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High-performance liquid chromatographic method to quantify total cysteine excretion in urine
No full text
DETERMINATION OF CREATININE IN SERUM AND URINE BY A RAPID LIQUID-CHROMATOGRAPHIC METHOD
No full textWe 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 (±6.7)%. 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’searth alkaline
picrate methods
EVALUATION OF AN AUTOMATIC GAS-CHROMATOGRAPHIC SYSTEM FOR THE IDENTIFICATION OF BACTERIAL INFECTIVE AGENTS
No full textThe 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-C2o) 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 onlyfrom 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
identications ofgenus and species are concernedfor 238 cases.
The major advantages of this computerized system are a reduction
in the time required to obtain thejnal results, the elimination of
human errors by using the autosampler and a better inter-laboratory
comparability ofresults owing to a higher degree ofobjectivity. On
the other hand, the limited throughput ofMIS (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
Quantification of gentamicin in Mueller-Hinton agar by high-performance liquid chromatography
No full textThe 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
Urine pyridinium cross-links determinations by Beckman Cross Links Kit
No full textIncreased understanding of bone turnover has led to the development of several biochemical tests of bone metabolism. Among the biochemical indexes of bone resorption is measurement of urinary excretion of pyridinoline (Pyr) and deoxypyridinoline (Dpyr), molecules that cross-link the collagen chains and are released into the systemic circulation after the breakdown of mature bone collagen (1)(2)(3). Because they are not metabolized in vivo, they are excreted directly into urine in free (∼40%) and peptide-bound forms (∼60%) (4). Methods to measure cross-links in urine involve mainly two technical approaches: HPLC analysis, which, after hydrolytic and purification steps, allows the determination of the total forms of cross-links (5)(6)(7), and monoclonal antibody immunoassay methods able to quantify the sum of free Pyr and Dpyr or only the free Dpyr form (8)(9). The quantification of total or free cross-links forms provides, in any case, similar clinical information (9)(10).
Here we report the evaluation of the Cross LinksTM HPLC kit recently introduced by Beckman Labs. to quantify the total forms of Pyr and Dpyr. We compared the performance of this procedure with the Chrom- LinksT
CHROMATOFOCUSING AND ISOELECTRIC-FOCUSING IN IMMOBILIZED PH GRADIENTS COMPARED FOR CHARACTERIZATION OF HUMAN-HEMOGLOBIN VARIANTS
No full textWe compared the performance of two highly resolving methods,
chromatofocusing (CRF) and isoelectrifcocusingin
immobilized pH gradients (IPGF), for the separation of human
hemoglobin vanants. Lysates containing 13 different
hemoglobins, including variants of clinical and geographical
importance, and four electrophoretically “silent” variants (Hb
Brockton,Hb Cheverly,Hb KOIn,and Hb Waco) were analyzed.
Both techniques showed a good intrarun precision(CV
= 0.87% for CRF, 0.27% for IPGF) and high and similar
resolving power (0.010 pH units, with the pH gradients used
in this work). The use of an ultranarrow IPGF range (pH
7.15-7.35; pH gradient = 0.019 pH/cm) allowed the resolution
between Hb Brockton,Hb KOIn,and Hb A. Insome cases
(Hb D-Los Angeles, Hb F, Hb Waco), the variants were
separated from Hb A in different orders, depending on which
technique was used, probably because of the different analytical
principles of the two methods. As a second-level test,
both procedures are informative for characterization of human
hemoglobin variants
ENZYMATIC-SYNTHESIS OF [METHYL-(H-2)3] CREATININE
No full text2-Amino-1,5-dihydro-1-[methyl-H-2(3)] 4H-imidazol-4-one ([methyl-H-2(3)] creatinine) was obtained by acidification and heating (120-degrees-C, 120 min) of the [methyl -H-2(3)] N-amidinosarcosine (creatine) synthesised by methylation of guanidoacetate (GA) with S-adenosyl-[methyl-H-2(3)) L-methionine ([methyl-H-2(3)] AdoMet) in the presence of rat liver guanidoacetate methyltransferase (GAA-MT). Isotopic enrichment was 94.7 %
Capillary electrophoresis for protein analysis: separation of human growth hormone and human insulin molecular forms
No full textThe capillary zone electrophoresis (CZE) technique was evaluated for separation and quantification of human growth hormone (hGH), human insulin (hI), and proinsulin. Three different molecular forms of biosynthetic hGH (20K, 22K, 44K), methionyl-hGH, biosynthetic hI, and proinsulin, were studied. The hormones were separated with uncoated capillaries, and various analytical conditions were tested (different buffers, ionic strength, pH). The samples were introduced both at positive pressure and electrokinetically, and the voltage applied was varied in each case. Linearity, repeatibility, and limit of detection (0.8 microM for hGH and 1.3 microM for insulin) were determined. Different purification steps were tried in order to find a suitable preanalytical procedure for hGH and hI purification from plasma. Recovery rates from 65 to 80% were obtained
Is the direct quantitation of antibiotics in agar by high-performance liquid chromatography useful?
No full textThe direct quantification of antibiotics in agar allows one to study the quality of the agar matrix, the kinetics of diffusion
and the bacteria–antibiotic interaction. Mueller–Hinton 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)
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