Contribution of high-performance liquid chromatography to the identification of some Corynebacterium species by comparison of their corynomycolic acid patterns.

Reverse-phase high-performance liquid chromatography of corynomycolic acids provided a specific pattern for each Corynebacterium species studied. These data suggest that a fast and reproducible procedure is now available for bacteriological identification at the genus and at the species level of corynomycolic-acid-containing bacteria. Mass spectrometry analysis of post-column collected fractions provided the order of elution of some corynomycolic acids and isomers and showed the high specificity of the chromatographic assay which could be used for the routine bacteriological identification of some species belonging to the genus Corynebacterium.

Toxin content, phallotoxin and amatoxin composition of Amanita phalloides tissues.

The toxin content and composition of Amanita phalloides tissues were determined in three specimens at two carpophore development stages. The carpophore was subdivided into six parts, namely, the cap, gills, ring, stipe, volva and bulb. To our knowledge, this is the first report of such an investigation on the ring and the bulb. Substantial differences in the tissue toxin content were revealed. The ring displayed a very high amount of toxins, whereas the bulb had the lowest toxin content. Compositional differences in relation to the nature of the tissue were also noted. The highest amatoxin content was found in the ring, gills and cap, whereas the bulb and volva were the richest in phallotoxins. Furthermore, variability in the toxin composition was observed. The differences in the distribution of individual toxins in the tissues might be related to the carpophore developmental stage.

[Determination of enoximone and its principle metabolite in serum and urine using high pressure liquid chromatography]. / Dosage de l'enoximone et de son principal métabolite dans le sérum et l'urine par chromatographie liquide à haute performance.

We developed a high performance liquid chromatography method for the monitoring of enoximone and its main metabolite in serum and urine. Samples handling involves a unique chemical extraction step by ethylacetate. Serum needs at first to be deproteinized by acetonitril. The chromatographic separation is realized on a reversed phase analytical column by gradient elution with acetonitrile. Quantification is by U.V. absorbance at 365 nm. We compared our new method with the method so far considered as reference one. When specificity, accuracy and linearity of both procedures are similar, we greatly enhanced the detection limit [(5 ng/ml for (E) and (SE)] and the practicability: ease of use, rapidity and lower cost.

High-performance liquid chromatography of antibiotics.

High-performance liquid chromatographic (HPLC) monitoring of antimicrobial agents has recently become more widely used, and represents an interesting alternative to other methods. The methodology is characterized by good specificity and accuracy, and it is applicable to almost all antibiotics. This review first describes the successive steps to investigate for the development of an HPLC method for a new antibiotic, and how to make use of it. Particular emphasis is put on the problems related to the standardization of sample preparation and to the development of mobile phases for use with different molecules belonging to the same class. The second part of the review describes one or more HPLC techniques for a representative antibiotic of each major class.

Amoxicillin and clavulanic acid concentrations in gingival crevicular fluid.

The beta-lactams are bactericidal antibiotics, but some of them may be inactivated by bacterial beta-lactamases which destroy the beta-lactam ring. The inactivation of amoxicillin by beta-lactamases of gram negative anaerobic bacteria can be circumvented by the addition of clavulanic acid, a beta-lactamases inhibitor. Thus, most of these bacteria are susceptible to this combination. The aim of this study was to investigate the concentrations of amoxicillin and clavulanic acid in gingival crevicular fluid (GCF). These concentrations were measured in 20 patients with rapidly progressive periodontitis 1 h after a dose of 500 mg (1 tablet Augmentin) on day 0 and 1 h after the 10th intake on day 3. For the sampling of GCF, Periopapers were introduced in 16 gingival sites per subject and time. The GCF volumes collected were estimated using the Periotron 6000. A high performance liquid chromatography method has been developed for the determination of amoxicillin and clavulanic acid in microsamples (1 to 10 microliters) of GCF. The concentrations of amoxicillin and clavulanic acid were respectively, 14.05 micrograms ml-1 and 0.40 microgram ml-1 at day 0, 13.93 micrograms ml-1 and 0.37 microgram ml-1 at day 3. Effective levels of amoxicillin and clavulanic acid, well above the minimal inhibitory concentrations of some susceptible periodontal anaerobes (P. intermedia) involved in destructive periodontal diseases, are achieved following the multiple administration of amoxicillin combined with clavulanic acid.

Determination of vancomycin in human serum by high-pressure liquid chromatography.

A rapid, accurate, reverse-phase high-pressure liquid chromatographic procedure for vancomycin quantitation in human serum, cerebrospinal fluid, and peritoneal fluid was developed. This procedure involves a simple chemical extraction of the antibiotic and is suitable for each of these body fluids. The column and mobile phase used provided a good resolution of the vancomycin peak with a retention time of 6.1 min. The precision of the assay was within the requirement for a daily routine clinical application. Coefficients of variation for within-day reproducibility were 5.80 and 6.28%, respectively, for samples at 50 and 25 micrograms/ml, and for between-day reproducibility they were 11.4 and 11.1%, respectively. No interference was found with respect to beta-lactam and aminoglycoside antibiotics and many other currently used drugs, indicating a good specificity for the procedure. The detection limit of 100 ng/ml has proven to be sufficient for monitoring drug levels in serum obtained after usual dosages. Drug levels in 112 clinical serum specimens assayed by high-pressure liquid chromatography were regressed against the levels obtained for the same samples by radioimmunoassay and fluorescent polarization immunoassay. Correlation coefficients were 0.945 and 0.967, respectively, and were highly significant (alpha less than 0.001).

[HPLC, RIA, FPIA. Evaluation of 3 methods for the assay of vancomycin]. / HPLC, RIA, FPIA. Evaluation de trois méthodes de dosage de la vancomycine.

We describe a rapid and accurate high performance liquid chromatographic (HPLC) method for vancomycin quantitation. This method is then compared with two immunoassays, RIA and FPIA. The chemical extraction step needed for HPLC is simple and rapid. Both conventional reversed phase HPLC and high speed reversed phase HPLC were tested. The mobile phase was a mixture of aqueous ammonium acetate and acetonitrile. Specificity of the HPLC assay was good. Serum levels in 112 clinical specimens assayed by HPLC were regressed against the levels obtained for the same samples by both RIA and FPIA. The correlation was good (RIA : r = 0.945; FPIA : r = 0.967). Considering the disadvantages of RIA, HPLC and FPIA emerge as the methods of choice.

Determination of fosfomycin in biological fluids by capillary electrophoresis.

A capillary electrophoresis method was developed for the determination of the antibiotic fosfomycin in serum, cerebrospinal fluid and aqueous humor. The technique uses indirect UV detection and the working buffer includes an organic cation to improve fosfomycin mobility. The electrophoretic time of migration is less than 7 min in both fluids. The limit of quantification is 2.5 and 1 microgram/ml in serum and aqueous fluids, respectively (signal-to-noise ratio = 3). The method was validated in serum and water over the concentration range 2.5-200 micrograms/ml. The calibration graph for serum was linear with a correlation coefficient r = 0.999. At a fosfomycin concentration of 2.5 micrograms/ml in serum, the intra- and inter-day precisions (coefficients of variation) were 5 and 5.2%, respectively. The mean recovery in serum was 94.5% (S.D. = 2.4%).

Simultaneous assay for amatoxins and phallotoxins in Amanita phalloides Fr. by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method is described that allows the simultaneous determination of up to eight amatoxins and phallotoxins. The method identifies both neutral toxins (alpha- and gamma-amanitin, phalloidin, phallisin and phalloin) and acidic toxins (beta-amanitin, phallacidin and phallisacin). Toxins were separated, identified and determined by gradient elution with 0.02 M aqueous ammonium acetate-acetonitrile and simultaneous monitoring of the absorbances at 214 and 295 nm. The assay was successfully applied to the analysis of the toxins in a crude extract of Amanita phalloides. The limit of detection for each toxin was 10 ng/ml of extraction medium. The assay was further validated by analysing the toxin content in Galerina marginata, a species containing only amatoxins. This relatively simple method should be suitable for the detection of amatoxins and phallotoxins in almost any species of mushrooms.

High-performance liquid chromatographic method for determination of ciprofloxacin in biological fluids.

A simple and precise high-performance liquid chromatographic procedure has been developed for the determination in biological fluids of ciprofloxacin, a new, with extended antibacterial spectrum, quinoline carboxylic acid. The work-up procedure involves a chemical extraction step followed by isocratic chromatography on a reversed-phase analytical column, with ultraviolet detection. The detection limit for blood levels is 10 ng/ml. The calibration curve is linear from this detection limit to 10 microgram/ml. The statistical analysis of the correlation made between this assay and an agar diffusion procedure during a pharmacokinetic study suggests the existence of one or more active metabolites which could be mainly excreted in the bile.

Determination of alpha-amanitin and beta-amanitin in human biological fluids by high-performance liquid chromatography.

A high-performance liquid chromatographic assay of alpha-amanitin and beta-amanitin in human serum, urine, or stomach washings is described. Sample preparation involves a chemical step with deproteinization and organic solvent treatment, and a selective cleanup and concentration step on reversed-phase prepacked cartridges. Separations are performed on a reversed-phase analytical column under isocratic conditions with uv detection at 280 nm. The method allows the quantitation of alpha- and beta-amanitin separately with a detection limit of 10 ng/ml for both toxins.