UV - spectrophotometric (UV) and high performance liquid chromatographic methods (HPLC) were described in determining amoxicillin and clavulanic acid in pharmaceutical preparations. Spectrophotometrically, amoxicillin was determined by measuring the absorbance values at 320 nm in buffer-CuS04 solution (pH 5.2) for amoxicillin and at 313 nm in imidazol solution (pH 6.8) for clavulanic acid. Beer's Law was obeyed in range 12 - 32mg. ml-1 for amoxicillin and 12 - 22 mg. ml-1 for clavulanic acid. The simultaneous HPLC method depended upon using a reserved phase RP18 column at ambient temperature with a mobile phase consisting of methanol - phosphate solution pH 4.4 (4:96) at a flow rate 1ml.min-1. Quantitation was achieved by UV detection at 220 nm. Calibration curve was linear over the concentration range 50-150 mg. ml-1 for amoxicillin and 15-35 mg. ml-1 for clavulanic acid. Both spectrophotometry and HPLC method showed good linear correlation, precision and accuracy. The obtained results from spectrophotometry and HPLC method showed no significant difference. Both methods were successfully applied to the assay of commercial tablets. The procedures were rapid, simple and suitable for quality control applications
Pharmaceutical Preparations ; Amoxicillin ; Clavulanic Acid

A simple, rapid, sensitive, precise and accurate high-performance liquid chromatographic (HPLC) method with ultraviolet detection at wavelength of 205nm has been validated for the simultaneous determination of amoxicillin and clavulanic acid in human serum. Serum samples were deproteinized with methanol. The separation of 2 obtained compounds was achieved using a reversed phase C8 column and a mobile phase, consisting of acetonitrile-phosphate solution. The calibration curves were linear over the concentration range of 0.625 – 10 g.ml-1 for amoxicillin and 0.3125 – 5 g.ml-1 for clavulanic acid. The quantitative limits are 0.625 and 0.3125 g.ml-1 for amoxicillin and clavulanic acid. Analytical recoveries from human serum ranged from 93 to 96% for both components. This method was fully validated. It allows the simultaneous assay of amoxicillin and clavulanic acid in biomedical applications. This method has been successfully applied to a pilot pharmacokinetic study in 3 healthy volunteers after a single oral administration of amoxicillin and clavulanic acid combination
Amoxicillin ; Clavulanic Acid ; Serum ; Chromatography, High Pressure Liquid

BACKGROUND: This study was designed to evaluate the ability of the Vitek and MicroScan ESBL test by comparing with NCCLS ESBL phenotypic confirmatory test by disk diffusion and to know the frequency of ESBL producers in the Seoul Veterans Hospital. METHODS: A total of 1,261 isolates(Escherichia coli 705, Klebsiella pneumoniae 502, K. oxytoca 54) from 883 patients were included in ESBL screening test by Vitek (494 strains) and MicroScan (767 strains). After excluding repetitive isolates from same patients, NCCLS ESBL confirmatory test was performed for 197 ESBL screening positives and 184 ESBL screening negatives. RESULTS: The overall frequency of ESBL screening positives was 22.3% (by MicroScan 26.2%, by Vitek 15.6%), and that of NCCLS ESBL positives was 18.9%(18.3% in E. coli, 21% in K. pneumoniae). MicroScan and Vitek ESBL test showed 100% and 92.3% sensitivity, 77.1% and 95.5% specificity, respectively. Among the 158 NCCLS ESBL positives, 17.7% showed clavulanic acid effect in cefotaxime only, 10.1% in ceftazidime only, and 72.2% in both. MicroScan Neg ComboPanel Type 21 test revealed that 91.4% of suspicious ESBL producers flagged by one or two antimicrobials were erroneous. In contrast, 96.2% of strains flagged by all five antimicrobials were correct. CONCLUSION: Suspicious ESBL producers by MicroScan showing three or four antimicrobial flags should be retested by NCCLS ESBLconfirmatory test. But strains with two or less flags and strains with all 5 flags can be reported as Non-ESBL producers and ESBL producers, respectively.
Cefotaxime ; Ceftazidime ; Clavulanic Acid ; Diffusion ; Hospitals, Veterans ; Humans ; Klebsiella pneumoniae ; Mass Screening ; Sensitivity and Specificity ; Seoul

To elucidate the role of plasmid-mediated AmpC-type B-lactamases in clinical practice, cefoxitin-resistant isolates of E. coli (19 strains) and K. pneumoniae (7 strains) from three hospitals in Korea were studied. All of the 26 isolates produced at least one j3-lactamase and 16 (62%) isolates produced AmpC-type B-lactamases poorly inhibited by clavulanic acid. In 16 such isolates, 4 kinds of AmpC enzymes were detected; the pI 8.0 AmpC enzyme in 11 isolates, the pI 8.9 in 3 isolates of E. coli, the pI 8.5 in 1 isolate of E. coli, and the pI 7.8 in 1 isolate of K pneumoniae. The pI 8.0 and 7.8 AmpC enzymes had an apparent molecular mass of 38 kDa and the pI 8.5 and 8.9 AmpC enzymes had a molecular mass of 35 kDa. Cefoxitin resistance was transmissible in six E. coli and three K pneumoniae strains due to a common AmpC-type B-lactamase with a pl of 8.0. This enzyme was confirmed to be CMY-1 B-lactamase by Southern blotting and PCR analysis. Four E. coli isolates produced large amounts of AmpC-type j3-1actamase. They were chromosomal AmpC hyperproducers carrying some alterations in the promoter and attenuator regions of the ampC chromosomal gene. The pI 7.8 AmpC enzyme is currently under study. In conclusion, this study showed that the CMY-1 plasmid-mediated cephamycinase play an important role in cephamycin resistance of K. pneumoniae and E. coli clinical isolates in Korea.
beta-Lactamases* ; Blotting, Southern ; Cefoxitin ; Clavulanic Acid ; Korea ; Pneumonia* ; Polymerase Chain Reaction

At the Venereal Disease Clinic of Choong-ku Public Health Center in Seoul ]03 male patients with uncomplicated gonococcal urethritis were allocated randomly into one of 2 treatment regimens and 101 patients were followed. All 51 patients, including PPNG infections, treated with clavulanate-potentiated amoxycillin, 375 mg, PO, t.i.d. for 5 days recovered(100%), Two(4%) of 50 patients treated with clavulanate-potentiated amoxycillin, 3. 25g, PO plus probenecid, lg, PO failed to recover. These cases were 2 of 25 Penicillinase Froducing Neisseria gonorrhoeae(PPNG) infections(failure rate of 8%) and all 25 non-PPNG infections recovered(100%). It is suggested that both of these clavulanate-potentiated amoxycillin regimens ha.ve similarly good effect with minimal side effects in the treatment of gonococcal urethritis and, because of high rate of PPNGs among circulating N, gonorrhoeae, they can be recommended as the first line treatment for gonorrhoa ir Korea.
Amoxicillin* ; Clavulanic Acid* ; Humans ; Korea ; Male* ; Neisseria ; Penicillinase ; Probenecid ; Public Health ; Seoul ; Sexually Transmitted Diseases ; Urethritis*

OBJECTIVETo establish a chromatography-based method for simultaneous analysis of the concentrations of amoxicillin and clavulanate potassium in human blood.

METHODSWith 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;.

RESULTSThe 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.

CONCLUSIONThis method is simple, accurate, sensitive, specific and reproducible for analyzing the concentrations of amoxicillin and clavulanate potassium simultaneously in human plasma.

BACKGROUND: Accurate detection of extended spectrum beta-lactamase (ESBL) is important because ESBLs producing organisms may appear susceptible to oxyimino-beta-lactams in standard susceptibility tests, but are considered to be clinically resistant to these drugs. Conventional antimicrobial susceptibility test methods do not reliably detect ESBL production. Molecular techniques and NCCLS broth dilution method, which detect ESBL production, may be time consuming, expensive and technically difficult to perform. The purpose of this study is to evaluate the clinical usefulness of NCCLS ESBL phenotypic confirmatory test by disk diffusion method. METHODS: For 96 Escherichia coli and 49 Klebsiella pneumoniae isolates collected between December 2000 to February 2001, double disk synergy test, NCCLS ESBL screening and phenotypic confirmatory test by disk diffusion test were performed. The ESBL producer was defined as organism showed an increase in the zone diameter of > or = 5 mm for either antimicrobial agent such as cefotaxime and ceftazidime tested in combination with clavulanic acid versus its zone when tested alone. RESULTS: The sensitivity of NCCLS ESBL phenotypic confirmatory test were as follows: cefotaxime/clavulanic acid disk; 100% in K. pneumoniae and 83% in E. coli, and ceftazidime/clavulanic acid disk; 94% in K. pneumoniae and 67% in E. coli, respectively. Among the organisms with positive to NCCLS ESBL phenotypic confirmatory test, the detection rate of antimicrobial agents in double disk synergy test were as follows: K. pneumoniae; cefotaxime (84%), aztreonam (74%), and ceftazidime (52%), and E. coli; cefotaxime (44%), ceftazidime (44%), and aztreonam (39%). CONCLUSIONS: The NCCLS ESBL phenotypic confirmatory test by disk diffusion method is easy, rapid, and sensitive method, suitable for routine use in the clinical laboratory.
Anti-Infective Agents ; Aztreonam ; beta-Lactamases* ; Cefotaxime ; Ceftazidime ; Clavulanic Acid ; Diffusion ; Escherichia coli* ; Escherichia* ; Klebsiella pneumoniae* ; Klebsiella* ; Mass Screening ; Pneumonia

BACKGROUND: The therapeutic option is limited for the infections caused by organisms producing plasmid- mediated AmpC beta-lactamases, increasingly identified worldwide. Two sporadic patients with bacteremia caused by K. pneumoniae possessing an unusual inducible beta-lactam resistant phenotype were found in a university hospital. RESULTS:We conducted antibiotic susceptibility test according to NCCLS guideline. Also, we characterized beta-lactamase by isoelectric focusing. RESULTS: DHA-1 gene conferred the resistant phenotype. The patients had experienced treatment failure when treated with extended-spectrum cephalosporin. For the isolates the cephalosporin resistance was induced by clavulanic acid (and cefoxitin). CONCLUSION: Theses results suggest that the extended-spectrum cephalosporins might not provide optimal therapeutic option for inducible DHA-1-producing K. pneumoniae infection, even when the pathogens are susceptible in vitro.
Bacteremia ; beta-Lactamases* ; Cephalosporin Resistance ; Cephalosporins ; Clavulanic Acid* ; Humans ; Isoelectric Focusing ; Klebsiella pneumoniae* ; Klebsiella* ; Phenotype ; Pneumonia ; Treatment Failure

BACKGROUND: The therapeutic option is limited for the infections caused by organisms producing plasmid- mediated AmpC beta-lactamases, increasingly identified worldwide. Two sporadic patients with bacteremia caused by K. pneumoniae possessing an unusual inducible beta-lactam resistant phenotype were found in a university hospital. RESULTS:We conducted antibiotic susceptibility test according to NCCLS guideline. Also, we characterized beta-lactamase by isoelectric focusing. RESULTS: DHA-1 gene conferred the resistant phenotype. The patients had experienced treatment failure when treated with extended-spectrum cephalosporin. For the isolates the cephalosporin resistance was induced by clavulanic acid (and cefoxitin). CONCLUSION: Theses results suggest that the extended-spectrum cephalosporins might not provide optimal therapeutic option for inducible DHA-1-producing K. pneumoniae infection, even when the pathogens are susceptible in vitro.
Bacteremia ; beta-Lactamases* ; Cephalosporin Resistance ; Cephalosporins ; Clavulanic Acid* ; Humans ; Isoelectric Focusing ; Klebsiella pneumoniae* ; Klebsiella* ; Phenotype ; Pneumonia ; Treatment Failure

BACKGROUND: Amoxicillin/clavulanate (A/C) is a combination of a broad spectrum -lactam antibiotic amoxicillin and the potent -lactamase inhibitor clavulanate. A/C 7:1 combination is known to be equal in its clinical efficacy and to have less gastrointestinal adverse effects compared to conventional A/C 4:1 combination. We estimated in vitro antimicrobial activities of the 7:1 combination (AMOCLA Duo) and the conventional 4:1 combination against clinical bacterial isolates known to be the major causes of acute otitis media or sinusitis. METHODS: Total 183 strains isolated from clinical specimens of patients at Seoul National University Hospital were tested for minimal inhibitory concentraion (MIC). Streptococcus pneumoniae and Haemophilus influenzae were tested by microdilution broth method and other bacterial species by agar dilution method according to the recommendations of National Committee for Clinical Laboratory Standards (NCCLS). RESULTS: AMOCLA Duo was compared with the 4:1 combination in respect to MIC50, MIC90 and MIC range. For total 183 strains (30 methicillin-susceptible Staphylococcus aureus, 30 methicillin-sus-ceptible Staphylococcus epidermidis, 25 penicillin-susceptible S. pneumoniae, 42 penicillin-resistant S. pneumoniae, 33 H. influenzae and 23 Moraxella catarrhalis), mean MICs did not show statistically significant difference between the 2 combinations but they did for H. influenzae and M. catarrhalis. CONCLUSIONS: As for the total test strains, in vitro antimicrobial activity of AMOCLA Duo was equal to that of the conventional 4:1 combination. For each species, H. influenzae and M. catarrhalis showed significant difference between mean MICs of the 2 combinations but other species did not. We do not suppose, however, that in case of H. influenzae this difference is of practical and clinical significance according to the NCCLS interpretive criteria for MIC. Although M. catarrhalis showed statistically very significant difference of MICs, this difference can be clinically solved due to the higher dose of amoxicillin in AMOCLA Duo.
Agar ; Amoxicillin* ; Bacteria* ; Clavulanic Acid* ; Haemophilus influenzae ; Humans ; Influenza, Human ; Moraxella ; Otitis Media ; Pneumonia ; Seoul ; Sinusitis ; Staphylococcus aureus ; Staphylococcus epidermidis ; Streptococcus pneumoniae