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1.
A new method for the determination of a guanosine—malonaldehyde adduct, β- -ribofuranosylpyrimido[1,2-a]purin-10(3H)-one (GMA), in rat and human urine is described. The method involves rapid pretreatment using, in sequence, polyamide, ion-exchange and reversed-phase cartridges; determination is by means of high-performance liquid chromatography with a thiobarbituric acid reactor in series with a fluorescence detector. This device can quantitatively determine the adduct at the sub-picomole level. This rapid, selective and sensitive method is suitable for the determination of guanine—malonaldehyde adducts in biological samples, such as human and rat urine. A semi-preparative method for the extraction and purification of these adducts from rat urine and for their identification by mass spectrometry and high-performance liquid chromatography with ultraviolet detection is also reported.  相似文献   

2.
A method for the determination of catecholamine sulfoconjugate isomers (CA-S) in urine was developed. The photo-induced fluorogenic reaction of CA-S with ethylenediamine reported previously was applied to the postcolumn labeling of HPLC for sensitive and selective detection. Special equipment for the reaction was made with a uv-irradiation lamp and a reaction coil of Teflon tubing inside a temperature-controlled reaction box. Lower determination limits of this system were 1 to 2 pmol. Urine samples pretreated with small ion-exchange resin columns were subjected to HPLC. Peaks corresponding to CA-S were identified quantitatively by two different separation methods. Thus, all six CA-S were first detected in the urine of normal individuals. The excretion rates of dopamine 3-sulfate, dopamine 4-sulfate, norepinephrine 3-sulfate, norepinephrine 4-sulfate, epinephrine 3-sulfate, and epinephrine 4-sulfate were 420 +/- 240, 98 +/- 55, 86 +/- 95, 15 +/- 14, 18 +/- 7, and 3 +/- 1 ng/min (+/- SD), respectively (n = 5).  相似文献   

3.
Quantitation of proteolytic enzymes using N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide has been adapted to flow-injection analysis. This procedure has been developed using two different proteases: subtilisin and chymotrypsin. For both enzymes the influence of substrate concentration on spectrophotometric response has been studied. The assay is based on the merging zones technique combined with a washing step. Results are obtained in less than 15 s and samples may be run at a rate of 90/h with good reproducibility. A linear relation between peak heights and enzyme concentrations was observed for 0-0.15 Anson unit/liter of subtilisin and for 0-30 mg/liter of a commercial preparation of chymotrypsin. The method requires only small sample volumes, and the consumption of the chromogenic substrate is reduced to a minimum by using intermittent pumping.  相似文献   

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A simple, rapid, precise and eco-friendly ion chromatography (IC) method for the determination of hippuric acid (HA) in human urine was proposed in this paper. The separation was carried out an anion exchange column with 2.0 mmol L?1 Na2CO3 + 2.0 mmol L?1 NaHCO3 as mobile phase at the flow-rate 0.7 mL min?1. A suppressed conductivity detector was used and the detection limit was 1.0 μg L?1(S/N = 3) for hippuric acid. The analysis time for one run was 30 min under the optimized IC condition. The recovery of hippuric acid was 93.2–98.0% while the relative standard deviation (RSD) was 1.4–2.3% by seven measurements.  相似文献   

6.
It was found that carbofuran enhances the chemiluminescence reaction between sodium sulphite and Ce(4+) in sulphuric acid, and this formed the basis of a flow-injection system with chemiluminescence detection for determination of carbofuran. Under optimum conditions, the enhanced chemiluminescence intensity was linear, with the concentration of carbofuran in the range 8 x 10(-8)-1.0 x 10(-5) g[sol ]mL, with a detection limit of 2.84 x 10(-8) g[sol ]mL (3 s[sol ]k). The proposed method was applied to the analysis of carbofuran in cabbage, with satisfactory results. Copyright (c) 2005 John Wiley & Sons, Ltd.  相似文献   

7.
Zhao S  Wang J  Ye F  Liu YM 《Analytical biochemistry》2008,378(2):127-131
A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K3[Fe(CN)6]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10−4 M luminol added to the CE running buffer and 1.0 × 10−4 M K3[Fe(CN)6] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10−7 M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10−7 to 3.0 × 10−5 M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.  相似文献   

8.
Incubation with a mixture of Sepharose-bound peptidases was shown to result in the quantitative release of amino acids from certain peptides and S-aminoethylated proteins. Subtraction of the low background values of amino acids generated by the enzymes enables amino acid ratios of corticotrophin-(1-24)-tetracosapeptide to be determined with a standard deviation on repeat digestions of 3-5%. Good values were obtained for amino acids that are completely or partially destroyed on acid hydrolysis, i.e. tryptophan, tyrosine, serine, asparagine and glutamine. Experiments with peptides containing d-amino acids showed that the enzyme mixture is stereospecific and could therefore be used to detect the presence of d-residues in peptides. The enzyme mixture completely hydrolyses peptide fragments obtained after Edman degradation and should therefore be useful for determining sequences of peptides containing acid-labile amino acid residues. The activities of the bound enzymes were unaltered over a period of 7 months and they provide a simple, reproducible procedure for the quantitative determination of amino acids in peptides and proteins containing l-amino acids.  相似文献   

9.
Determination of muramic acid in automated amino acid analysis   总被引:7,自引:0,他引:7  
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Melamine was found to be the etiological factor for the urinary stones epidemic in infants and young children in China in 2008. Urine level of melamine and its analog cyanuric acid may be useful markers for the evaluation of toxic effects. Liquid chromatography tandem mass spectrometry methods for the individual determination of melamine and cyanuric acid in human urine are described. Using isotope labeled internal standards during liquid–liquid extraction, the method was fully validated by verifying specificity, linearity, LLOQ, intra- and inter-assay precision and accuracy, matrix effect, recovery and stability. Calibration curves with good linearity (r = 0.9999) over the concentration range from 10 to 5000 ng/ml, intra-assay precision <10% and inter-assay precision <15%, accuracy between 93.0 and 111.6% were obtained with multiple reaction monitoring mode for melamine and cyanuric acid in human urine. The methods were successfully applied to the analysis of urine samples collected from 86 infants and 110 adults.  相似文献   

12.
Acetylsalicylic acid (Aspirin) is rapidly metabolized to salicylic acid (salicylate) and other compounds, including gentisic acid and salicyluric acid. Monitoring of salicylate and its metabolites is of toxicological, pharmacological and biomedical interest. Three capillary electrophoresis (CE) methods featuring alkaline aqueous buffers, laser-induced fluorescence (LIF) detection and no solute extraction or derivatization have been explored. A competitive binding, electrokinetic capillary-based immunoassay is developed that recognizes the presence of salicylate and gentisic acid in urine. Differentiation of the two compounds, however, is problematic. With appropriate ultraviolet excitation, many salicylate-related compounds are fluorescent so that CE with direct urine injection and LIF detection permits the determination of salicylate, gentisic acid and salicyluric acid. Using a HeCd laser with 325 nm produces interference-free monitoring of all three compounds. Using 257 nm excitation from a frequency doubled Ar ion laser, native fluorescence of an endogenous urinary compound that co-migrates with gentisic acid is observed. With wavelength-resolved fluorescence detection, however, the two substances are distinguished. Furthermore, this technique, with comparison to literature data, permits the putative assignment of several peaks to other salicylate metabolites, namely glucuronide conjugates of salicylate and salicyluric acid. All three CE-LIF techniques have been applied to toxicological patient urines and urines collected after ingestion of 500 mg acetylsalicylic acid. CE results compare favorably with those obtained by a commercial fluorescence polarization immunoassay and by a conventional photometric assay.  相似文献   

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The mixture of chromium, nicotinic acid and the amino acids glycine, glutamic acid and cysteine which stimulates the rate of CO2 production in a yeast bioassay system was subjected to the separation scheme based on ion-exchange chromatography which has been used to separate the chromium- containing fractions in brewer's yeast, [S.J. Haylock, P.D. Buckley and L.F. Blackwell, J. Inorg. Biochem., 18, 195 (1983)]. Four chromium-containing fractions (C2 to C5) were obtained by salt gradients and two further fractions (G1 and G2) were obtained using a pH gradient. All were amino acid-containing complexes of chromium and all except C5 also contained nicotinic acid. However, none of the isolated chromium fractions showed any activity in a yeast bioassay. On the basis of previous work, the activity of the original mixture was attributed to the presence of an oxygen-coordinated trans chromium(III)-dinicotinate complex. Biologically- inactive chromium complexes such as Cr(glu)2(H2O)+2 and Cr(gly)2(H2O)+2 after elution by ammonium hydroxide from Dowex 50W-X12 cation- exchange columns, stimulated the rate of CO2 production in the yeast bioassay. Elution with other bases, such as lithium hydroxide, potassium hydroxide and sodium hydroxide led to inactive fractions in all cases. A warning is therefore given that the use of ammonium hydroxide-elution of ion-exchange columns to isolate glucose tolerance factor fractions from biological samples (such as brewer's yeast) can lead to active fractions which do not relate to the native material.  相似文献   

15.
For use in clinical studies, a fast and sensitive assay method was developed for the determination of nifedipine in human plasma samples. The assay method is based on tandem mass spectrometry detection (HPLC–MS–MS). The effect of flow injection as well as HPLC separation on the results of the nifedipine determination were evaluated. The limit of quantification is 0.5 ng/ml and the accuracy (as determined by spiking recovery) was found to be good.  相似文献   

16.
A new amino acid has been isolated from the normal human urine. The chemical structure of the amino acid was determined to be alpha-hydroxy-beta-keto-gamma-aminobutyric acid based on its physical properties involving NMR, infrared and mass spectra, as well as chemical degradation and synthesis. In six healthy adults the urinary contents of the new amino acid were 3.2--4.5 mumol/24 h.  相似文献   

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20.
A mixture of eighteen protein amino acids was heated in sea water medium enriched with transition metal ions. Small granules were obtained as precipitates. Both dialyzable polymers and undialyzable polymers were obtained from the supernatant. Dialyzable polymers yielded mainly Glu, Asp, Ser, and Thr on hydrolysis; undialyzable polymers (C, 29.45; H, 3.87; N, 4.87; and ash, 31.5 wt%) yielded Thr, Asp, Glu, Gly, Leu, Ser, Lys, Pro, His, Phe, and a few unidentified ninhydrin positive peaks after acid hydrolysis. Five wt% of the undialyzable polymers con-ist of acid-hydrolyzable protein amino acids.  相似文献   

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