Application of MEKC for determination of ticarcillin and clavulanic acid in Timentin intravenous preparation.

A micellar electrokinetic chromatography method for simultaneous assay of ticarcillin and clavulanic acid in Timentin i.v. injection preparation was developed. This method ensures excellent separation of both components of Timentin preparation. The validation of the method was performed, and specificity, reproducibility, precision and accuracy were confirmed. The detection and quantitative limits for Timentin were established in the concentrations 0.04 and 0.08 mg/ml, respectively. The elaborated technique was compared with two methods routinely used-UV and high performance liquid chromatography (HPLC). The obtained results and their statistical analysis proved the same precision of all methods, however, no significant differences were observed between CE and HPLC.

Simultaneous determination of amoxycillin and clavulanic acid in pharmaceutical products by HPLC with beta-cyclodextrin stationary phase.

A simple, rapid and accurate method for simultaneous determination of amoxycillin and clavulanic acid using HPLC with beta-cyclodextrin stationary phase was developed. It involves the use of tetraethylammonium acetate (TEAA) as an additive reagent, methanol-buffer solution (pH 4.5) (35:65; v/v) as the mobile phase, detection at 225 mm and chromatogram within 12 min. Linearity and precision of the internal standard method have been obtained. Recoveries ranged from 99.25 to 105.63% for amoxycillin in the synthetic mixture. For clavulanic acid it was from 99.50 to 101.64%. This method is convenient and reproducible for analyses of these two components in different dosage forms.

Stability of amoxicillin-clavulanate in BACTEC medium determined by high-performance liquid chromatography and bioassay.

The stabilities of amoxicillin (16 micrograms/ml) and clavulanate (8 micrograms/ml), alone and in combination in BACTEC medium (Middlebrook 7H12B medium), were determined by high-performance liquid chromatography (HPLC) and bioassay. By HPLC, the half-life of amoxicillin (trihydrate and sodium) in combination with clavulanate in nonradiolabelled 7H12B medium was 6.7 days, whereas the half-life of clavulanate in combination with amoxicillin was 2.0 days. By bioassay, the half-lives of amoxicillin trihydrate and clavulanate in radiolabelled 7H12B medium were comparable (7 and 2 days, respectively) to those determined by HPLC. When clavulanate was tested alone, the half-life was determined to be 1.88 days by HPLC and 1.87 days by bioassay. The relatively short half-life of clavulanate can be adjusted by a procedure of "topping up," or adding one-half the concentration of clavulanate every second day, in order to allow accurate amoxicillin-clavulanate MIC testing with the BACTEC mycobacterial susceptibility system.

Simultaneous determination of the binary mixtures of cefsulodin and clavulanic acid by using first-derivative spectrophotometry.

Two-component mixtures of cefsulodin and clavulanic acid were analysed by a first-derivative spectrophotometric method using a zero-crossing technique of measurement. The relative ease offered by this derivative technique for the quantification of these drugs, with closely overlapping spectral bands, was clearly demonstrated. As the absorption band of clavulanic acid closely overlaps with that of cefsulodin, both direct and derivative spectrophotometric methods have been investigated and evaluated by an exhaustive statistical analysis of the experimental data. The first-derivative spectrophotometric method was found to be more rapid, accurate and reproducible. The procedure does not require any separation step. The calibration graphs were linear in the range 2.0-56.0 micrograms ml-1 for cefsulodin and 2.0-28.0 micrograms ml-1 for clavulanic acid. The lower detection limits of cefsulodin and clavulinic acid (P 0.05 level) were calculated to be 0.16 and 0.24 microgram ml-1, respectively. Mixtures of cefsulodin and clavulanic acid in ratios of 1:4-7:2 were satisfactorily resolved. Both components were also determined in physiological solutions used to prepare intravenous infusions of these antibiotics.

Penetration of amoxycillin/clavulanic acid into bronchial mucosa with different dosing regimens.

BACKGROUND: The efficacy of an antibiotic is related to its concentration at the site of infection. Previous studies of the concentrations of amoxycillin and clavulanic acid (co-amoxiclav) in respiratory secretions or whole lung tissue have suffered from methodological problems. The concentration of amoxycillin and clavulanic acid was determined in bronchial mucosal biopsy samples obtained at bronchoscopy following five different dosing regimens. METHODS: Bronchial biopsy and serum samples were obtained from 50 patients undergoing diagnostic bronchoscopy. Ten patients each received 375 mg, 625 mg, 750 mg, and 3.25 g oral, and 1.2 g intravenous co-amoxiclav 1-3 hours before bronchoscopy. The concentrations of clavulanic acid and amoxycillin were determined by high performance liquid chromatography using a microbore column, solid phase extraction, and preconcentration to improve sensitivity tenfold over previous methods. RESULTS: Concentrations of both clavulanic acid and amoxycillin in bronchial mucosa were dose related and were well above the MIC90 of co-amoxiclav for the common bacterial respiratory pathogens including Haemophilus influenzae, Micrococcus catarrhalis and Streptococcus pneumoniae for all dosing regimens. Mean mucosal levels were 200% and 118% of the corresponding serum levels for amoxycillin and clavulanic acid respectively. CONCLUSIONS: Amoxycillin and clavulanic acid are concentrated in bronchial mucosa and, even at the lowest dose of 375 mg orally, are likely to produce tissue levels in the lung sufficient to inhibit all the common community acquired respiratory pathogens.

Stopped-flow photometric determination of clavulanic acid in pharmaceutical and serum samples.

The stopped-flow mixing technique was used to develop a simple, fast kinetic method for the determination of clavulanic acid by reaction with imidazole. Whereas the conventional method requires about 12-15 min for equilibrium to be reached, kinetic measurements can be made within a few seconds. The calibration graph was linear over the range 1-40 microgram ml-1 of clavulanic acid and the detection limit achieved was 0.3 microgram ml-1. The precision and selectivity of the method are reported. The results obtained by applying the proposed method to the analysis of pharmaceutical and serum samples show how easily it can be adapted for routine analyses.

Liquid chromatographic determination of amoxycillin and clavulanic acid in pharmaceutical preparations.

A liquid chromatographic method for the simultaneous determination of amoxycillin and potassium clavulanate in tablet and suspension preparations is presented. The method specifies reversed phase column and a buffered mobile phase (CH3OH + KH2PO4-buffer pH 6 + H2O, 15:1:84) isocratically at a rate of 1.0 ml min-1, with detection at 235 nm. The suitability of the chromatographic system developed is tested using replicate injections of the sample and standard preparations. The observed relative standard deviations (RSDs) were within 2%. Recovery experiments conducted utilizing the proposed method gives results of 101.5% +/- 1.72 (n = 6) and 101.22% +/- 1.93 (n = 6) for amoxycillin in tablets and powder for oral administration, respectively. Similarly, recovery experiments for clavulanic acid gave results of 100.33 +/- 1.90 (n = 6) and 99.61 +/- 1.32 (n = 6) in the tablets and suspension powder, respectively. Comparison of the proposed method with the USP method proved it to be satisfactory. The statistical F- and t-tests observed, indicated that there were no significant differences between the two methods regarding precision and accuracy.

[Stability of beta-lactamase inhibitors and beta-lactam antibiotics in parenteral formulations as well as in body fluids and tissue homogenates. Comparison of sulbactam, clavulanic acid, ampicillin and amoxicillin]. / Stabilität von beta-Lactamase-Inhibitoren und beta-Lactam-Antibiotika in parenteralen Darreichungsformen sowie in Körperflüssigkeiten und Gewebehomogenaten. Vergleichende Untersuchung von Sulbactam, Clavulansäure, Ampicillin und Amoxicillin.

Stability of beta-Lactamase Inhibitors and beta-Lactam Antibiotics in Parenteral Formulations as Well as in Body Fluids and Tissue Homogenates/Comparative studies with sulbactam, clavulanic acid, ampicillin and amoxicillin. The beta-lactamase inhibitors and the beta-lactam antibiotics are markedly different in chemical stability. The comparative examination of 4 different infusion solutions at 4 degrees C, 25 degrees C and 37 degrees C gives the following sequence of decreasing stability: sulbactam (CAS 68373-14-8), ampicillin (CAS 69-53-4), amoxicillin (CAS 61336-70-7) and clavulanic acid (CAS 58001-44-8). It is particularly striking that the two beta-lactamase inhibitors, sulbactam and clavulanic acid, behave very differently. Sulbactam is also much more stable than clavulanic acid to incubation at 37 degrees C in body fluids or in tissue homogenates. The differences in the stability of the individual drugs should be born in mind during clinical use of combination formulations such as sulbactam/ampicillin (Unacid) and clavulanic acid/amoxicillin.

Polarographic determination of clavulanic acid.

A method is proposed for the determination of clavulanic acid by differential pulse polarography. The electroactive product was obtained by hydrolysis in sulphuric medium. It shows a reduction peak, that can be used analytically, at -0.75 V (vs SCE). The optimum conditions for the polarographic signal were determined and a study was made of the different parameters affecting the electrochemical process. A polarographic procedure is proposed for the determination of clavulanic acid in a concentration range of 8.0 X 10(-6) -1.4 X 10(-4) M. The detection limit is about 2 x 10(-6) M and the relative standard deviation is 1.1%. The method was applied to the determination of clavulanic acid in the presence of amoxicillin.

Determination of clavulanic acid by a sensitive HPLC method.

A new HPLC method is described for the estimation of clavulanic acid. It should be of use for further studies on the penetration of clavulanic acid into body tissues and fluids. It utilizes standard HPLC equipment and UV detection, but provides at least ten-fold increased sensitivity (less than 0.008 mg/l) over previously published methods by using solid phase extraction with imidazole derivatization and subsequent pre-concentration of the sample, and a microbore chromatographic column with a conventional detection system.

Antibiotic persistence and tolerance in the lactating sheep following a course of intramammary therapy.

The commercial use of sheep for the production of milk and milk products is attractive to farmers actively diversifying their dairy interests due to the impact of the quota system. As intensification of milking increases, flock sizes will enlarge and the incidence of ovine mastitis will inevitably increase. The pharmaceutical industry and the veterinary practitioner will be required to provide advice and data upon the performance of currently available bovine intramammary preparations for the sheep. This study produces evidence to confirm that one available bovine intramammary preparation, when infused into milking sheep, produced a withholding time approximately three times as long as that defined for the cow. Following a course of three infusions over a period of 24 hours after consecutive milkings, milk was not acceptable for human consumption or for the production of cheese and yoghurts until 136 hours following the final infusion. This situation is likely to be representative of that which will occur with other intramammary products used in the ovine species following infusion with bovine intramammary preparations.

Efficacy of amoxycillin/clavulanic acid in experimental Bacteroides fragilis/Escherichia coli mixed infections.

The efficacy of amoxycillin/clavulanic acid was compared with those of metronidazole, cefuroxime, metronidazole/ampicillin, metronidazole/gentamicin and metronidazole/cefuroxime, in experimental mixed infections produced in mice by subcutaneous inoculation of amoxycillin-resistant strains of Bacteroides fragilis and Escherichia coli. The combination of metronidazole/ampicillin failed to inhibit the growth of E. coli, and exerted only a transient effect on the numbers of Bact. fragilis in the groin abscesses. In contrast, amoxycillin/clavulanic acid prevented the development of the infection, eliminating both organisms. Metronidazole and cefuroxime, alone and in combination, were less effective than amoxycillin/clavulanic acid in inhibiting the growth of the infecting organisms. These results demonstrate the clinical potential of amoxycillin/clavulanic acid in prophylaxis, or in the therapy of mixed aerobe/anaerobe infections.

Tissue and serum concentrations of amoxycillin and clavulanic acid in patients having reconstructive vascular surgery.

Fifteen patients received amoxycillin/clavulanic acid combination intravenously at anaesthetic induction as part of a three-dose schedule of antibiotic prophylaxis for vascular surgery. After 30 min, the mean serum concentration of amoxycillin was 39.9 mg/l and clavulanic acid was 9.3 mg/l. Mean amoxycillin tissue concentrations in fat, 5.1 mg/l; vein, 7.5 mg/l; and artery, 4.8 mg/l, were lower than serum values. Mean clavulanic acid concentrations were: fat, 0.7 mg/l; vein, 1.2 mg/l; and artery, 1.6 mg/l. At the end of surgery, the serum amoxycillin level was always greater than 6 mg/l and clavulanic acid greater than 2 mg/l, which were both still within the therapeutic range. Concentrations in fat had declined by the end of the surgery ranging from 0.76 to 5.2 mg/l for amoxycillin and from 0.03 to 0.6 mg/l for clavulanic acid, there being almost none detected in four cases. Serum and tissue concentrations of both compounds 30 min after injection were sufficient to inhibit the growth of 97% of pathogenic organisms recovered from the skin of 135 vascular surgical patients. In a few cases tissue levels of the compounds were below inhibitory concentrations for coliforms and anaerobes at the time of skin closure, although they were still effective against most Gram-positive organisms. Amoxycillin/clavulanic acid combination is suitable antibiotic prophylaxis for vascular surgery.

[Direct determination of clavulanic acid in biological fluids using HPLC]. / Détermination directe de l'acide clavulanique dans les liquides biologiques par HPLC.

The so far described HPLC methods for clavulanic acid (CA) monitoring needed post-column derivatization with imidazole, resulting in poorly practicable methods. We propose here a direct determination of CA in human biological fluids with a ion-pairing technology using the bathochromic shift of tetrabutylammonium bromide (TBAB). The separation is performed on a reversed phase analytical column (250 X 4.6 mm) with the following mobile phase: 10% acetonitrile in 1 mM TAB and 20 mM ammonium acetate (pH = 5). The U.V. detection is at 214 nm. Serum and bile are prepared with acetonitrile and methylene chloride, and urines are diluted 1/10 prior the analysis. Retention time of CA is 8.4 min. Detection limit for bile and serum is 0.1 mg/l and 5 mg/l for urines. Within and between-day reproducibility is respectively 5.4% and 7.2% for serum and bile, and 4.7% and 6.8% for urine. This method may be suitable for clinical routine analysis and pharmacokinetic studies.

[A simple enzyme assay for the simultaneous determination of penicillin derivatives and clavulanic acid in biological fluids]. / Ein einfaches enzymatisches Verfahren zur simultanen Bestimmung von Penicillinderivaten und Clavulansäure in biologischen Flüssigkeiten.

A simple enzymatic assay for simultaneous determinations of amino-/ureidopenicillins and clavulanic acid concentrations in various body fluids is described; no pretreatment of the samples is required. The assay is based on the competitive inhibition of the chromogenic compound Padac by clavulanic acid in the presence of Proteus vulgaris beta-lactamase. Detection limit for clavulanic acid amounts 0.02 mg/l. Interferences with other antibiotics such as aminoglycosides, macrolides, quinolones etc. can be ruled out.

High-performance liquid chromatographic assay of clavulanate in human plasma and urine by fluorimetric detection.

A high-performance liquid chromatographic method with fluorimetric detection has been developed for the determination of clavulanate in human urine and plasma. Clavulanate in plasma samples was ultrafiltered using YMT membrane and reacted with benzaldehyde in phosphate buffer solution (pH 3.8) at 100 degrees C for 20 min. Clavulanate in urine samples was filtered with a polyacrylate membrane after ten-fold dilution, and reacted under the same conditions as those for plasma samples. The fluorescent product thus formed from clavulanate was separated from ordinary components of plasma and urine on a reversed-phase C18 column followed by fluorimetric detection (lambda ex = 386 nm, lambda em = 460 nm). The within- and between-run precisions were of the order of 4.02% (n = 10) and 6.23% (n = 5) for plasma samples at a level of 0.67 microgram/ml. The detection limit was as low as 10 ng/ml in plasma samples with a 50-microliter injection. Coexisting ticarcillin, amoxicillin or 1-amino-4-hydroxybutan-2-one (which is a metabolite of clavulanate in rat and dog) did not interfere in the clavulanate assay.