the therapeutic monitoring of these antibiotics due to the superiority of specificity shown by this method. This assay method can be routinely used for the daily plasma assays of patients treated with these antibiotics in the context of therapeutic monitoring., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Antibiotics are considered emerging pollutants which indiscriminate use has led to the development of antibiotic-resistant bacteria, while their improper disposal has caused adverse effects to the environment and human health. Thus, the development of devices or techniques capable of detecting antibiotics with high sensitivity, low detection limits, and reasonable cost becomes of prime importance. In this work, an electronic tongue (e-tongue) based on molybdenum disulfide (MoS 2 ) and graphene oxide (GO) was developed and employed to detect four distinct antibiotics, namely cloxacillin benzathine, erythromycin, streptomycin sulfate, and tetracycline hydrochloride. The five sensing units of the e-tongue were obtained using the drop-casting method to modify gold interdigitated electrodes with MoS 2 and GO. Using Principal Component Analysis to process the experimental data allowed the e-tongue to recognize samples contaminated with distinct antibiotics at varied concentrations from 0.5 to 5.0 nmol L -1 . Analyses with real samples were also performed using river water and human urine and the electronic tongue was able to differentiate the samples at a nanomolar level. The proposed system represents a sensitive and low-cost alternative for antibiotic analyses in different liquid media., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Nowadays, the abuse of antibiotics in aquaculture has generated considerable problems for food safety. Therefore, it is imperative to develop a simple and selective method for monitoring illegal use of antibiotics in aquatic products. In this study, a method combined molecularly imprinted membranes (MIMs) extraction and liquid chromatography was developed for the selective analysis of cloxacillin from shrimp samples. The MIMs was synthesized by UV photopolymerization, and characterized by scanning electron microscope, Fourier transform infrared spectra, thermo-gravimetric analysis and swelling test. The results showed that the MIMs exhibited excellent permselectivity, high adsorption capacity and fast adsorption rate for cloxacillin. Finally, the method was utilized to determine cloxacillin from shrimp samples, with good accuracies and acceptable relative standard deviation values for precision. The proposed method was a promising alternative for selective analysis of cloxacillin in shrimp samples, due to the easy-operation and excellent selectivity., (Copyright © 2018. Published by Elsevier Ltd.)

There is an increasing demand for rapid, sensitive, and low cost analytical methods to routinely screen antibiotic residues in food products. Conventional detection of antibiotics involves sample preparation by liquid-liquid or solid-phase extraction, followed by analysis using liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis (CE), or gas chromatography (GC). The process is labor-intensive, time-consuming, and expensive. In this study, we developed a new analytical method that combines magnetic molecularly imprinted polymer (MMIP)-based sample preparation with surface-enhanced Raman spectroscopy (SERS)-based detection for quantitative analysis of cloxacillin in pig serum. MMIP microspheres were synthesized using a core-shell technique. The large loading capacity and high selectivity of the MMIP microspheres enabled efficient extraction of cloxacillin, while the magnetically susceptible characteristics greatly simplified sample handling procedures. Low cost and robust SERS substrates consisting of vertical gold capped silicon nanopillars were fabricated and employed for the detection of cloxacillin. Quantitative SERS was achieved by normalizing signal intensities using an internal standard. By coherently combining MMIP extraction and silicon nanopillar-based SERS biosensor, good sensitivity toward cloxacillin was achieved. The detection limit was 7.8 pmol. Cloxacillin recoveries from spiked pig plasma samples were found to be more than 80%.

Penicillins are typical of complex ionic samples which likely contain large number of degradation-related impurities (DRIs) with different polarities and charge properties. It is often a challenge to develop selective and robust high performance liquid chromatography (HPLC) methods for the efficient separation of all DRIs. In this study, an analytical quality by design (AQbD) approach was proposed for stability-indicating method development of cloxacillin. The structures, retention and UV characteristics rules of penicillins and their impurities were summarized and served as useful prior knowledge. Through quality risk assessment and screen design, 3 critical process parameters (CPPs) were defined, including 2 mixture variables (MVs) and 1 process variable (PV). A combined mixture-process variable (MPV) design was conducted to evaluate the 3 CPPs simultaneously and a response surface methodology (RSM) was used to achieve the optimal experiment parameters. A dual gradient elution was performed to change buffer pH, mobile-phase type and strength simultaneously. The design spaces (DSs) was evaluated using Monte Carlo simulation to give their possibility of meeting the specifications of CQAs. A Plackett-Burman design was performed to test the robustness around the working points and to decide the normal operating ranges (NORs). Finally, validation was performed following International Conference on Harmonisation (ICH) guidelines. To our knowledge, this is the first study of using MPV design and dual gradient elution to develop HPLC methods and improve separations for complex ionic samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: The co-existence of malaria with bacterial infections is common in the tropics, hence the concurrent use of antimalarials and antibiotics., Objective: This study aimed to investigate the effect on pharmacokinetics and antimicrobial activity of co-administration of quinine and combined ampicillin-cloxacillin., Methods: In total, 14 healthy adults received single oral doses of ampicillin-cloxacillin combination alone and with quinine in a randomized crossover manner. Urine samples collected at predetermined intervals over 48 h were analysed. The effect of quinine on minimum inhibitory concentrations (MICs) of ampicillin and cloxacillin were determined against Staphylococcus aureus by agar diffusion, agar dilution, and broth dilution., Results: Quinine significantly reduced the rate and extent of excretion of ampicillin and cloxacillin (p < 0.0002). The total amounts of ampicillin and cloxacillin excreted unchanged (Du(∞)) alone were 217.10 ± 53.82 and 199.0 ± 64.29 mg versus 126.40 ± 50.63 and 135.20 ± 52.24 mg, respectively, with quinine. Respective maximum excretion rates (dDu/dt max) for ampicillin and cloxacillin were 43.55 ± 19.41 and 77.64 ± 29.65 mg/h alone versus 18.01 ± 8.52 and 53.16 ± 20.72 mg/h with quinine. This indicates a significant reduction in Du(∞)and dDu/dt max by 41.78 and 58.65 % for ampicillin and 32.06 and 31.53 % for cloxacillin. Conversely, the disposition of quinine was unaffected by ampicillin-cloxacillin (p > 0.1). The MIC of antibiotics alone versus with quinine, respectively, were 0.11 ± 0.04 and 0.78 ± 0.1 µg/ml for ampicillin, and 0.18 ± 0.1 and 0.92 ± 0.4 µg/ml for cloxacillin, with a five- to sevenfold increase (p > 0.01); indicating a decrease in antimicrobial activity by quinine., Conclusions: Quinine therefore, reduced the bioavailability and the antimicrobial activity of ampicillin-cloxacillin upon co-administration, which may have therapeutic implications. Caution is required with the co-administration of these medicines.

A novel chemiluminescence (CL) system is introduced based on the oxidation of carminic acid by KMnO4 in acidic conditions. CdS quantum dots (QDs) were synthesized using a facile hydrothermal method which efficiently enhanced the intensity of the CL system. A possible mechanism for the proposed system is presented using the kinetic curves, CL spectra, photoluminescence (PL), and ultraviolet-visible (UV-Vis) analysis. The emission intensity of the KMnO4-carminic acid-CdS QDs system was quenched in the presence of a trace level of cloxacillin. Based on this quenching effect, a novel and sensitive flow injection CL method was developed for determining cloxacillin concentrations. At optimal experimental conditions, the decreased CL intensity had a good linear relation with the cloxacillin concentration in the range of 0.008 to 22.0 mg L(-1). The detection limit (3σ) was 5.8 µg L(-1). The precision of the method was calculated by analyzing samples containing 4.0 mg L(-1) of cloxacillin (n=11), and the relative standard deviations (RSD%) were 2.08%. The feasibility of the method is also demonstrated for determining cloxacillin concentrations in environmental water samples and a pharmaceutical formulation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Ampicillin exhibited a negative and a positive cotton effects on the circular dichroism (CD) spectra in the wavelength range of 200-280nm. Cloxacillin showed a positive cotton band peaking at 228nm. Three sensitive, precise and accurate CD spectroscopic methods have been developed for the determination of ampicillin and cloxacillin. Method A was used for the determination of ampicillin in presence of cloxacillin by measuring ellipticity at 206nm. Method B and C were employed to determine ampicillin and cloxacillin based on evaluation of ellipticity at 233nm and 228nm, respectively. Methods A, B and C showed linearity in the concentration range of 10-40μgmL(-1), 5-40μgmL(-1) ampicillin and 10-80μgmL(-1) cloxacillin, respectively. The method A was successfully applied to the determination of ampicillin in commercial dosage forms containing equivalent amount of cloxacillin., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A simple, linear gradient, rapid, precise and stability-indicating RP-UPLC method was developed for the determination of Cloxacillin Sodium in its bulk form and formulation. Ultra performance liquid chromatography, a most promising advancement in a world of chromatography, reduces analysis time, increases reliability through higher resolution, sensitivity and selectivity as well as used as an economic method due to reducing solvent consumption. A chromatographic separation of a drug as well as its degradants was achieved using Waters acquity BEH, 2.1 × 100 mm, 1.7 μm C18 column with gradient of mobile phase A: phosphate buffer, pH 6.8 and mobile phase B: methanol:acetonitrile (75:25). The drug and degradants were monitored at a detection wavelength of 225 nm with a flow rate of 0.35 mL/min and an injection volume of 10 µL. The temperature of the column and auto sampler compartments was at 30°C and 25°C ± 1°C, respectively. The retention time of the drug was ∼6.9 min. The resolution of the drug and degradant peak was >1.5 in all cases. Force degradation of CLOX SOD was carried under alkaline, acidic, oxidative, thermal, photo degradation conditions and it was analyzed by the proposed method. The drug degrades under alkaline, acidic and oxidative conditions but was stable in temperature and light. A developed method was validated as per ICH guidelines using validation parameters such as precision, linearity and range, limit of quantification, specificity, assay and robustness., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In this paper, a rapid and sensitive flow-injection chemiluminescence (flow-CL) system was developed for the determination of cloxacillin sodium in environmental water samples and pharmaceutical preparations. The method was based on the enhancement effect of cloxacillin sodium on the CL reaction of luminal-H₂O₂-CuO nanosheets (NSs) in alkaline medium. The CuO nanosheets were synthesized using a green sonochemical method. The physical properties of the synthesized CuO nanosheets were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM) analyses. The influences of various experimental factors such as H₂O₂, NaOH, luminol and CuO nanosheets concentrations were investigated. Under the optimum conditions, the enhanced CL intensity was linearly related to the concentration of cloxacillin sodium in the range of the 0.05-30.00 mg L(-1) with a correlation coefficient of 0.995. The corresponding detection limit (3σ) was calculated to be 0.026 mg L(-1). The relative standard deviation (RSD) of the developed method was 2.21% with 11 repeated measurements of 4.00 mg L(-1) cloxacillin sodium. Also, a total analysis time per sample was 30 s which confirmed the rapidity of the proposed method. The analytical applicability of the proposed CL system was assessed by determining cloxacillin sodium in spiked environmental water samples and pharmaceutical preparation. Furthermore, the possible mechanism of CL reaction was discussed., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The aim of the study was to evaluate the interference of acid and alkaline detergents employed in the cleaning of milking equipment of caprine dairy farms on the performance of microbial tests used in antibiotic control (BRT MRL, Delvotest MCS, and Eclipse 100). Eight concentrations of commercial detergents, five acid (0-0.25%) and five alkaline (0-1%) were add to antimicrobial-free goat's milk to evaluate the detergent effect on the response of microbial inhibitor tests. To evaluate the effect of detergents on the detection capability of microbial tests two detergents at 0.5 ml/l (one acid and one basic) and eight concentrations of four β-lactam antibiotics (ampicillin, amoxicillin, cloxacillin and benzylpenicillin) were used. Milk without detergents was used as control. The spiked samples were analysed twelve times by three microbial tests. The results showed that the presence of acid detergents did not affect the response of microbial tests for any of the concentrations tested. However, at concentrations equal to or greater than 2 ml/l alkaline detergents positive results were found in microbial tests (16.7-100%). The detection limits of the screening tests for penicillins were not modified substantially by the presence of detergents. In general, the presence of acid and alkaline detergents in goat's milk did not produce a great interference in the microbial tests, only high concentrations of detergents could cause non-compliant results, but these concentrations are difficult to find in practice if proper cleaning procedures are applied in goat dairy farms.

In the present study we developed and validated a liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay for the determination of flucloxacillin in human plasma and microdialysis samples and cloxacillin in microdialysis samples, using oxacillin as the internal standard for the assay. The samples were separated on a UPLC BEH C18,1.7 μm column (2.1x50mm) and analyzed by a tandem-quadrupole mass spectrometer in multiple reaction monitoring mode using an electronspray ionization interface. For flucloxacillin the method was demonstrated to be accurate and precise in the linearity range of 1-30 mg/L in plasma and 0.05-5.0 mg/L for microdialysate with a regression coefficient (r) of 0.9986 and 0.9989 in plasma and microdialysate respectively. For cloxacillin it was accurate and precise in the range of 0.1-5.0 mg/L for microdialysate with a regression coefficient of 0.9972. The method presents a high sensitivity for flucloxacillin (lower limit of quantification of 1 mg/L for plasma and 0.05 mg/L for microdialysis samples) combined with a low within- and between-day variation (<5.0% for flucloxacillin and cloxacillin in microdialysis samples and <6.5% for plasma samples of flucloxacillin). The validation experiments for the microdialysis probes showed a relative recovery of 85.5% for flucloxacillin at a flow rate of 1.0 μL/min. The results justify the use of this assay for clinical studies for measuring free unbound tissue concentrations of flucloxacillin in patients with a Staphylococcus aureus bacteremia.

A validation study designed to meet the requirements of the AOAC Research Institute and the U.S. Food and Drug Administration, Center for Veterinary Medicine (FDA/CVM) was conducted for a receptor and antibody-based, immunochromatographic method (BetaStar Plus) for detection of beta-lactam antibiotic residues in raw, commingled bovine milk. The assay was found to detect amoxicillin, ampicillin, ceftiofur, cephapirin, cloxacillin, and penicillin G at levels below the FDA tolerance/safe levels, but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments (NCIMS). Results of the part I (internal) and part II (independent laboratory) dose-response studies employing spiked samples were in close agreement. The test was able to detect all six drugs at the approximate 90/95% sensitivity levels when presented as incurred residues in milk collected from cows that had been treated with the specific drug. Selectivity of the assay was 100%, as no false-positive results were obtained in testing of 1031 control milk samples. Results of ruggedness experiments established the operating parameter tolerances for the BetaStar Plus assay. Results of cross-reactivity testing established that the assay detects certain other beta-lactam drugs (dicloxacillin and ticarcillin), but it does not cross-react with any of 30 drugs belonging to other classes. Abnormally high bacterial or somatic cell counts in raw milk produced no interference with the ability of the test to detect beta-lactams at tolerance/safe levels.

An ion-paired extraction (IPE) has been developed for the analysis of penicillin antibiotics (penicillin G, oxacillin and cloxacillin) in beef and milk samples using tetrabutylammonium bromide (TBABr) as ion-pairing agent and binary water-acetonitrile as extractant. The factors affecting the IPE efficiency were optimized including solution pH, volume of acetonitrile (ACN), concentration of TBABr and electrolyte salt (NH(4))(2)SO(4). The optimum IPE conditions were 10 mmol L(-1) phosphate buffer pH 8, 2 mL of ACN, 6 mmol L(-1) of TBABr and 2.5 mL of saturated ammonium sulfate. Under the HPLC condition: an Xbridge™ C18 reversed-phase column, isocratic elution of 5 mmol L(-1) phosphate buffer (pH 6.6) and acetonitrile (75:25, v/v) and a flow rate of 1 mL min(-1), with UV detection at 215 nm, the separation of three penicillins was achieved within 10 min. Under the selected optimum conditions, the enhancement of 21-53 folds compared to that without preconcentration and limits of detection (LODs) of 1-2 ng mL(-1) were obtained. Good reproducibility was achieved with RSD<2% for retention time and <5% for slope of calibration curves. The average recoveries higher than 85% were obtained. The proposed IPE-HPLC method has shown to be high efficient preconcentration and analysis method for penicillin residues in beef and milk with LOD lower than the maximum residue limits., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The study examined combined photo-Fenton-SBR treatment of an antibiotic wastewater containing amoxicillin and cloxacillin. Optimum H(2)O(2)/COD and H(2)O(2)/Fe(2+) molar ratio of the photo-Fenton pretreatment were observed to be 2.5 and 20, respectively. Complete degradation of the antibiotics occurred in one min. The sequencing batch reactor (SBR) was operated at different hydraulic retention times (HRTs) with the wastewater treated under different photo-Fenton operating conditions (H(2)O(2)/COD and H(2)O(2)/Fe(2+) molar ratio). The SBR performance was found to be very sensitive to BOD(5)/COD ratio of the photo-Fenton treated wastewater. Statistical analysis of the results indicated that it was possible to reduce the Fe(2+) dose and increase the irradiation time of the photo-Fenton pretreatment. The best operating conditions of the combined photo-Fenton-SBR treatment were observed to be H(2)O(2)/COD molar ratio 2, H(2)O(2)/Fe(2+) molar ratio 150, irradiation time 90 min and HRT of 12h. Under the best operating conditions, 89% removal of sCOD with complete nitrification was achieved and the SBR effluent met the discharge standards., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A previously reported multiresidue method for the analysis of 11 important beta-lactams (amoxicillin, ampicillin, cefazolin, cephalexin, cloxacillin, desfuroylceftiofur cysteine disulfide (DCCD), deacetylcephapirin, dicloxacillin, nafcillin, oxacillin, and penicillin G) in bovine kidney has been further streamlined. The method is based on a simple extraction using acetonitrile-water (4:1, v/v), followed by dispersive solid-phase extraction clean-up with C(18) sorbent, concentration of an extract aliquot, and filtration of the final extracts using syringeless filter vials, which are used for the sample introduction in the liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. The recoveries have been improved by adding the internal standard [(13)C(6)]sulfamethazine to the homogenized sample before the extraction step, which enabled a proper control of the volume changes during the sample preparation. Average recoveries of fortified samples were 87-103% for all beta-lactams, except for DCCD, which had an average recovery of 60%. Based on the results of the stability study and LC mobile phase tests, methanol has been eliminated from the entire method, including the LC-MS/MS analysis. The best overall LC-MS/MS (electrospray positive ionization) performance was achieved by using 0.1% formic acid as an additive in both parts of the mobile phase, in water and in acetonitrile. To prevent carry-over in the LC-MS/MS analysis, the LC method was divided into two parts: one serving as an analytical method for injection of the sample and elution of the analytes and the other one, starting at a highly organic mobile phase composition, being dedicated for injection of a solvent, washing of the system, and equilibration of the column to the initial conditions of the analytical method. In this way, a blank solvent is injected after each sample, but these in-between injections contribute minimally to the overall sample throughput.

A weak ion exchange monolithic column prepared by modifying the GMA-MAA-EDMA (glycidyl methacrylate-methacrylic acid-ethylene glycol dimethacrylate) monoliths with ethylenediamine was applied to remove matrix compounds in biological fluid. Using this monolithic column, on-line clean-up and screening of oxacillin and cloxacillin in human urine and plasma samples had been investigated. Chromatography was performed by reversed-phase HPLC on a C(18) column with ultraviolet detection at 225 nm. Results showed that the ion exchange monolithic column could be used for deproteinization and retaining oxacillin and cloxacillin in human urine and plasma, which provided a simple and fast method for assaying drugs in human urine and plasma.

Ampicillin and cloxacillin were degraded together under different stress test conditions prescribed by International Conference on Harmonization. The samples so generated were used to develop a stability-indicating high performance liquid chromatographic (HPLC) method for the two drugs. The drugs were well separated from degradation products using a reversed-phase (C-18) column and a mobile phase comprising of acetonitrile:phosphate buffer (pH 5.0), which was delivered initially in the ratio of 15:85 (v/v) for 1 min, then changed to 30:70 (v/v) for next 14 min, and finally equilibrated back to 15:85 (v/v) from 15 to 20 min. Other HPLC parameters were: flow rate, 1 ml/min; detection wavelength, 225 nm; and injection volume, 5 microl. The method was validated for linearity, precision, accuracy, specificity and selectivity. It was also compared with the assay procedures given in British Pharmacopoeia for individual drugs. Similar results were obtained, indicating that the proposed single method allowed selective analysis of both ampicillin and cloxacillin, in the presence of their degradation products formed under a variety of stress conditions. The developed procedure was also applicable to the determination of instability of the drugs in commercial products.

The aim of present study was to evaluate the application of a hydrophilic matrix tablet capable of polyion complex (PIC-tablet) to a controlled-release device for highly water-soluble drugs. The PIC-tablet was prepared from a mixture of dextran sulfate and [2-(diethylamino)ethyl] dextran chloride, and diltiazem hydrochloride was used as a model drug. Release tests revealed that the drug release was sustained even in 50% drug loading and was influenced by ionic strength but not by pH in medium. The drug release mechanism was thus investigated from the viewpoint of drug micelle forming property. The micelle forming ability of diltiazem was examined by the conductivity method, and was found to be influenced by ionic strength but not by pH value in accordance with the release tests. The results suggested that the drug's micelle interacted with the polyionic matrix. Further studies were conducted using metoprolol tartrate and thiamine hydrochloride as cationic drugs and sodium cloxacillin and sodium salicylic acid as anionic ones. The release profiles of the micelle-forming drugs metoprolol tartrate and sodium cloxacillin were also suppressed in spite of different solubility or opposite ionic charge from diltiazem hydrochloride. These findings demonstrated that the PIC-tablet is a promising device for oral controlled release delivery of water-soluble drugs with good micelle-forming ability.

A quantitative method for the determination of four penicillin antibiotics, amoxicillin (AMO), oxacillin (OXA), cloxacillin (CLO), and dicloxacillin (DICLO), has been developed. Separation was achieved on an Inertsil ODS-3 (250 x 4 mm, 5 microm) column after selective extraction of penicillin drugs from biological matrices by means of SPE. Gradient elution with a mobile phase consisting of 0.1% TFA (pH 1) and ACN, and PDA detection with monitoring at 240 nm was applied. Salicylic acid (5 ng/microL) was used as the internal standard. RP-8 Adsorbex Merck cartridges provided high absolute recoveries (98-101%). The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, and sensitivity. Repeatability (n = 8) and between-day precision (n = 8) revealed RSD <10%. Recoveries from biological samples ranged from 91 to 103%. The detection limits were estimated as 3.3 ng for AMO, OXA, and CLO, and 6.6 for DICLO in blood plasma. LOD in whole blood and urine was 6.6 ng. Injection volume was 20 microL. The method was applied to commercially available AMO containing pharmaceuticals and spiked biological matrices. The method was also applied to biological samples after AMO oral administration, where the drug was successfully identified and quantified.

Objective: To determine whether half-udder intramammary infusion of cloxacillin results in transfer of cloxacillin from treated to untreated mammary gland quarters within nonlactating cows, and, if so, at what concentrations, and to determine whether selection of ipsilateral versus diagonal-contralateral quarters for treatment affects cloxacillin transfer among quarters., Animals: 20 Holstein-Friesian cows from a dairy herd., Procedures: A within-cow half-udder comparison trial was used in which 2 of 4 mammary gland quarters (ipsilaterally or diagonally) received an intramammary infusion of cloxacillin on day 1 of the nonlactating period. Three days later, milk samples were taken from all untreated quarters and high-pressure liquid chromatography was used to detect and quantify milk cloxacillin concentrations., Results: Cloxacillin was detected in 25% of all untreated mammary gland quarters. Mean cloxacillin concentration in untreated quarters was below minimum inhibitory concentrations for targeted mastitis pathogens. No significant difference in cloxacillin concentrations was found in the ipsilateral or diagonal treatment groups., Conclusions and Clinical Relevance: Within-cow half-udder comparison trials are valid for antimicrobial trials in nonlactating cows, although transfer of antimicrobials does occur in trace concentrations. Ipsilateral or diagonal-contralateral treatment designs perform similarly. This type of design is economical for researchers, although care must be taken to account for within-cow clustering of mammary gland quarter data.

A simple, quick and sensitive capillary electrophoretic technique has been developed for the pharmaceutical analysis of isoxazolylpenicillins (oxacillin, cloxacillin and dicloxacillin) at trace levels for the first time. This method comprises large-volume sample stacking using the electroosmotic flow (EOF) pump (LVSEP), separation using 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as selective complex-forming background electrolyte additive, and direct UV detection. A complete resolution was achieved in the optimal background electrolyte containing 5.2 mM HP-beta-CD. LVSEP was successfully applied in their determinations to improve the sensitivity, where the EOF in the buffer zone was suppressed by using an acidic buffer with pH 3.6. The detection limits of the current technique were found to be 2.0 microg/L for each of the isoxazolylpenicillins based on the signal-to-noise ratio of 3. The curves of peak response versus concentration were linear from 5.0 to 400.0 microg/L with regression coefficients of 0.9982, 0.9986 and 0.9976, respectively. The interaction of isoxazolylpenicillins with HP-beta-CD was discussed. The association constants for complexes of HP-beta-CD with isoxazolylpenicillins were determined by electrophoretic method. The obtained association constants were 27.3, 34.9, and 48.5 M(-1), respectively, being proportional to their hydrophobic properties and steric hindrances. A simple and easy-manipulative sample preparation method was developed and validated by analyzing commercially available milk samples. It was found that with current sample preparation process and instrumentation system, 0.1 mL of milk sample is enough for the analysis of isoxazolylpenicillins to meet European Union (EU) guideline of 30 microg/kg.

A simple and rapid method for the simultaneous determination of five penicillins (ampicillin, penicillin G, penicillin V, oxacillin and cloxacillin) in muscle, liver and kidney tissues using high-performance liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) was developed. Mass spectral acquisition was done in the negative ion mode by applying selected reaction monitoring (SRM). The five penicillins were extracted with water, and the extracted solution was cleaned up on a C18 cartridge. Phenethicillin was added as an internal standard, and the extract was diluted with water for injection into the LC-ESI-MS/MS. The recoveries of the five penicillins were in the range of 77.3-99.8% from muscle, liver and kidney fortified at 10-250 ng/g. The detection limits for ampicillin were 6 ng/g in muscle and kidney and 15 ng/g in liver. For penicillin G and penicillin V, the detection limits were 2 ng/g in muscle and kidney and 5 ng/g in liver. For oxacillin and cloxacillin, the detection limits were 4 ng/g in muscle and kidney and 10 ng/g in liver. Twenty-three muscle, fourteen liver and twenty-two kidney samples from the markets were analyzed by this method. No penicillins were detected in any sample.

An analytical system was developed for detection of antibiotic residues in bovine milk. The method is based on competitive fluorescent immunoassays in glass capillary tubes (U.S. Patent No. 5,624,850). The system consists of an assay cartridge containing 4 glass capillaries, a reagent tray with 4 wells of dried reagents, and a Parallux processor, which processes the assay, reads fluorescent output, and reports test results. Minimum sensitivity for detection of 6 beta-lactam antibiotics in bovine milk was determined to be penicillin-G, 3.2 ppb; ampicillin, 2.9 ppb; amoxicillin, 3.6 ppb; cloxacillin, 7.4 ppb; cephapirin, 16.3 ppb; and ceftiofur, 33.7 ppb. The assay system was also specific and sensitive for detection of incurred residues at U.S. Food and Drug Administration tolerance levels: penicillin-G, 5 ppb; ampicillin, 10 ppb; amoxicillin, 10 ppb; cloxacillin, 10 ppb; cephapirin, 20 ppb; and ceftiofur, 50 ppb. There was no interference in detection of minimum sensitivity levels of antibiotic by the presence of somatic cells at approximately 1 x 10(6) cells/mL. Milk containing 3 x 10(6) cells/mL bacteria commonly found in mastitic milk also showed no interference when tolerance levels of antibiotic were present. There was no detectable interference on results by a wide variety of non-beta-lactam drugs.

A high-performance liquid-chromatographic method has been developed for the determination of five penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, and dicloxacillin) at trace levels in commercially available milk samples. This method comprises extraction of the lipids with ethyl acetate, clean-up and concentration on a C-18 solid-phase extraction column, and derivatization with 1,2,4-triazole and mercury(II) chloride solution, pH 8, at 65 degrees C for 10 min. The derivatized compounds are eluted from a C-2 column with a mobile phase containing acetonitrile and phosphate buffer loaded with sodium thiosulfate and tetrabutylammonium hydrogen sulfate as ion-pairing reagent. The limit of determination was found to be 4 microg L(-1) milk for benzylpenicillin and 10 microg L(-1) for the others. This meets EU criteria according to decision No. 93/256/EEC.

A high-performance liquid chromatographic (HPLC) method based on solid-phase extraction (SPE) was developed for determination of amoxicillin, penicillin G (benzylpenicillin), ampicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin in muscle, liver and kidney tissues of pigs and cattle. The compounds were extracted in aqueous solution by precipitation of organic materials with a mixture of sulphuric acid and sodium tungstate. The extract was cleaned up by SPE on a divinylbenzene-co-N-vinylpyrrolidone polymeric sorbent. Further clean-up was performed by liquid-liquid partition with diethyl ether. The extract was derivatised with benzoic anhydride and 1,2,4-triazole mercury (II) reagent. Chromatography was performed by reversed-phase gradient HPLC on a C18 column with ultraviolet detection at 323 nm. The limits of detection estimated by a conservative model were in the range 8.9-11.1 microg/kg for amoxicillin, penicillin G, ampicillin, oxacillin, cloxacillin and nafcillin and 18.3-20.9 microg/kg for dicloxacillin. The mean recovery range was 66-77% for amoxicillin, 73-75% for penicillin G, 81-82% for ampicillin, 73-76% for oxacillin, 74-75% for cloxacillin, 66-72% for nafcillin and 58-65% for dicloxacillin.

A method for the analysis of degraded products of amoxycillin, ampicillin and cloxacillin in drug formulations, obtained as a result of their base hydrolysis is described. Simultaneous spectrophotometric and volumetric determinations of the antibiotic is based on the neutralization of the degraded product by dilute hydrochloric acid to get a pH approximately 2 to be conducive for redox titration using potassium iodate as titrant. A red purple colour is developed in carbon tetrachloride at the end point. Spectrophotometry is done after separating the organic layer and measuring the absorbance of red-purple colour at lambda(max) 520 nm. The pathways of different degraded products and their oxidation mechanism is described on IR, TLC and UV spectroscopic studies.

A multiresidue analytical method was developed for the simultaneous determination of benzylpenicillin (PCG), phenoxymethylpenicillin (PCV), oxacillin (MPIPC), cloxacillin (MCIPC), nafcillin (NFPC) and dicloxacillin (MDIPC) in meat. The method involves the use of an ion-exchange cartridge for sample clean-up followed by ion-pair high-performance liquid chromatography with ultraviolet detection. The recoveries of PCG, PCV, MPIPC, MCIPC, NFPC and MDIPC from pork muscle spiked at levels of 0.5, 0.1 and 0.05 mg/kg were in the range of 77-90, 73-95 and 80-93% with coefficients of variation of 0.5-1.7, 1.6-4.4 and 3.2-6.6%, respectively. For beef muscle spiked at levels of 0.5, 0.1 and 0.05 mg/kg, the recoveries of these compounds were 83-92, 71-86 and 77-90% with coefficients of variation of 1.7-4.4, 2.6-7.0 and 3.9-6.4%, respectively. The detection limits for each penicillin were 0.02 mg/kg in meat.

Fusidic acid and cloxacillin were studied in patients who underwent joint aspiration for noninfectious disorders. Nine patients were given oral 500 mg fusidic acid tid for 72 h, the last dose being given 4, 8 or 12 h before the joint aspiration. Cloxacillin was administered in a single 2 g iv dose to 9 patients, 0.5, 4 or 8 h before the aspiration. Bactericidal activity was determined against five isolates each of methicillin-susceptible and methicillin-resistant Staphylococcus aureus. Satisfactory activity (> or = 1:3) was detected in the serum in patients who received fusidic acid, while in the synovial fluids titres reflected borderline effectiveness (c. 1:2). Despite drug concentrations and excellent MICs, fusidic acid demonstrated markedly lower inhibitory and bactericidal activity against S. aureus than did cloxacillin.

A recently developed multiresidue method using gradient liquid chromatographic conditions for analysis of residues of beta-lactam drugs in bovine muscle tissues after precolumn derivatization with acetic anhydride was modified to permit isocratic analysis. The modification included replacing the acylating reagent, acetic anhydride, with benzoic anhydride and increasing sample size for extraction from 2 to 3 g. The modifications have reduced analysis time per sample from > 1 h to about 22 min without loss in detection sensitivities for the beta-lactams, resulting in a significant increase in the number of samples that can be analyzed in 1 day from 1 to 8. The modifications make the multiresidue LC method more suitable than the original method for use in regulatory programs for routine analysis of these veterinary drug residues in bovine muscle tissues.

Antibiotic residues in animal tissues can be detected by various screening tests based on microbial inhibition. In the 7-plate assay used by the U.S. Department of Agriculture's Food Safety and Inspection Service (FSIS), penicillinase is incorporated into all but one plate to distinguish beta-lactam antibiotics from other types. However, beta-lactams such as cloxacillin and the cephalosporins are resistant to degradation by penicillinase. They may not be identified as beta-lactams by this procedure, and thus, they may be identified as unidentified microbial inhibitors (UMIs). However, these penicillinase-resistant compounds can be degraded by other beta-lactamases. The present study describes an improved screening protocol to identify beta-lactam antibiotics classified as UMIs. A multiresidue liquid chromatographic procedure based on a method for determining beta-lactams in milk was also used to identify and quantitate residues. The 2 methods were tested with 24 tissue FSIS samples classified as containing UMIs. Of these, 3 contained penicillin G, including one at a violative level, and 5 contained a metabolite of ceftiofur. The others were negative for beta-lactam antibiotics.

A liquid chromatographic method with UV detection at 325 nm was developed for simultaneous determination of amoxicillin, ampicillin, penicillin G, and cloxacillin residues in bovine muscle tissue as their mercaptide derivatives. The penicillins are extracted from bovine tissues with 0.1 M phosphate buffer (pH 8.5), cleaned up on a t-C18 Sep-Pak cartridge, and eluted with 2 mL acetonitrile. After the acetonitrile in the eluate is evaporated to dryness, the residue is dissolved in 200 microL (40 + 60, v/v) acetonitrile-phosphate buffer (pH 6.5) and derivatized with acetic anhydride and mercuric chloride in the presence of 1,2,4-triazole at 65 degrees C for 30 min. Gradient analysis on a Spherisorb 5 microns ODS(2) (octadecyl silane) analytical column using a binary mobile phase consisting of acetonitrile and 0.10 M phosphate buffer (pH 6.5) in the presence of 0.0157 M sodium thiosulfate at 1 mL/min permits determination of each intact penicillin in bovine muscle tissue at > or = 10 ppb with recoveries > or = 72%. This laboratory method provides detection sensitivities equivalent to those of rapid tests used for screening beta-lactam drug residues in bovine tissue samples for regulatory enforcement.

Under the auspices of the International Dairy Federation's Group E503, a collaborative study of the Delvotest SP multiplate microbial inhibitor test was performed to gain information about the detection limits of 2 antimicrobial agents (a beta-lactam and a sulfa compound), the variation of test results between users and 2 batches of the test, and the reasons for deviating results. Lyophilized milk samples spiked with various concentrations of cloxacillin or sulfamethoxazole were analyzed. Each substance/concentration combination was tested with each of 2 test batches 14 or 15 times per participating laboratory. Test results were to be read by more than one person and reported on separate forms. Results were obtained from 29 laboratories, which included 5 with no experience and 11 with limited experience with this test. Detection limits for cloxacillin (22.5 or 30 micrograms/kg, depending on batch) and sulfamethoxazole (45 micrograms/kg) were established from dose-response curves. A small difference in cloxacillin detection levels between the 2 test batches was observed. Analyses of samples gave almost unanimous results (> or = 95%). Of deviating results, defined as anomalous results (1.4% of readings), half could be attributed to human errors and half to procedural errors.

Stability-indicating high-performance liquid chromatography analytical procedures were developed for specific determination of four isoxazolyl penicillins during degradation under neutral and accelerated acid/alkali conditions. The chromatographic conditions were set so that the drug peak was well separated from the peaks of the degradation products. Peak homogeneity of the resolving drug peak was assessed by the shape of the ratio chromatogram. Good and reproducible separations were achieved on a reversed-phase column using a mobile phase consisting of acetonitrile and a solution of 20 mM potassium dihydrogen phosphate plus 10 mM tetramethylammonium chloride in water (adjusted to pH 5). Optimal separations for all four drugs were achieved within the range of 15-21% organic modifier in the mobile phase. The detection wavelengths were 220 nm and 240 nm. The stability-indicating nature of the methods was confirmed by the linearity of the pseudo-first order plots. The utility of dual-wavelength chromatography in establishment of stability-indicating assays is highlighted.

A high-performance liquid chromatographic method has been developed for the determination of isoxazolylpenicillins (oxacillin, cloxacillin and dicloxacillin) residues in milk. This method involves extraction of the penicillins from milk with phosphate buffer pH 8, deproteinization by acidification with sulfuric acid followed by cleanup and concentration on a C18 solid-phase extraction column and reaction with 1,2,4-triazole and mercury(II) chloride solution pH 9.0 at 65 degrees C. The derivatized compound is eluted on a C8 column with a mobile phase containing acetonitrile, methanol and phosphate buffer (pH 6.5, 0.1 mol l(-1)) loaded with sodium thiosulfate and ion-pairing tetrabutylammonium hydrogenosulphate. The detection limit of the method is 2 microg l(-1) for oxacillin, 3 microg l(-1) for cloxacillin and 5 microg l(-1) for dicloxacillin in milk and the three penicillins have been quantified down to 15 microg l(-1) in line with the EU criteria of the directive No. 93/256/EEC.

The feasibility of a technique to confirm the presence of residues from seven beta-lactam antibiotics in bovine milk has been demonstrated. The technique makes use of electrospray ionization and tandem ion trap mass spectrometry. Residues are first extracted from milk by reversed-phase solid phase extraction. Target analytes are separated by on-line reversed-phase liquid chromatography and ionized in the electrospray interface. The product ion mass spectra are acquired following collision-induced dissociation of protonated molecules. Confirmation is based on comparison of full scan spectra between unknowns and bona fide standards. The feasibility of this technique has been demonstrated for the six beta-lactams currently approved for use in lactating dairy cattle (penicillin G, ampicillin, amoxicillin, cloxacillin, cephapirin and ceftiofur) and a drug not approved for animal use, cefazolin. The technique has been applied to control milk fortified at 5 ng/mL of penicillin G and 10 ng/mL of the other six drugs.

A rapid and sensitive high-performance liquid chromatographic method is described for the determination of cloxacillin residues in milk and serum. Only a clean-up step after deproteinization is necessary before the analysis. The chromatographic system involves the use of a C18 column and ultraviolet absorbance detection at 225 nm. The mobile phase was acetonitrile-0.02 M KH2PO4 (21:79) at pH 5. Recoveries for cloxacillin were 83.5 and 75.7% in serum and milk, respectively. Detection limits (10 ng/ml in milk and 50 ng/ml in serum) were below the stipulated European Union maximum residue limits for cloxacillin. Thus, the described method showed the same accuracy, precision and sensitivity as the microbiological assays but without interferences caused by other drugs commonly used in therapy. Analysis of different blood and milk samples obtained at different times from dairy cows treated with an intramammary dose of cloxacillin benzatine showed undetectable cloxacillin levels both in milk and blood samples.

A micellar electrokinetic capillary chromatographic (MECC) method for quantitation of penicillin G in medicated premix feeds was compared with the AOAC microbiological cylinder plate method. Feed was extracted according to the AOAC method, enabling comparison of both methods with the same extract. The MECC method detected penicillin G within 20 min, using a 150 mM sodium dodecyl sulfate (SDS)-20 mM sodium borate run buffer and a UV photodiode array detector at 210 nm. Recoveries from 5 replicates of medicated feed containing 117 g penicillin G/kg and then spiked to give a concentration of 176 g/kg were 98.4-108%, with a relative standard deviation (RSD) of 3.76% for the MECC method, and 102-104% with RSD of 1.06% for the AOAC microbiological cylinder plate method. Using a 5-point calibration curve, the MECC method detected and quantitated penicillin G in medicated premix feeds within 3 h, versus 24 h for the AOAC microbiological cylinder plate method.

A high-performance liquid chromatographic method based on C18 solid-phase extraction and ultraviolet detection at 323 nm of analytes derivatized with benzoic anhydride and 1,2,4-triazole mercuric chloride solution was developed for the simultaneous determination of amoxicillin, penicillin G (benzylpenicillin), ampicillin, oxacillin, cloxacillin and dicloxacillin residues in raw milk. The detection limit of the method was 1.3 microg/l for penicillin G; 1.4 microg/l for amoxicillin, oxacillin and cloxacillin, 1.5 microg/l for ampicillin and 2.7 microg/l for dicloxacillin. The mean recovery was 95-102% for amoxicillin, penicillin G and ampicillin, 92-98% for oxacillin and cloxacillin and 87-94% for dicloxacillin, measured by using an internal standard. The relative repeatability standard deviation was 4-9% on level 4-15 micro/l, respectively, 2-7% on level 30-40 microg/l.

A method was developed for determining benzylpenicillin and cloxacillin in animal tissues. Samples are extracted with acetonitrile, and the extract is cleaned up on a C18 solid-phase extraction (SPE) cartridge, derivatized, and quantitated by liquid chromatography with UV detection at 325 nm. The method was validated on spiked bovine kidney, liver, and muscle tissues. Kidney was spiked at 0.01, 0.05, and 0.20 mg/kg; liver and muscle were spiked at 0.20 mg/kg. Recoveries were 75-100% , with coefficients of variation of 2-7%. The method was further validated on kidney, liver, and muscle tissues from 2 sheep dosed with Aquacaine G suspension (containing benzylpenicillin). Mean levels of benzylpenicillin in these tissues ranged from 0.02 to 4.06 mg/kg, with coefficients of variation of replicate analyses between 3 and 7%. The limit of detection was approximately 0.005 mg/kg for benzylpenicillin and cloxacillin in kidney, liver, and muscle tissues. This method was used to study the stability of both spiked and incurred residues of benzylpenicillin in ovine liver during storage at -20 degrees C for 3 months. Assuming the rate of loss follows a first-order kinetic decay, the mean half-life of benzylpenicillin in liver is 62 days for spiked tissues and 71 days for tissues with incurred residues.

Polyclonal antibodies were raised against isoxazolyl penicillins in rabbits after immunization with a cloxacillin-human serum albumin conjugate. The antisera were tested in direct and indirect competitive enzyme immunoassays (EIAs), using glucose oxidase or horseradish peroxidase conjugates of oxacillin, cloxacillin, or dicloxacillin, respectively, as the labelled antigen. The relative cross-reactivities of each test system with oxacillin, cloxacillin, and dicloxacillin, determined from the amount of antibiotic required for 50% inhibition of labelled antigen binding, were dependent on the antibiotic used as the labelled antigen. Other beta-lactam antibiotics did not cross-react in these test systems. In a direct EIA using a cloxacillin-horseradish peroxidase conjugate, cloxacillin and dicloxacillin in milk were detected at levels of 10 and 30 ng ml-1; the average recoveries at these levels were 102 and 84%, respectively.

To ensure human food safety, the European Union has defined maximum residue limits (MRLs) for veterinary drug residues in food products. Analytical methods need to be developed to confirm the presence of drugs at the MRL level. A method using particle beam liquid chromatography-mass spectrometry was developed for the confirmation of oxacillin, cloxacillin and dicloxacillin in bovine muscle at the maximum residue limit of 300 micrograms kg-1. Beta-lactams were extracted from tissues with ethyl acetate under slightly acidic conditions and separated on a C18 bonded silica column with a methanol-aqueous formic acid (2%) solution-acetonitrile mobile phase. Negative ion chemical ionization with methane as the reagent gas was used to identify the compounds. The specificity required for a regulatory confirmation procedure was achieved by monitoring five fragment ions for each compound (selected ion monitoring mode): m/z 183, 198, 213, 214 and 241 for oxacillin; m/z 196, 248, 250, 275 and 277 for cloxacillin; m/z 165, 230, 232, 274 and 276 for dicloxacillin.

A high-performance liquid chromatographic (HPLC) method with automated cleanup by on-line solid phase extraction was developed for the determination of oxacillin, cloxacillin and dicloxacillin in bovine muscle tissue. The penicillins were extracted from the matrix with acetonitrile, transferred to phosphate buffer and automatically trapped on a RP-18 solid phase extraction column. After on-line elution onto the analytical HPLC-column, penicillins were quantified by UV (lambda = 225 nm) after isocratic separation. Mean recoveries at the MRL (0.3 mg/kg) were 92% for oxacillin, 92% for cloxacillin and 86% for dicloxacillin. The coefficients of variation for the three penicillins in the range of 0.15-0.6 mg/kg were 13.8%, 11.0%, and 7.3%, respectively. The applicability of this method for the determination of cloxacillin in milk was demonstrated.

This report contributes to a preliminary investigation of high-performance liquid chromatographic (HPLC)-mass spectrometric (MS) methods for confirming beta-lactam antibiotic residues in bovine milk. Initial work for each antibiotic evaluated the collisional activated dissociation (CAD) spectra that could be generated between the capillary and skimmer in the electrospray (ESP) interface. The drugs show various characteristic fragmentation, mostly within the beta-lactam ring and the amide group. Response for a particular compound in a given solvent can vary drastically. Usually, the more organic component in the solvent, the higher the ESP response. In many cases use of acetonitrile also results in slightly better ion currents than for methanol when comparing equal percentages of either organic solvent in water. The ESP response of most of the tested antibiotics can be enhanced by the addition of formic acid or acetic acid to the mobile phase methanol-water (1:1). In general, the negative ion spectra are lower in intensity, exhibiting an [M-H]- ion and producing less fragmentation at higher CAD voltages as compared to positive ion spectra. An isocratic reversed-phase HPLC method for the separation of a mixture of five common beta-lactam antibiotics was developed using acetic acid as a mobile phase additive and optimized for detection with a new ESP HPLC-MS interface. A post-column split ratio of 70:1 for the eluent from a 150 x 2 mm I.D. column was chosen to provide the required lower flow-rate (approximately 4 microliters/min). The limit of detection for the simultaneous determination of these antibiotics was estimated to be 100 ppb. Electrospray HPLC-MS could be used to confirm these antibiotics for quantities down to about 100 pg entering the mass spectrometer. Multiresidue analysis with microbore HPLC-ESP-MS has the advantage that no post-column splitting of the eluent is required and all of the analyte (on-column injected) will be transferred into the ESP interface. Preliminary work showed good mass spectrometric sensitivity down to the level of regulatory interest, but chromatographic separation efficiency must be improved.

A high performance liquid chromatography method, using spectrophotometric detection (254 nm) has been developed for the analysis of cloxacillin in an oily suspension. Analysis was performed by isocratic elution with 0.02M potassium dihydrogen phosphate solution, methanol, 50/50 (V/V). The method was specific, linear, accurate (99.8 +/- 1.4%) and precise (less than 1.0%). A stability study of cloxacillin in the formulation, showed that about 10% degradation occurred for 6 months storage at room temperature, when lyophilized sodium cloxacillin was used. On the contrary, when sodium cloxacillin obtained by precipitation from non aqueous solvent was used, about 6% degradation appeared in the drug for 3 years storage in the same conditions.

The objective was to develop confirmatory high-performance liquid chromatographic methods for penicillin residues in animal tissues with detection limits of less than or equal to 10 ng/g. A previously described procedure was modified by using a larger sample size and isocratic analysis. Tissues (15 g) were blended with 45 ml of water and 20 ml of homogenate were mixed with 40 ml acetonitrile and filtered. The filtrate (30 ml) was mixed with 10 ml of 0.2 M H3PO4 and extracted with methylene chloride. The combined methylene chloride layers were mixed with acetonitrile and hexane, washed with two 4-ml portions of water and then extracted with four 1-ml portions of 0.01 M phosphate buffer (pH 7). The combined buffer extracts were concentrated to 1 ml under reduced pressure. Analysis was isocratic during 0.01 M phosphate buffer (pH 7)-acetonitrile with proportions 85:15 (penicillin G), 82:18 (penicillin V) or 78:22 (cloxacillin). A polystyrene-divinylbenzene copolymer column, 150 x 4.6 mm I.D. (Polymer Labs. PLRP-S), was used with a flow-rate of 1 ml/min and detection at 210 nm. The presence of penicillins was confirmed by treating a duplicate sample with penicillinase. Recoveries were greater than 90% in most instances. Detection limits were 5 ng/g in muscle and higher in liver and kidney. The procedure is a simple and sensitive method for confirming the presence of penicillins in animal tissues.

A spectrophotometric method is described for the assay of ampicillin in the presence of cloxacillin in pharmaceutical preparations. It involves the reaction of hydrolysed ampicillin with formaldehyde in an acidic phosphate buffer (pH 2.5) and measurement of the absorbance of the product at 373 nm after dilution with 2 M sulphuric acid. The method is selective for ampicillin in the presence of cloxacillin. The absorbance had a linear relationship with concentration for the range studied (5-35 micrograms ml-1). By this method the ampicillin content of combined preparations of ampicillin and cloxacillin has been successfully determined in capsules, injections and a syrup.

Concentrations of cloxacillin in plasma and deep thoracic muscle tissue were measured in 10 patients who underwent elective coronary bypass surgery or valve replacement. One g of cloxacillin was administered after the induction of anaesthesia and 1 g cloxacillin was added to the oxygenator pump priming fluid before the start of the procedure. Blood and tissue samples were obtained before, during and after cardiopulmonary bypass. The relation between unbound plasma concentrations and total tissue contents of the drug was calculated. It was shown that measurement of the free plasma concentration may provide fairly reliable information on the free concentrations of cloxacillin in the tissues, and that determination of tissue contents may therefore not be necessary. Due to the administration of the second dose of cloxacillin at the start of cardiopulmonary bypass free tissue contents were just adequate in most patients. However, to obtain adequate tissue concentrations after bypass it is recommended that a third dose of the antibiotic be administered before the end of the operation.