64 research outputs found
Evaluation of an automatic gas chromatographic system for the identification of bacterial infective agents
The potential clinical application of gas chromatography to
microbial identifcation was evaluated. A completely automated
system, the MIS (Microbial Identification System; Hewlett-
Packard) can analyse and identify pure strains by comparison of
their cellular fatty acids patterns (C9-C20) with the reference
parameters stored in a library. Three hundred and sixty-seven
strains were tested, comparing the gas chromatographic results
with those obtained by the traditional microbiological methods in
the bacteriology laboratory of our Institute. A standardized
extractive procedure was followed to obtain the fatty acid methyl
esters (FAMEs), but some modifications to the recommended
procedure were introduced in the bacterial growth procedures:
colonies harvested not only from the recommended growth media but
also from selective media routinely used in the bacteriology
laboratory were successfully examined. These modifications did not
influence the results but improved the ease for the user; good
agreement with the comparison method was observed as far as
identifications of genus and species are concerned for 238 cases.
The major advantages of this computerized system are a reduction
in the time required to obtain the final results, the elimination of
human errors by using the autosampler and a better inter-laboratory
comparability of results owing to a higher degree of objectivity. On
the other hand, the limited throughput of MIS (only 40 samples in
24 h) prevents its use in a large routine laboratory; this technology
is appropriate in emergency cases, in taxonomic studies and as a
confirmatory method
Plasma mitomycin C concentrations determined by HPLC coupled to solid-phase extraction
The aim of this study was to set up a method for quantification of plasma mitomycin C (MMC) concentrations during intravesical chemotherapy delivered in the presence of local bladder hyperthermia (HT). In comparison with existing methods, this assay, characterized by relative simplicity and efficiency, resulted in the facilitation of performance with nondedicated instrumentation or nonspecialized staff. Purification from plasma matrix was carried out by solid-phase extraction under vaccuum. The purified drug was then collected directly into the vials of the HPLC autosampler. Chromatographic analysis was performed on a reversed-phase C18 column with water:acetonitrile (85:15 by vol) as the mobile phase and the UV detector set at 365 nm. The use of porfiromycin as internal standard provided a method with good within-day precision (CV 6.0% at 5 micrograms/L, n = 6), linearity (0.5-50 micrograms/L), and specificity. The lower limit of detection (< or = 0.5 microgram/L) proved to be suitable for plasma pharmacokinetics monitoring in two tested patients treated with MMC + HT for superficial bladder cancer
Quantitative cytometry of MHC class I digestion from living cells
Digestion of crude membrane preparations with papain releases the extracellular portion of major histocompatibility complex (MHC) class I molecules. MHC class I molecules are integral membrane glycoprotein complexes formed by the noncovalent association of 2 invariant molecules, the heavy chain and the beta2-microglobulin (beta2-m), to a wide array of peptides. The cleaved soluble moiety retains the antigenic properties of the intact membrane-bound complex. Here we show that MHC class I digestion may be carried out on living cells, and we quantitate the surface expression of MHC complexes by a combined cytometric/high performance liquid chromatographic (HPLC) approach. Papain digestion results in time- and dose-dependent disappearance of membrane MHC-associated-fluorescence as detected by FACS analysis with MHC-specific monoclonal antibodies (mAbs). beta2-m and peptides became detectable by HPLC analysis and western blotting in the digestion buffer and were quantitated by comparison with purified standards. The cytometric assessment of the digestion allows one to simultaneously monitor efficacy and toxicity of the treatment. The procedure we describe allows to selectively retrieve by affinity chromatography MHC from the cell membrane, avoiding any contamination due to intracellular, "immature" MHC molecules
OPEN-CHAIN PEPTIDES OBTAINED BY ACIDIC HYDROLYTIC CLEAVAGE OF CYCLOSPORINE-A
Hydrolysis of cyclosporin A (CsA) was studied in order to clarify the still undefined point of attack of the acidic degradation. Among ether extractable and water-soluble products formed from CsA in HCl, two open-chain peptides were isolated by high-performance liquid chromatography which were identified as the deca- and nonapeptides deriving from CsA through the hydrolytic cleavage of amino acid residue 11 and both residues 11 and 10, respectively. Identification was carried out by fast atom bombardment tandem mass spectrometry
Is the direct quantitation of antibiotics in agar by high-performance liquid chromatography useful?
The direct quantification of antibiotics in agar allows one to study the quality of the agar matrix, the kinetics of diffusion
and the bacteria\u2013antibiotic interaction. Mueller\u2013Hinton agar (MHA) plates from three manufacturers were tested using
HPLC and the disc diffusion test of ceftazidime (CAZ). Notable differences in the chromatographic profiles of MHA plate extracts from OXOID, DID and Becton Dickinson (BD) were shown, with a higher CAZ concentration after 24 h at 6 mm in BD P. aeruginosa inoculated plates (5.1+/-1.7 mg/ ml, n=6) vs. OXOID and DID (1.6+/-0.3 mg/ ml, n=12). BD plates gave also a different inhibition zone diameter (26+/-0.5 mm, n=3) with respect to DID and OXOID (29+/-0.5 mm, n=3)
High-performance liquid chromatographic purification and capillary electrophoresis quantification of the chemokine stromal cell-derived factor-1
Chemokines are members of the chemotactic cytokines family implicated in various immunoregulatory functions. The CXC-chemokine stromal cell derived factor-1 (SDF-1alpha) was purified from the culture medium of murine bone marrow stromal cell line (MS-5) by affinity and reversed-phase liquid chromatography. Yield and purity were assessed by capillary electrophoresis (CE) with reference to the human SDF-1alpha from recombinant DNA technology. CE technique was useful to evaluate the purity of human SDF-1alpha from chemical synthesis and to resolve murine and human SDF-1alpha, differing by only one amino acid. Chemotactic activity of the murine SDF-1alpha was tested on the basis of CE quantification
DETERMINATION OF CREATININE IN SERUM AND URINE BY A RAPID LIQUID-CHROMATOGRAPHIC METHOD
We describe an HPLC ion-pair procedure for rapid and
specific evaluationof creatinineinserum and urine. We used
a 15 cm x 4.6 mmODS column with a 50/50 (by vol) mixture
of sodium decanesulfonic acid (10 mmol/L, pH 3.2) and
methanoland measuredabsorbance at 236 nm. Serum (100
tL) or 30-fold-diluted urine (100 L) was added to 400 pL of
acetone. After centrifugation,the supemates (300 L) were
dried, reconstituted with the mobile phase, and injected into
the HPLC. Assay precision was tested for concentrations of
10, 29, and 130mg/Land yielded, respectively, 3.1%, 2.1%,
and 1.1% for within-dayCV and 2.8%, 2.1%, and 2.2% for
totalCV. Analytical recovery was 102 (\ub16.7)%. Unearitywas
demonstrated in the 0-200 mg/L range for serum and 0-3.5
g/L range for urine (r 0.999). The detection limit for creatmine
(signal-to-noiseratio = 3) was 0.5 mg/L. We used
cimetidinefor internalstandardization.Correlationwasgood
between this procedureand the Jaff#{k2i3n3e}tic,the enzymatic
(creatinineamidohydrolase),and the Fuller\u2019searth alkaline
picrate methods
Quantification of gentamicin in Mueller-Hinton agar by high-performance liquid chromatography
The aim of this study was to optimise a method for gentamicin determination in an agar matrix and to investigate if and how agar composition can affect the gentamicin diffusion kinetics during the agar diffusion tests for antibiotics sensitivity. Gentamicin was separated by RP-HPLC and detected at 365 nm after pre-column derivatization with 1-fluoro-2,4-dinitrobenzene. Recovery (> or = 79%), linearity (r2 > or = 0.997) and sensitivity (1 microg/ml) were assessed using four different agar matrices. The kinetics of gentamicin diffusion tested on BioMerieux and DID manufacturers' products showed in uninoculated agar plates significant differences that were even more pronounced in the presence of Pseudomonas aeruginosa metabolism
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