Direct determination of 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid in urine by high-performance liquid chromatography with amperometric detection

The improvement of high-performance liquid chromatographic analysis with electrochemical detection for urinary homovanillic acid is described. The method permits the chromatographic resolution of authentic homovanillic acid from coeluting interfering compounds in human and nonhuman primate, and rat urine. The electrochemically derived results are compared with post-column derivatized fluorescence results, and quality-control checks necessary to maintain assay precision in automated analysis are described.     

Strategy of glycerolipid separation and quantitation by complementary analytical techniques : Plenary lecture

Although improved systems for chromatographic resolution continue to be developed there is good reason to believe that no single method will be capable of complete separation of all lipid mixtures including the geometric, positional and stereochemical isomers in each molecular species. Furthermore, the chromatographic systems giving the highest resolution usually yield the least complete recoveries of components and require separate procedures of quantitation. It is therefore necessary to develop appropriate strategies that yield the required resolution as a result of consecutive application of complementary analytical techniques. At the present time, the original combination of thin-layer and gas— liquid chromatography has been joined by the combination of thin-layer and liquid, and liquid and gas—liquid chromatography with both liquid and gas—liquid chromatography being frequently coupled to mass spectrometry with computerized data processing. Internal standardization with hydrogen flame ionization provides a simple quantitative detection for gas chromatography, while mass spectrometry serves a similar purpose in liquid chromatography, although a much more extensive calibration may be required for quantitation. Special advantages for both separation and quantitation of most neutral lipid mixtures are derived from enzymic and chemical modification of the samples prior to chromatography. With imaginative work-up of samples, superior qualitative and quantitative results can frequently be obtained by appropriate combination of chromatographic techniques of limited resolving power.     

Methods for analyzing peptides and proteins on a chromatographic timescale by electron-transfer dissociation mass spectrometry

Advancement in proteomics research relies on the development of new, innovative tools for identifying and characterizing proteins. Here, we describe a protocol for analyzing peptides and proteins on a chromatographic timescale by coupling nanoflow reverse-phase (RP) liquid chromatography (LC) to electron-transfer dissociation (ETD) mass spectrometry. For this protocol, proteins can be proteolytically digested before ETD analysis, although digestion is not necessary for all applications. Proteins 相似文献   

Purification and biochemical characterization of alpha 2-adrenergic receptor from the rat adrenocortical carcinoma

The alpha 2-adrenergic receptor was purified from rat adrenocortical carcinoma 494 by an affinity chromatographic step using a novel para-aminoclonidine-sepharose resin followed by a gel-permeation high performance liquid chromatographic step. The iodinated receptor protein was homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by high performance liquid chromatography. Both SDS-PAGE and high performance liquid chromatographic studies revealed that Mr of the protein was 64,000, suggesting the monomeric nature of the receptor protein. The purified protein showed the typical binding characteristics of alpha 2-adrenergic receptor.     

Direct liquid introduction LC/MS microbore experiments for the analysis of nucleoside material present in human urine

In order to obtain detection limits low enough for the analysis of nucleoside material in biological samples, a direct liquid introduction (liquid chromatographic/mass spectrometric DLI) system was upgraded by inserting a self-built desolvation chamber between the DLI probe and the ion source and by switching to microbore high-performance liquid chromatography (HPLC) on a C-18 column. The system was evaluated for the analysis of pure nucleoside and 2'-deoxynucleoside compounds in CH3OH + H2O (80/20) and for the analysis of nucleoside mixtures which were separated using 0.01 M ammonium formate + methanol (97:3) as eluant. The detection of structurally important fragment ions in the lower mass region which could not be observed before and an enhanced sensitivity are considered to be the main improvements. In combination with an appropriate clean-up procedure the system was evaluated for the DLI liquid chromatographic/mass spectrometric analysis of some nucleosides present in a human urine sample. Pseudouridine and 5,6-dihydrouridine were detected and identified.     

Simultaneous high-performance liquid chromatographic determination of salicylates in whole blood, plasma and isolated erythrocytes

A method using liquid—liquid extraction has been developed for the isolation of acetylsalicylic acid and its metabolites, salicylic, gentisic or possibly salicyluric acids, from whole blood, isolated erythrocytes and plasma. Methylene chloride proved to be the best of the organic solvents tested. For whole blood and isolated erythrocytes it was necessary to carry out haemolysis prior to their extraction. The high-performance liquid chromatographic conditions for the quantitation of acetylsalicylic acid and its metabolites from samples of whole blood, erythrocytes and whole plasma were optimized. Separation was performed using reversed-phase chromatography on Separon SGX C₁₈ and ultraviolet detection at 236 nm. A mixture of methanol—water (80:100, v/v) was the mobile phase, acidified with perchloric acid to pH 2.5.     

An automated method for the quasi-continuous analysis of degradation and transfer products during the enzymatic hydrolysis of oligosaccharides

A method is presented which allows for the automated quasi-continuous analysis of the degradation and transfer products developing during the enzymatic hydrolysis of oligosaccharides. A liquid chromatographic system is integrated into the bypass of a small batch reactor which makes it possible to take oligomer spectra without any manual sample processing being necessary. The time intervals between analyses are substantially reduced by making use of an overlapping analysis technique. Postcolumn derivatization with an orcinol sulfuric acid reagent gives a high sensitivity for carbohydrates. The great potential of this method is demonstrated for the characterization of a beta-glucosidase (pI 8.4) from Trichoderma reesei QM 9414 and an alpha 1,4-glucan glucohydrolase from Aspergillus niger with cellotetraose and maltohexaose as examplary substrates.     

Utilization of different methodologies for the characterization of Hb Hasharon heterozygotes

Hb Hasharon has an electrophoretic mobility similar to that of Hb S in cellulose acetate and a mobility between Hb S and C at acid pH. In high-performance liquid chromatography, Hb Hasharon shows a distinct chromatographic profile and retention time. The origin of this variant is a mutation in codon 47 (GAC --> CAC) of the alpha2-globin gene, resulting in the replacement of asparagine by histidine during the translation process. Ten blood samples from individuals suspected of being Hb Hasharon carriers were analyzed. In addition to classic laboratory tests and high-performance liquid chromatography, molecular analysis by polymerase chain reaction with restriction fragment length polymorphism designed in the laboratory was performed to confirm this mutation. The study of these cases showed that a combination of classical and molecular methodologies is necessary in the diagnosis of hemoglobinopathies for a correct hemoglobin mutant identification. The accurate identification of hemoglobin variants is essential for genetic counseling and choice of therapy.     

Fast determination of ochratoxin A in serum by liquid chromatography: comparison with enzymic spectrofluorimetric method

A rapid high-performance liquid chromatographic method for the determination of low concentrations of ochratoxin A in serum is described. The extraction procedure was simple and short, and liquid chromatographic analysis was carried out isocratically on a reversed-phase C₁₈ column, with methanol—water—acetic acid (30:70:1) as mobile phase and fluorescence detection (excitation at 336 nm, emission at 465 nm). The examined concentration range, 5–50 ng/ml ochratoxin, the recovery method was 87–94%, compared with 62–67% for the enzymic spectrofluorimetric method. The high-performance liquid chromatographic method was faster because the extraction procedure was shorter, and more sensitive so that small sample volumes could be used.     

Analysis of enzymatic steroid conversions by high pressure liquid chromatography.

Enzyme catalyzed introduction of the 1–2 double bond into a steroid can be monitored through spectrophotometric changes accompanying electron acceptor reduction or through paper or thin-layer chromatographic analysis of the reaction product. The spectrophotometric method is not applicable to cases in which the oxidized form of the electron acceptor is continually regenerated. In studying such cases, we have found high pressure liquid chromatography (HPLC) to be a method of direct analysis more convenient than paper chromatography or tlc. Use of a water based eluant and a reverse phase column for the HPLC analysis allows direct injection of a sample of the aqueous reaction solution after acidification, and no extraction with an organic solvent is necessary.     

Determination of N-acetylation phenotype using caffeine as a metabolic probe and high-performance liquid chromatography with either ultraviolet detection or electrospray mass spectrometry

A rapid, sensitive method using liquid chromatography–electrospray mass spectrometry (LC–ES-MS) was developed and evaluated for the simultaneous quantitative determination of caffeine metabolites 1U, 1X and AAMU in human urine. This method involved a simple dilution of urine samples. The chromatographic separation was achieved on a C₁₈ reversed-phase column using a gradient of acetonitrile in 2 mM, pH 3.0 ammonium formate as mobile phase. After ionisation in an electrospray source, mass spectrometric detection was performed in the negative ion, selected ion monitoring mode. This method yielded acceptable accuracy and precision within the range 0.25–50 μg/ml. This analytical method was applied to investigate the N-acetylator phenotype of HIV-infected patients and compared with high-performance liquid chromatography with UV detection. Its specificity was better, which appeared to be absolutely necessary to prevent errors in metabolic ratios and phenotype interpretation.     

Correlation between high-performance liquid chromatography and automated fluorimetric methods for the determination of dopamine, 3, 4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid in nervous tissue and cerebrospinal fluid

The correlation between automated fluorimetric methods and high-performance liquid chromatography is described for the determination of homovanillic acid and 5-hydroxyindoleacetic acid in cerebrospinal fluid, and for dopamine, 3, 4-dihydroxyphenylacetic acid and homovanillic acid in striata of rat brain. The automated fluorimetric methods for 3, 4-dihydroxyphenylacetic acid and homovanillic acid showed a good correlation with the high-performance liquid chromatographic methodology. The fluorimetric determination for dopamine was somewhat less reliable than the high-performance liquid chromatographic assay. The fluorimetric assay for 5-hydroxyindoleacetic acid correlated poorly with the chromatographic method.     

Direct connection of high-speed liquid chromatograph (equipped with gel permeation column) to atomic absorption spectrophotometer for metalloprotein analysis: Metallothionein

A high-speed liquid chromatograph equipped with a gel permeation column (Toyo Soda TSK GEL SW 3000) was directly connected to an atomic absorption spectrophotometer for rapid analysis of metalloproteins and applied for analysis of metallothionein. Although the column used in the present study exhibits a nonselective adsorption for metals attached to biopolymers with low stability constants, the analytical system was shown to be practically useful, especially for metalloproteins with high stability constants. Metallothioneins were separated into two isometallothioneins on the column, indicating that the column has both gel chromatographic and ion-exchange chromatographic properties.     

Liquid Chromatographic Procedure for Fermentation Product Analysis in the Identification of Anaerobic Bacteria

High-performance liquid chromatography with a cation-exchange resin-packed column was used to determine fermentation products of several known and unknown Clostridium species. The column was operated at 30°C, and isocratic elution was done with 0.013 N H₂SO₄. Sample preparation for high-performance liquid chromatographic analysis required only membrane filtration. Glucose and formate were readily determined. Quantitative results were easily obtained. Chromatograms of eight unknown strains could be matched with chromatograms of at least one of the type culture strain chromatograms. In some cases, additional testing was necessary before identification could be made. The same conclusions were reached by parallel testing with gas chromatography to determine fermentation products. High-performance liquid chromatography is simple to apply and, under some conditions, is faster than gas chromatography for fermentation product analysis.     

Determination of bufuralol and its major metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of bufaralol, a benzofuran analogue, in plasma is described.The unchanged drug, the major metabolites and an internal standard are extracted from plasma, purified by back-extraction steps and thereafter separated using a reversed-phase liquid chromatographic system. The detection is carried out by means of a fluorescence detector and an UV detector connected in series. The sensitivity of the assay for the unchanged drug and the major metabolite is about 1 ng/ml plasma using a 0.5 ml specimen per analysis and the relative standard deviation of the whole assay lies in the range ± 4–5%.The procedure was successfully used to determine plasma levels in volunteers following a single oral dose of 40 mg of bufaralol. The results obtained using the new high-performance liquid chromatographic method were compared with those determined by another method which combines gas chromatography with mass fragmentography, and it was found that these two sets of results coincided quite well.     

Determination of rat liver triglycerides by gas–liquid chromatography and reversed-phase high-performance liquid chromatography

Rats fed with a fat-free or an olive oil-rich diet were employed to compare the response of two chromatographic techniques in the determination of rat liver triglyceride (TG) molecular species composition. Gas–liquid chromatography (GLC) on polarizable liquid phase and reversed-phase high-performance liquid chromatography (RP-HPLC) have been commonly employed for TG analysis, obtaining a similar number of chromatographic peaks when used for animal tissue TG determination. In the present study similar results were achieved with regard to most relevant chromatographic peaks, however, important differences were found in the content of minor TGs. Indeed, RP-HPLC permitted separation of long chain polyunsaturated fatty acids, which were not detected by GLC, while the latter technique reported a higher number of myristoyl-containing TG species. RP-HPLC analysis reported a greater number of TGs, with more similarity to a random composition, made up from the liver fatty acid composition. Therefore, it was concluded that utilization of both techniques would be helpful for liver TG analysis as the use of only one of them does not provide a complete profile of liver TGs. Nevertheless RP-HPLC seems to be more useful for this purpose since revealed a more extensive profile.     

Purification of human B cell growth factor

Human B cell growth factor (BCGF, 12,000 to 14,000 daltons) has been purified from lectin-stimulated, peripheral blood mononuclear cell-conditioned medium. The purification procedure involves a series of column chromatographic steps incorporating ion exchange, affinity binding, and gel filtration. This procedure is centered around a relatively high yield single chromatographic step, for the removal of co-eluting cytokines from BCGF, that is based on differential binding characteristics to the weak ion-exchange matrix, hydroxylapatite. Reverse-phase high-pressure liquid chromatographic separation on a C18-Bondapak column effectively separates the BCGF and TCGF moieties, yet is characterized by poor yields. High-pressure liquid chromatographic procedures on anion exchange and size exclusion provided the final purification step for BCGF, at an analytical level, resulting in a single band with a m.w. of 12,000 on a SDS-polyacrylamide gel.     

High-performance liquid chromatographic determination of the lecithin/sphingomyelin ratio in amniotic fluid

A high-performance liquid chromatographic (HPLC) procedure has been developed for the separation of phospholipids commonly found in amniotic fluid. The chromatographic separation was achieved on a 25-cm column packed with LiChrosorb DIOL (10 μm). A 3-cm column packed with silica was fitted between the injector and the DIOL column to provide complete separation of lecithin (L) and sphingomyelin (S) from the remaining amniotic fluid phospholipids. The eluted phospholipids were quantitated employing an ultraviolet absorption detector set at 203 nm. The new HPLC separation described herein has improved the resolution and peak sharpness of L and S. Furthermore, phosphatidyl glycerol and phosphatidyl inositol were completely separated and quantitated. Amniotic fluid L/S ratios determined by this technique have been compared to those of an established thin-layer chromatographic procedure.     

Simple and selective high-performance liquid chromatographic method for estimating plasma quinidine levels

A reversed-phase, high-performance liquid chromatographic method employing fluorescence detection is described for the rapid quantification of plasma levels of quinidine, dihydroquinidine and 3-hydroxyquinidine. It involves protein precipitation with acetonitrile followed by direct injection of the supernatant into the chromatograph. For the preparation of plasma standards, pure 3-hydroxyquinidine was isolated from human urine by a simplified thin-layer chromatographic procedure. The mobile phase for the chromatography was a mixture of 1.5 mM aqueous phosphoric acid and acetonitrile (90:10) at a flow-rate of 2 ml/min. The intra-assay coefficient of variation for the assay of quinidine and 3-hydroxyquinidine over the concentration range 2.5–20 μmole/l was < 1% for both. Interassay coefficients of variation for quinidine (10 μmole/l) and 3-hydroxyquinidine (5 μmole/l) were 3.5% and 4.0% with detection limits of 50 and 25 μmole/l respectively. The method correlated well (r² = 0.96) with an independently developed gas—liquid chromatographic—nitrogen detection assay for quinidine which also possessed a high degree of precision. (Intra-assay coefficient of variation 3.6% at 20 μmole/l). As expected, comparison of the high-performance liquid chromatographic assay with a published protein precipitation—fluorescence assay showed poor correlation (r² = 0.78).     

Chromatographic procedures for determination of cannabinoids in biological samples, with special attention to blood and alternative matrices like hair, saliva, sweat and meconium

This paper reviews chromatographic procedures for determination of cannabinoids in biological samples. Special attention was focused on blood and alternative matrices like hair, saliva, sweat and meconium. Papers published from 1998 to the early beginning of 1999 were taken into consideration. Gas chromatographic and liquid chromatographic procedures with different detectors (e.g. mass spectrometer or diode array) were considered. Basic information about the biosample assayed, sample preparation, work-up, gas chromatography column or liquid chromatography column and mobile phase, detection mode, reference and validation data are summarized in tables.