Mass spectrometric identification and quantification of the antibiotic clavulanic acid in broiler chicken plasma and meat as a necessary analytical tool in finding ways to increase the effectiveness of currently used antibiotics in the treatment of broiler chickens.

Clavulanic acid is a molecule with antimicrobial effect used in several livestock species treatment. Its inclusion in the treatment of infectious diseases of broilers requires determination of pharmacokinetic and pharmacodynamic parameters in order to determine the appropriate dosage for broilers and ensure safety of chicken products for human health. The present study describes the optimisation of analytical LC-MS/MS method for identification and quantification of clavulanic acid in broiler chicken plasma and meat. The limit of detection and the limit of quantification for the developed method were 3.09 µg·L-1 and 10.21 µg·L-1 for plasma and 2.57 µg·kg-1 and 8.47 µg·kg-1 for meat. The recoveries of the developed plasma and tissue extraction procedure were > 105.7% and > 95.6%, respectively. The achieved coefficient of variation of within-run precision ranged from 2.8 to 10.9% for plasma and from 6.5 to 8.5% for meat. The pharmacokinetic experiment was performed in 112 Ross broiler chickens assigned into time interval groups ranging from 10 min to 24 h in accredited animal facilities. Administered dose of clavulanic acid was 2.5 mg·kg-1 according to the manufacturer's recommendations. The pharmacokinetic parameters obtained from the experiment are as follows: Cmax = 1.82 ± 0.91 mg·L-1, Tmax = 0.25 h, T1/2 = 0.87 h, Kel = 0.80 ± 0.04 h-1, AUC0-∞ = 2.17 mg·h ·L-1.

Rapid and Simultaneous Quantitation of Amoxicillin and Clavulanic Acid in Human Plasma and Urine by Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry and Its Application to a Pharmacokinetic Study.

Rapid, accurate and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods were developed and validated for the simultaneous quantitation of amoxicillin and clavulanic acid in human plasma and urine samples. Amoxicillin and clavulanic acid in both plasma and urine were extracted using a solid-phase extraction method. The compounds were separated on an Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm). Ampicillin was used as the internal standard (IS) in plasma, while amoxicillin-d4 and sulbactam were used as ISs in urine. The lower limit of quantitation was 0.0500 and 0.0250 µg/mL for amoxicillin and clavulanic acid in plasma, and 0.0500 µg/mL for both analytes in urine. The established methods were validated in terms of selectivity, precision, accuracy, linearity, matrix effect, recovery, carryover, interaction, dilution integrity and stability, and successfully applied to a pharmacokinetic study of amoxicillin sodium and clavulanate potassium (10:1) injection in healthy volunteers.

Simultaneous quantification of cefpodoxime proxetil and clavulanic acid in human plasma by LC-MS using solid phase extraction with application to pharmacokinetic studies.

A simple, rapid and selective high performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS) method was developed and validated for the simultaneous estimation of cefpodoxime proxetil (CDPX) and clavulanic acid (CA) in human plasma. Extraction of samples was done by solid phase extraction technique (SPE) and chloramphenicol used as internal standard. Chromatographic separation was carried out on a reverse phase Princeton SPHER C18 (150mm×4mm i.d., 5µm) column using mixture of methanol: acetonitrile: 2mM ammonium acetate (25:25:50, v/v, pH 3.5) at 0.8mL/min flow rate. Detection was performed on a single quadrupole MS by selected ion monitoring (SIM) mode via APCI source. The calibration curve was linear within the concentration range, 0.04-4.4µg/mL and 0.1-10.0µg/mL for CDPX and CA respectively. Pharmacokinetic parameters of tablet (CDPX 200mg, CA 125mg) were evaluated. Cmax, Tmax, T1/2, elimination rate constant (Kel), AUC0-t, and AUC0-∞ of tablet were 2.13±0.06µg/mL, 2h, 3.05±0.15h, 0.24±0.37h(-1), 6.81±0.14µg h/mL and 7.72±0.23µg h/mL respectively for cefpodoxime (CP), 5.34±0.28µg/mL, 2h, 2.73±0.25h, 0.26±0.31h(-1), 15.37±0.16µg h/mL and 16.59±0.53µg h/mL respectively for CA.

[Simultaneous determination of serum concentrations of amoxicillin and clavulanate potassium in human blood using high-performance liquid chromatography].

OBJECTIVE: To establish a chromatography-based method for simultaneous analysis of the concentrations of amoxicillin and clavulanate potassium in human blood. METHODS: With paracetamol as the internal control, human plasma samples, after treatment with methanol for protein sedimentation and centrifugation, were loaded for analysis with high-performance liquid chromatography (HPLC). HPLC analysis was carried out using a C18 column (5 µm, 4.6 mm×150 mm) with the mobile phase of acetonitrile-PBS (0.05 mol/L) of 10:90 (pH 2.3), UV detection wavelength of 220 nm, flow rate of 1.0 ml/min, and column temperature of 25 degrees celsius;. RESULTS: The retention time of acetaminophen for potassium clavulanate, amoxicillin sodium and the internal control was 5.3, 7.2, and 8.5 min, respectively, and no interference by the endogenous impurities in the plasma samples was found. Amoxicillin sodium showed a good linearity within the concentration range of 0.52-4.16 µg/ml (r(2)=0.9996), and potassium clavulanate had a good linearity within the range of 0.266-2.14 µg/ml (r(2)=0.9998). The minimum detectable concentrations of amoxicillin sodium and potassium clavulanate were 0.065 µg/ml and 0.066 µg/ml, respectively. The relative recoveries of amoxicillin sodium were 95.9%-96.5% (n=5), and those of clavulanate potassium were 92.5%-98.8% (n=5); the intra- and inter-day RSD of amoxicillin sodium was 1.84%-6.4% and 2.1%-7.8%, as compared to that of potassium clavulanate of 3.57%-8.6% and 1.8%-9.1%, respectively. CONCLUSION: This method is simple, accurate, sensitive, specific and reproducible for analyzing the concentrations of amoxicillin and clavulanate potassium simultaneously in human plasma.

[Simultaneous determination of amoxicillin and clavulanic acid in the human plasma by high performance liquid chromatography-mass spectrometry (UPLC/MS)].

BACKGROUND/AIM: Quantitative analysis of amoxicillin and clavulanic acid in biological matrices requires sensitive and specific methods which allow determination of therapeutic concentration in 1 g/mL range. Analytical methods for determination of their concentrations in body fluids described in literature include high performance liquid chromatography coupled to UV detector (HPLC-UV) and liquid chromatography-mass spectrometry (LC-MS). The aim of this study was to develop sensitive and specific ultra performance liquid chromatography/mass spectrometry (UPLC/MS) method which could be used for the spectral identification and quantification of the low concentrations of amoxicillin and clavulanic acid in the human plasma. METHOD: A sensitive and specific UPLC/MS method for amoxicillin and clavulanic acid determination was developed in this study. The samples were taken from the adult healthy volunteers receiving per os one tablet of amoxicillin (875 mg) in combination with clavulanic acid (125 mg). RESULTS: Plasma samples were pretreated by direct deproteinization with perchloric acid. Quantification limit of 0.01 microg/ml for both amoxicillin and clavulanic acid was achieved. The method was reproducible day by day (RSD < 7%). Analytical recoveries for amoxicillin ranged from 98.82% to 100.9% (for concentrations of 1, 5 and 20 microg/mL), and recoveries for clavulanic acid were 99.89% to 100.1% (for concentrations of 1, 2 and 5 microg/mL). This assay was successfully applied to a pilot pharmacokinetic study in healthy volunteers after a single-oral administration of amoxicillin/clavulanic combination. The determined plasma concentrations of both amoxicillin and clavulanic acid were in the range of the expected values upon the literature data for HPLC-UV and LC-MS methods. CONCLUSION: The described method provided a few advantages comparing with LC/MS-MS method. The method is faster using running time of 5 minute, has lower limit of quantification (LOQ) and it could be used in pharmacokinetic studies of both amoxicillin and clavulanic acid.

Disposition and oral bioavailability of amoxicillin and clavulanic acid in pigs.

The pharmacokinetic properties of amoxicillin and clavulanic acid were studied in healthy, fasted pigs after single intravenous (i.v.) and oral (p.o.) dosage of 20 mg/kg of amoxicillin and 5 mg/kg of clavulanic acid. The plasma concentrations of the drugs were determined by validated high-performance liquid chromatographic methods and the pharmacokinetic parameters were calculated by compartmental and noncompartmental analyses. After i.v. administration of the two drugs, plasma concentration-time curves were best described by a three-compartmental open model for amoxicillin and a two-compartmental open model for clavulanic acid. Amoxicillin (with a t(1/2 gamma) = 1.03 h and a clearance of 0.58 L/h.kg) and clavulanic acid (with a t(1/2 beta) of 0.74 h and a clearance of 0.41 L/h.kg) were both rapidly eliminated from plasma. Both drugs had apparently the same volume of distribution of 0.34 L/kg. After p.o. administration of the two drugs, a noncompartmental model was used. Elimination half-lives of amoxicillin and clavulanic acid were not significantly different, i.e. 0.73 and 0.67 h respectively. The mean maximal plasma concentrations of amoxicillin and clavulanic acid were 3.14 and 2.42 mg/L, and these were reached after 1.19 and 0.88 h respectively. The mean p.o. bioavailability was found to be 22.8% for amoxicillin and 44.7% for clavulanic acid.

Simultaneous determination of amoxicillin and clavulanic acid in human plasma by isocratic reversed-phase HPLC using UV detection.

A simple, rapid and sensitive isocratic reversed phase HPLC method with UV detection using internal standard has been developed and validated for simultaneous determination of amoxicillin and clavulanic acid in human plasma. The assay enables the measurement of amoxicillin and clavulanic acid for therapeutic drug monitoring with a minimum quantification limit of 15 and 30 ng ml(-1), respectively. The method involves simple, one-step extraction procedure and analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100 mm x 4.6mm) column with an isocratic mobile phase consisting of 0.02 M disodium hydrogen phosphate buffer-methanol (96:4, v/v) adjusted to pH 3.0. The wavelength was set at 228 nm. The coefficients of variation for inter-day and intra-day assay were found to be less than 9.0%.

Determination of clavulanic acid in calf plasma by liquid chromatography tandem mass spectrometry.

A method for the quantification of clavulanic acid in calf plasma using high-performance liquid chromatography combined with electrospray ionization (ESI) mass spectrometry, operating in the negative ionization mode (LC-MS/MS), is presented. Sample preparation includes a simple and fast deproteinization with acetonitrile and a back-extraction of the acetonitrile with dichloromethane. Chromatography is performed on a reversed-phase PLRP-S polymeric column using 0.05% formic acid in water and acetonitrile. The limit of quantification is 25 ng/ml, which is lower than other published methods using ultraviolet (UV), fluorimetric or mass spectrometric detection. The limit of detection is calculated to be 3.5 ng/ml. The stability of clavulanic acid was demonstrated according to The Guidelines of Bioanalytical Method Validation of The Food and Drug Administration (FDA): freeze and thaw stability, short-term stability, long-term stability, stock solution stability and postpreparative stability. The method is used in a pharmacokinetic and bioequivalence study of amoxycillin/clavulanic acid formulations in calves.

Pharmacokinetic and pharmacodynamic assessment of co-amoxiclav in the treatment of melioidosis.

OBJECTIVES: We conducted a prospective pharmacokinetic study of oral co-amoxiclav in patients with melioidosis to determine the optimal dosage and dosing interval in this potentially fatal infection. PATIENTS AND METHODS: Serial plasma concentrations were measured after administration of two 1 g tablets of Augmentin (1750 mg of amoxicillin and 250 mg of clavulanate) to 14 adult patients with melioidosis. Monte Carlo simulation was used to predict the concentration of each drug following multiple doses of co-amoxiclav at different dosages and dose intervals. The proportion of the dose-interval above MIC (T > MIC) was calculated from 10,000 simulated subject plasma concentration profiles together with chequerboard MIC data from 46 clinical isolates and four reference strains of Burkholderia pseudomallei. RESULTS: The median (range) observed maximum plasma concentrations of amoxicillin and clavulanate were 11.5 (3.3-40.2) mg/L and 5.1 (0.8-12.1) mg/L, respectively. The median (range) elimination half-lives were 94 (73-215) and 89 (57-140) min, respectively. Simulation indicated that co-amoxiclav 1750/250 mg given at 4, 6, 8 or 12 hourly dosing intervals would be associated with a T > MIC of < or = 50% in 0.7%, 2.8%, 8.6% and 33.2% of patients, respectively. Corresponding proportions for T > MIC of > or = 90% were 95.8%, 78.6%, 50.2% and 10.8%, respectively. CONCLUSIONS: The dosing interval for co-amoxiclav (750/250 mg) in melioidosis should not be greater than 6 h.

Simultaneous determination of amoxicillin and clavulanic acid in human plasma by HPLC-ESI mass spectrometry.

A simple, fast and sensitive high-performance liquid chromatography (HPLC)-mass spectrometric (MS) method has been developed for simultaneous determination of amoxicillin and clavulanic acid in human plasma using terbutaline as internal standard. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a C(8) reversed-phase column with formic acid-water-acetonirile (2:1000:100) and detected using electrospray ionization (ESI) mass spectrometry in negative selected ion monitoring (SIM) mode. The method was validated and successfully applied to analysis of amoxicillin and clavulanic acid in clinical studies. The limit of quantitation, 0.12 microg/ml for amoxicillin and 0.062 microg/ml for clavulanic acid, was five times lower than that of the published HPLC-UV method.

[Amoxicillin in tonsillectomy: preliminary data on the evaluation of serum and tissue concentrations]. / L'amoxicillina nella tonsillectomia: dati preliminari sulla valutazione delle concentrazioni sieriche e tessutali.

Amoxicillin was administered to 50 patients with chronic recurrent tonsillitis waiting for tonsillectomy. Group A (N=16) received 2.2 g of amoxicillin plus clavulanic acid with intravenous injection 10 minutes before tonsillectomy Group B (N=34) was treated with 3 doses of amoxicillin-clavulanic acid administered orally the day before surgery, plus one oral administration 2 hours before tonsillectomy. Antibiotic doses were established on patient's weight using maximum suggested. The measures were, estimated in serum and in tonsils using High Performance Liquid Chromatography, (HPLC). The data show better efficacy of intravenous administration than oral administration.

Simultaneous determination of cefatrizine and clavulanic acid in dog plasma by HPLC.

A rapid and specific high-performance liquid chromatographic method was developed and validated for the simultaneous determination of cefatrizine and clavulanic acid in the plasma of beagle dog. The sample pretreatment procedure involved reaction of clavulanic acid with 1,2,4-triazole, which readily produced a derivative with its maximum UV absorption at 314 nm. This derivative was separated in a reverse-phase C-18 column without being interfered by other components present in plasma. Cefatrizine, however, was not derivatized and, therefore determined directly at 269 nm. Sulfanilamide was used as an internal standard. The retention times of sulfanilamide, the derivative, and cefatrizine were, 3.5, 4.9, and 6.0 min, respectively. The assay showed linearity from 2 to 100 microg/ml for cefatrizine and from 1 to 50 microg/ml for clavulanic acid. Precision expressed as R.S.D. ranged from 4.2 to 18.2% for cefatrizine and 5.5 to 15.8% for clavulanic acid. Accuracy ranged from 97.9 to 120% (lower limit of quantitation) for cefatrizine and from 97.7 to 119.2% for clavulanic acid. Extraction efficiencies for cefatrizine, clavulanic acid, and internal standard from dog plasma averaged 79.8+/-5.8%, 84.8+/-6.2%, and 89.0+/-3.8%, respectively. This method was employed successfully to follow the time course of the concentration of cefatrizine and clavulanic acid in beagle dogs following oral administration of cefatrizine and clavulanic acid.

Simultaneous determination of ticarcillin and clavulanate in rabbit serum and tissue cage fluid by liquid chromatography.

A gradient elution HPLC method with a wavelength switch technique was developed to simultaneously analyze the beta-lactam ticarcillin and the beta-lactamase inhibitor clavulanate in rabbit serum and tissue cage fluid (TCF). A C18 reversed-phase column with a programmable UV detector changing the wavelength from 218 to 254 nm at 9 min was used for chromatographic separation. The mobile phase consisted of acetonitrile, phosphate buffer and tetrabutylammonium hydrogen sulfate by following a gradient elution program at a flow-rate of 1 ml/min. Sample processing was carried out with liquid-liquid extraction. Good linearity, recoveries, precision and accuracy were obtained. The ranges of the standard curves were 1-100 microg/ml for ticarcillin, and 0.2-20 microg/ml for clavulanate. This assay has been successfully applied to analyze ticarcillin and clavulanate in rabbit serum and tissue cage fluid samples from a pharmacokinetic study.

Variable absorption of clavulanic acid after an oral dose of 25 mg/kg of Clavubactin and Synulox in healthy dogs.

The aims of this investigation were to calculate the pharmacokinetic parameters and to identify parameters, based on individual plasma concentration-time curves of amoxicillin and clavulanic acid in dogs, that may govern the observed differences in absorption of both drugs. The evaluation was based on the data from plasma concentration-time curves obtained following a single dose in an open, randomized, two-way crossover study involving 24 male Beagle dogs treated with two Amoxi-Clav formulations (A Clavubactin and B Synulox, each with 200/50 mg). Plasma amoxicillin and clavulanic acid concentrations were determined using validated bioassay methods. The half-life of elimination of amoxicillin was 1.5 h (t1/2 = 1.52 +/- 0.19 h, Cmax = 11.4 +/- 2.74 microg/mL), and that of clavulanic acid 0.76 h (t1/2 = 0.71 +/- 0.23 h, Cmax = 2.06 +/- 1.05 microg/mL). There was a fivefold variation in the AUCt of clavulanic acid for both formulations, while the AUCt of amoxicillin varied by a factor of 2. The mean ratio of the AUCt amoxicillin : clavulanic acid was 12.7 +/- 3.65 for formulation A and 11.8 +/- 5.22 for formulation B (P = 0.51).

Simultaneous determination of amoxicillin and clavulanic acid in human plasma by HPLC with UV detection.

A simple and accurate high-performance liquid chromatographic method with ultraviolet detection at 220 nm has been validated for the simultaneous determination of amoxicillin and clavulanic acid in human plasma. Plasma samples were pretreated by direct deproteinization with methanol. A good chromatographic separation between both compounds was achieved using a reversed phase C8 column and a mobile phase, consisting of acetonitrile-phosphate solution-tetramethyl ammonium chloride solution. The calibration curves were linear over the concentration range of 0.625-20 mg l(-1) for amoxicillin and 0.3125-10 mg l(-1) for clavulanic acid with determination coefficients > 0.998. The method is accurate (bias < 7%) and reproducible (intra- and inter-day R.S.D. < 15%), with a quantitation limit of 0.625 and 0.3125 mg l(-1) for amoxicillin and clavulanic acid, respectively. Analytical recoveries from human plasma ranged from 91 to 102% for both components. This fully validated method, which allows the simultaneous measurement of amoxicillin and clavulanic acid in biological samples, is rapid (total run time < 10 min) and requires only a 100 microl sample. This assay is suitable for biomedical applications and was successfully applied to a pilot pharmacokinetics study in healthy volunteers after a single-oral administration of amoxicillin/clavulanic acid combination (500/125 mg).

Variable absorption of clavulanic acid after an oral dose of 25 mg/kg of Clavubactin and Synulox in healthy cats.

The aims of this investigation were to calculate the pharmacokinetic parameters and to identify parameters, based on individual plasma concentration-time curves of amoxicillin and clavulanic acid in cats, that may govern the observed differences in absorption of both drugs. The evaluation was based on the data from plasma concentration-time curves obtained following a single-dose, open, randomised, two-way crossover phase-I study, each involving 24 female cats treated with two Amoxi-Clav formulations (formulation A was Clavubactin and formulation was B Synulox; 80/20 mg, 24 animals, 48 drug administrations). Plasma amoxicillin and clavulanic acid concentrations were determined using validated bioassay methods. The half-life of elimination of amoxicillin is 1.2 h (t1/2 = 1.24 +/- 0.28 h, Cmax = 12.8 +/- 2.12 microg/ml), and that of clavulanic acid 0.6 h (t1/2 = 0.63 +/- 0.16 h, Cmax = 4.60 +/- 1.68 microg/ml). There is a ninefold variation in the AUCt of clavulanic acid for both formulations, while the AUCt of amoxicillin varies by a factor of two. The highest clavulanic acid AUCt values indicate the best absorption; all other data indicate less absorption. Taking into account that the amoxicillin-to-clavulanic acid dose ratio in the two products tested was 4:1, the blood concentration ratios may actually vary much more, apparently without compromising the products" high efficacy against susceptible microorganisms.

Bioequivalence study of a novel Solutab tablet formulation of amoxicillin/clavulanic acid versus the originator film-coated tablet.

With amoxicillin/clavulanic acid Solutab tablet, a new tablet formulation of amoxicillin/clavulanic acid (500/125), was developed. The aim of the present study was to demonstrate bioequivalence between the new tablet formulation, taken as an intact tablet and after prior dispersal, versus the originator product viz. Augmentan film-coated tablet. The study was performed in 48 healthy volunteers, according to an open, single-dose three-period, crossover design. Blood samples were taken prior to each administration and at 10 time points after dosing. Plasma concentrations of amoxicillin and clavulanic acid were determined by validated high performance liquid chromatography with UV detection. With regard to amoxicillin, the results were within the preset bioequivalence range of 0.8 to 1.25 for the ratios of the primary parameters AUC(0-t) and Cmax. In terms of clavulanic acid the 90% confidence intervals of the ratios for AUC(0-t) and Cmax versus the reference lay outside the predefined bioequivalence range of 0.75 to 1.33. This result, however, was mainly due to the large variability of the reference formulation compared to the amoxicillin/clavulanic acid Solutab tablet. Based on statistical indications that 3/48 subjects with extremely low levels on the reference formulation could be regarded as "outliers" and after excluding these subjects' data from the statistical analysis, results for clavulanic acid were within the predefined bioequivalence range of 0.75 to 1.33. Overall, the amoxicillin/clavulanic acid Solutab tablet provided, in comparison to the reference tablet, less variable levels of clavulanic acid, thus giving more appropriate protection to the available amoxicillin. Thirteen adverse events were reported post dosing by 7 subjects. There were no differences in incidence of adverse events between amoxicillin/clavulanic acid Solutab tablet taken intact or dispersed and Augmentan.

A fast and simple method for the determination of clavulanic acid in human plasma using derivatisation reaction kinetics.

A stopped-flow mixing technique is used to determine clavulanic acid in human plasma after plasma deproteinisation with acetonitrile and removal of the organic solvent by extraction with dichloromethane. The reaction kinetic profiles for the reaction between clavulanic acid and imidazole are determined by measuring the absorbance at 312 nm of the imidazole derivative. The reaction rates are proportional to the concentration of the clavulanic acid and by plotting reaction rates against clavulanic acid concentrations linear calibration curves could be constructed over the range 0.30-10.0 microg/ml.

Administration of ticarcillin in combination with clavulanic acid intravenously and intrauterinely to clinically normal oestrous mares.

Ticarcillin and clavulanic acid (potassium clavulanate) were administered to normal oestrous mares intravenously (i.v.) at a dose of 50 and 1.67 mg/kg for ticarcillin and clavulanate, respectively. In a crossover design, the same drugs were administered intrauterine (i.u.) at a dose of 12.4 and 0.4 mg/kg for ticarcillin and clavulanate, respectively. The i.u. dose was administered in 100 mL of saline solution. Endometrial tissue biopsies and plasma samples were collected after drug administration for the determination of ticarcillin and clavulanate concentrations by high-pressure liquid chromatography and pharmacokinetic calculations. After i.u. administration both drugs were poorly absorbed into the plasma. The ticarcillin half-life from tissue and plasma was short after i.v. administration. Although concentrations in tissue were higher after i.u. administration than i.v., concentrations of ticarcillin declined rapidly, which would necessitate frequent treatment in order to maintain drug concentrations above the minimum inhibitory concentrations (MIC) throughout the treatment period. Clavulanate concentrations in tissue were either low or persisted for only a short time after administration via either route. It appears that addition of clavulanate to the formulation for treatment of i.u. infections in mares is of questionable value based on these concentrations.