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1.
A high-performance liquid chromatographic method was developed for the determination of methylguanidine in biological fluids. Methylguanidine and the internal standard were isolated from plasma by cation-exchange solid-phase extraction prior to chromatographic analysis. Urine samples were diluted and injected directly onto the analytical column. Chromatographic separation was carried out on an Ultrasil cation-exchange column using a mixture of methanol and monochloroacetate (15/85, v/v) as the mobile phase. Postcolumn derivatization of methylguanidine was carried out using alkaline ninhydrin reagent and the resulting fluorescent product was detected on-line. The method was specific, sensitive, reproducible, and linear over a wide a range of concentrations. The lower limit of detection for methylguanidine in plasma and urine was 1 and 100 ng/ml, respectively. The method was successfully employed for quantification of the levels of methylguanidine in normal and uremic human subjects, normal dogs, and dogs with ischemic-induced acute or spontaneous chronic renal failure. 相似文献
2.
In this paper, we report a rapid, sensitive, and quantitative procedure to conduct disaccharide compositional analyses of keratan sulfates (KS) by means of high-performance liquid chromatography (HPLC) separation and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection of products generated by hydrolysis of this glycosaminoglycan with Bacillus sp. keratanase II or Escherichia freundii endo-beta-galactosidase. Following E. freundii endo-beta-galactosidase digestion of bovine corneal KS, the monosulfated disaccharide glcNAc6sbeta(1-->3)gal, accounting for approximately equals 95% nmol and 50% yield products, is produced. On the contrary, bovine corneal KS treated with endo-beta-N-acetylglucosaminidase (keratanase II) from Bacillus sp. generates two major products, the monosulfated disaccharide galbeta(1-->4)glcNAc6s ( approximately equals 50% nmol product) and the disulfated disaccharide gal6sbeta(1-->4)glcNAc6s ( approximately equals 40% nmol product) for over 90% nmol products. These disaccharides are separated and readily determined within 30 min by using a linear-gradient strong anion-exchange separation. A linear relationship was found for the two purified disaccharides over a wide range of concentrations, from approximately equals 108 pmol, 50 ng, to 2,160 pmol, 1,000 ng, for the disaccharide galbeta(1-->4)glcNAc6s, and from 92 pmol, 50 ng, to 1,840 pmol, 1,000 ng, for the disaccharide gal6sbeta(1-->4)glcNAc6s. HPLC analysis was applied to the quantitative and qualitative determination of KS produced by 3T3-J2 murine fibroblasts in the cell medium. The amount of KS was found to be 2.80+/-0.34 microg/ml/10(6) cells and composed of approximately equals 71% nmol of disaccharide galbeta(1-->4)glcNAc6s and 18% nmol of the disulfated disaccharide gal6sbeta(1-->4)glcNAc6s having approximately equals 1.20 sulfate groups/disaccharide. Our data illustrate that the HPLC procedure reported represents an improved approach for the quantitative and compositional microanalyses of KS, especially applicable to experimentation involving small amounts ( approximately 50 ng) of this glycosaminoglycan and in relation to its biological function and pathological importance. 相似文献
3.
Analysis of tamoxifen-DNA adducts by high-performance liquid chromatography using postcolumn online photochemical activation 总被引:1,自引:0,他引:1
Sharma M 《Biochemical and biophysical research communications》2000,273(1):40-44
Tamoxifen, a widely used nonsteroidal antiestrogen in the treatment of breast cancer, forms several metabolites. 4-Hydroxytamoxifen (4-OHTam), a metabolite found in the bloodstream, has much higher affinity for the estrogen receptor than tamoxifen itself. Oxidative activation of 4-OHTam induces DNA damage. DNA isolated from HL-60 cells exposed to 10 microM 4-OHTam in the presence of 1 microM hydrogen peroxide was digested enzymatically to release both normal and modified nucleosides. The modified nucleosides were enriched by butanol extraction. Using UV detection, HPLC analysis of the butanol extract from 200 microg DNA digest detected approximately 4 4-OHTam-dG adducts per 10(7) nucleotides (n = 3). Online postcolumn UV irradiation in HPLC and fluorescence detection improved the detection sensitivity by 3 x 10(2) times. Using 4-OHTam as an example, this report demonstrated for the first time the power of the technique to assay tamoxifen-DNA adducts directly in the DNA digest without relying on postlabeling. 相似文献
4.
Summary In order to analyse hydroxyproline (HYP) in urine, a high-performance liquid chromatographic method was modified. The primary amino groups were blocked with o-phthalaldehyde, and then the secondary amino groups were derivatized with 4-dimethylaminoazobenzene-4-sulphonyl chloride. In addition, the dabsylated samples were treated with ethyl acetate to obtain a simple elution profile in high-performance liquid chromatography. The dabsyl-HYP and -proline were eluted at 4.7 min and 8.0 min, respectively. The chromatographic analysis was completed within 10 min, including the time needed for reequilibration of the column. Using the present method, the concentration of HYP in urine was determined to 260 ± 6µmol/l. 相似文献
5.
Gehring TA Griffin B Williams R Geiseker C Rushing LG Siitonen PH 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2006,840(2):132-138
A liquid chromatographic (LC) method for determining 14 sulfonamide (SA) (sulfanilamide, sulfadiazine (SDZ), sulfathiazole, sulfapyridine, sulfamerazine (SMR), sulfamethazine (SMZ), sulfamethizole, sulfamethoxypyridazine, sulfachloropyridazine (SCP), sulfamonomethoxine, sulfadoxine, sulfamethoxazole, sulfadimethoxine (SDM), and sulfaquinoxaline (SQX)) residues in edible catfish, shrimp and salmon tissues was developed and validated at 5, 10 or 20 ng g(-1). The method was then used to determine residues in tissues of catfish, shrimp and salmon dosed with six selected sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfachloropyridazine, sulfadimethoxine and sulfaquinoxaline). All assays were within U.S. Food and Drug Administration guidelines for recovery and intra-assay variability. The method was developed to determine possible sulfonamide residues in aquacultured catfish, shrimp and salmon produced for food. 相似文献
6.
Acetylcholine measurement by high-performance liquid chromatography using an enzyme-loaded postcolumn reactor 总被引:2,自引:0,他引:2
Choline oxidase and cholinesterase were found to retain their activity for 1-2 weeks at room temperature while adsorbed to a commercially available anion-exchange cartridge. These enzymes convert acetylcholine to H2O2. Acetylcholine can be measured in tissue extracts by separation at pH 7 on a polymeric reverse-phase high-performance liquid chromatography column, conversion of acetylcholine to H2O2 on a postcolumn enzyme-loaded anion-exchange cartridge, and electrochemical detection of the H2O2 formed. 相似文献
7.
8.
A reversed-phase ion-pair HPLC method for separating hyaluronic acid oligomers, using a polymeric C18 column at alkaline pH, is described. As the concentration of the ion-pairing agent tetrabutylammonium hydroxide increased, over the range of 0.01 to 0.06M, the capacity factors (k') of tetra- to dodecasaccharide decreased. The change in k', for each increment in pairing agent, increased with oligomer molecular weight. When changing mobile phase pH from 7 to 8, k' dramatically decreased and remained unchanged from pH 8 to 11. The isocratic separation was optimized to resolve tetrato dodecasaccharide at pH 9.0 in under 19 min. The postcolumn derivatizing agent 2-cyanoacetamide reacted with the reducing N-acetylglucosamine end groups of hyaluronic acid oligomers to yield reaction products that were monitored at 27 nm. In a series of control experiments using decasaccharide and N-acetylglucosamine, it was found that maximum product formation took place at pH 9 and was greatly influenced by borate buffer concentration. The optimum concentration for 2-cyanoacetamide was 0.33% and a temperature of 100 degrees C gave the best signal to noise ratio for the postcolumn reaction. The method is linear and reproducible, and has a lower limit of detection for tetrasaccharide of 20 ng (25 pmol). This system is suitable for studying the degradation kinetics of purified hyaluronic acid oligomers by bovine testicular hyaluronidase. Extension of the method to fluorescent and electrochemical detection and its applicability to other glycosaminoglycans is discussed. 相似文献
9.
Susumu Honda Masaye Takahashi Seiko Shimada Kazuaki Kakehi Sigetake Ganno 《Analytical biochemistry》1983,128(2):429-437
Eight alditols were separated in ca. 80 min as their borate complexes by stepwise elution with three borate buffers on a column packed with Hitachi 2633 resin. The alditols in the eluate were derivatized automatically to colored, fluorescent products by applying sequential reactions of periodate oxidation and Hantzsch condensation, and the products were detected either photometrically or fluorimetrically. This automated method allowed simultaneous determination of 20–500 and 20–200 nmol amounts of alditols by photometric and fluorimetric monitorings, respectively. The lower limits of detection were ca. 2 and 0.5 nmol, respectively. The interference by aldoses was slight. Aldoses may be also determined as alditols by direct injection of aqueous solutions to which excess amounts of sodium borohydride have been added. This method was applied with success to urinary alditol assay and to molecular weight determination by end group analysis. 相似文献
10.
Direct detection of proteins in high-performance liquid chromatography electrochemistry (LCEC) is difficult. By using on-line, postcolumn photolysis, proteins now can be detected by LCEC at microgram per milliliter levels. The compatibilities of size exclusion chromatography (SEC), reversed-phase chromatography (RPC), ion-exchange chromatography (IEC), and hydrophobic interaction chromatography (HIC) with photolysis-electrochemical detection is described for proteins together with the analytical figures of merit. Inherent from the advantages of electrochemical detection, the method is sensitive and selective. 相似文献
11.
L.A. Berrueta B. Gallo F. Vicente 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1993,616(2)
A reversed-phase high-performance liquid chromatographic method for oxazepam in human urine samples has been developed. The sample preparation consists of an enzymatic hydrolysis with β-glucuronidase, followed by a solid-phase extraction process using Bond-Elut C2 cartridges. The mobile phase used was a methanol—water (60:40, v/v) mixture at a flow-rate of 0.50 ml/min. The column was a 3.5 cm × 4.6 mm I.D. C18 reversed-phase column. The detection system was based on a fluorescence post-column derivatization of oxazepam in mixtures of methanol and acetic acid. A linear range from 0.01 to 1 μg/ml of urine and a limit of detection of 4 ng/ml of urine were attained. Within-day recoveries and reproducibilities from urine samples spiked with 0.2 and 0.02 μg/ml oxazepam were 97.9 and 95.0 and 2.1 and 9.4%, respectively. 相似文献
12.
Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate 总被引:81,自引:0,他引:81
Methods for the quantitative derivatization of amino acids with phenylisothiocyanate and for the separation and quantitation of the resulting phenylthiocarbamyl derivatives by reverse-phase high-performance liquid chromatography are described. Phenylthiocarbamylation of amino acids proceeds smoothly in 5 to 10 min at room temperature. Coupling solvents, reagent, and some byproducts are removed by rotary evaporation under high vacuum, and the phenylthiocarbamyl derivatives are dissolved in 0.05 M ammonium acetate, pH 6.8, for injection onto the octyl or octadecylsilyl reverse-phase column. Columns are equilibrated with the same solvent and the effluent stream is monitored continuously at 254 nm for detection of the amino acid derivatives. Elution of all of the phenylthiocarbamyl amino acids is achieved in about 30 min utilizing gradients of increasing concentrations of ammonium acetate and acetonitrile or methanol. This approach to amino acid analysis offers select advantages, both with respect to methods which employ reverse-phase separation of prederivatized samples and to the classical ion-exchange procedure. All amino acids, including proline, are converted quantitatively to phenylthiocarbamyl compounds and these are stable enough to eliminate any need for in-line derivatization. Furthermore, results comparable in sensitivity and precision to those obtained by state-of-the-art ion-exchange analyzers may be generated with equipment that need not be dedicated to a single application. 相似文献
13.
A highly sensitive peptide mapping method using derivatization and fluorescence detection is described. Bovine cytochrome c was digested using a buffer compatible with the derivatization that followed. The derivatization was performed with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The peptide mapping of the tagged digest was conducted with both HPLC and capillary LC (CLC) systems. A capillary LC-electrospray ionization mass spectrometer (MS) was set up for measuring the molecular weights of the tagged peptides. Optimization was made of the conditions used for digestion, derivatization, and mapping. MS measurements of the tagged peptides suggested that there was only one derivatization product produced from all peptides (except one) and that all the identified peptides were fully tagged. Peptide mapping of the tagged digest reviews a larger number of peptides, covering almost the entire sequence. Peptide mapping of a 20 fmol amount of tagged digest was readily performed with the CLC system. By using derivatization and fluorescence detection, the sensitivity of peptide mapping could be improved 2000 times compared to that observed with uv detection of untagged peptides. 相似文献
14.
Zhang W Wan F Zhu W Xu H Ye X Cheng R Jin LT 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,818(2):227-232
Glutathione (GSH) and glutathione disulfide (GSSG) are important thiols, which provide defence against oxidative stress by scavenging free radicals or causing the reduction of hydrogen peroxide. The ratio GSH/GSSG is often used as a sensitive index of oxidative stress in vivo. In this paper, a direct electrochemical method using an electrode modified with functionalized carbon nanotubes as electrochemical detector (ED) for liquid chromatography (LC) was described. The electrochemical behaviors of GSH and GSSG on this modified electrode were investigated by cyclic voltammetry and it was found that the functionalized carbon nanotubes exhibited efficiently electrocatalysis on the current responses of GSH and GSSG. In LC-ED, both of the analytes showed good and stable current responses. The detection limit of GSH was 0.2 pmol on column and that of GSSG was 1.2 pmol on column, which were low enough for the analysis of real small samples. The method was sensitive enough to detect difference in concentration of GSH and GSSG in hepatocytes from animals with and without introduction of oxidation stress by glucose or hydrogenperoxide. 相似文献
15.
A new high-performance liquid chromatography procedure with a postcolumn reaction system for determination of free malondialdehyde (MDA) and other thiobarbituric acid-reactive substances (TBA-RS) in oxidized lipids in vitro has been developed. Using this procedure, both thermally oxidized methyl linoleate and the degradation products of methyl linoleate hydroperoxides revealed many kinds of lipophilic TBA-RS, but no free MDA was detected on the high-performance liquid chromatography. Similarly, oxidized methyl arachidonate also produced certain kinds of TBA-RS in the lipophilic phase and a small amount of free MDA in the hydrophilic phase. These results indicate that lipophilic TBA-RS produced in oxidized lipids in vitro are major TBA-RS and that the production of free MDA is small, even though the degree of lipid oxidation has previously been estimated as an MDA equivalent measured by the TBA colorimetric test. 相似文献
16.
Suo X Deng Y Hao A 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,819(1):191-196
A method based on a liquid-liquid extraction procedure followed by high-performance liquid chromatography (HPLC) coupled with UV-visible detection is described and validated for the determination of lauroyl-indapamide in rat whole blood. The blood sample was extracted with diethyl ether after the addition of 10% trifluoroacetic acid (aq.). The chromatographic separation was performed on a Chromasil ODS column, using methanol-acetonitrile-tetrahydrofuran-0.2% trifluoroacetic acid (170:20:15:38, v/v/v/v) as the mobile phase. The UV detection wavelength was set at 240 nm. The extraction recovery of lauroyl-indapamide was ranged from 76.5 to 82.6%, and the calibration curve had a good linearity in the range of 0.048-200 microg/ml (r = 0.9976). The method presents appropriate intra-day and inter-days repeatabilities, showing values below 7.4% in terms of the percentage of relative standard deviation (R.S.D.). The method proposed is simple, rapid and sensitive, being useful for pharmacokinetic studies in rats. 相似文献
17.
Feng CH Lin SJ Wu HL Chen SH 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,780(2):349-354
A simple and sensitive high-performance liquid chromatographic (HPLC) method is established for the trace determination of tobramycin in human plasma by derivatization. The method is based on the chemical derivatization of aminoglycoside antibiotic, tobramycin in human plasma, with 1-naphthyl isothiocyanate (NITC) in pyridine at 70 degrees C. After derivatization reaction, a methylamine/acetonitrile solution was added to the reaction mixture to eliminate the excess derivatizing agent and shorten the analysis time. The resulting derivative was separated using a Purospher STAR RP-18e column and a water-acetonitrile (50:50, v/v) mobile phase (detection at 230 nm). Optimization conditions for the derivatization of tobramycin were investigated by HPLC. The linear range for the quantitation of tobramycin in spiked plasma was over 0.93-9.34 mg/l; the detection limit (signal-to-noise ratio=3; injection volume, 10 microl) was about 0.23 mg/l. The relative standard deviation was less than 2.1% for intra-day assay (n=6) and 5.2% for inter-day assay (n=6) and relative recoveries were found greater than 99%. 相似文献
18.
Assay of biological thiols by a combination of high-performance liquid chromatography and postcolumn reaction with 6,6'-dithiodinicotinic acid 总被引:1,自引:0,他引:1
A simple and rapid method for the determination of nanomole levels of biological thiols is described. The analysis is based on the combination of reverse-phase high-performance liquid chromatography with a postcolumn reaction with 6,6'-dithiodinicotinic acid. Thiols, including cysteine, cysteamine, thiolhistidine, homocysteine, glutathione, penicillamine, ergothioneine, and thiouracil were separated by eluting with 33 mM KH2PO4 at pH 2.2. Glutathione, cysteine, cysteamine, homocysteine, and penicillamine were quantitatively determined with detection limits of 0.1 nmol, while the quantitative detection of thiolhistidine, ergothioneine, and thiouracil was not successful. The method was applied to the assay of glutathione in human erythrocytes and Escherichia coli. 相似文献
19.
A convenient method for the rapid and sensitive automated analysis of uronic acids, based on high-performance anion-exchange chromatography on a Hitachi 2633 column and photometric as well as fluorimetric postcolumn labeling with 2-cyanoacetamide, has been developed. This method allows the simultaneous determination of 1-1000 nmol of D-mannuronic and D-galacturonic acids and 5-1000 nmol of L-iduronic and D-glucuronic acids in approx 70 min with high precision by photometric monitoring. In fluorimetric monitoring the linearity range was 1-1000 nmol for all these uronic acids, but reproducibility was rather low at the lowest limit of linearity. Application of this method to the analysis of component uronic acids in some polyuronides has suggested the inadequacy of generally accepted conditions for hydrolysis. 相似文献
20.
T.K. Wang M.S. Fuh 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,686(2):6218
A simple procedure for the determination of amphetamine in urine with minimal sample preparation is described. This method involves direct addition of human urine to an acetone-dansyl chloride solution for simultaneous deproteinization and fluorescence derivatization. The derivatized amphetamine is then measured by HPLC with fluorescence detection. It eliminates the extraction procedures often required by other HPLC or GC methods. The effects of pH, temperature and reaction time on the derivatization reaction were investigated. The stability of amphetamine-dansyl chloride in different storage conditions was examined. The detection limit and linearity associated with this assay are discussed. 相似文献