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Determination of tocopherols, tocopherolquinones and tocopherolhydroquinones by gas chromatography-mass spectrometry and preseparation with lipophilic gel chromatography.

Lipophilic gel chromatography using Sephadex LH-20 helps in separating alpha-, beta- + -gamma, and delta-tocopherol and also in separation of their oxidation products e.g. the tocopherolquinones or other oxidation products. This preseparation can help to overcome analytical problems due to the complexity of synthesis mixtures of tocopherol oxidation procedures as well as in separation of complex physiological matrices. Determination of the preseparated tocopherols, tocopherolquinonesand tocopherolhydroquinones can then be achieved by means of GC-MS measurement of the free substances or their trimethylsilyl derivatives.

Chromatography, Gel↗

Solid-phase extraction-thin-layer chromatography-gas chromatography method for the detection of hazelnut oil in olive oils by determination of esterified sterols.

The sterol composition of extra virgin olive oil is very characteristic and, thus, has become a helpful tool to detect adulterations with other vegetable oils. Special attention has been addressed to the separate determination of the free and esterified sterol fractions, since both have different compositions and can thus provide more precise information about the actual origin of the olive oil. In the case of admixtures with small amounts of hazelnut oil, this approach can be extremely useful, because the similarity between the fatty acid compositions of both oils hampers the detection of the fraud. A hyphenated chromatographic method was developed for a sensitive and precise determination of esterified sterols in olive oils. The oil was subjected to silica solid-phase extraction (SPE) fractionation, cold saponification of the collected fraction and purification on silica TLC. The sterol band was then injected into an SPB-5 (30 m x 0.25 mm I.D., 0.25 microM film thickness) and the ratio [% campesterol x (% 7-stigmastenol)2]/(% 7-avenasterol) was calculated. The method was tested on extra virgin olive oil; good sterol recoveries and repeatability were obtained. The results were compared with another method. which has a different sample preparation sequence (silica column chromatography, hot saponification and silica TLC). Similar results were achieved with both methods; however, the SPE-cold saponification-TLC-capillary GC was faster, required less solvent and prevented sterol decomposition. The SPE-method was applied to an admixture with 10% of hazelnut oil and to a screening of 11 oils (husk oil, virgin and refined olive oils) from different Mediterranean countries.

Chromatography, Gas↗

Determination of biogenic amines as dansyl derivatives in alcoholic beverages by high-performance liquid chromatography with fluorimetric detection and characterization of the dansylated amines by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

A sensitive high-performance liquid chromatographic method for the simultaneous determination of 11 biogenic amines has been developed. The method involves addition of an internal standard (1,7-diaminoheptane), pre-column dansylation of the amines and subsequent solid-phase extraction of the derivatives through C18 cartridges. The dansylamides were separated on an Inertsil ODS-3 column (250 x 4 mm I.D., 5 microm) using a 35-min gradient elution with a binary system of acetonitrile-water, a flow-rate of 1 ml min(-1) and fluorescence detection at excitation and emission wavelengths of 320 and 523 nm, respectively. The identity of the dansyl derivatives was confirmed by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. Linearity of derivatization was obtained for concentrations ranging from 0.008 to 40.0 mg l(-1). The within- and between-day relative standard deviations ranged from 0.2 to 7.6% and 0.3 to 8.6%, respectively. The overall process was successfully applied to identify and quantify biogenic amines in white-, red- and Retsina Greek wines and Greek beers, after treatment with polyvinylpyrrolidone.

Alcoholic Beverages↗

Application of porous carbon for solid-phase extraction of dicarboxyimide fungicide residues from wines in combination with high-resolution capillary gas chromatography and gas chromatography-mass spectrometry.

Solid-phase extraction with a novel porous carbon sorbent CARB GR was used for the clean-up step of dicarboxyimide fungicides residues from variety of Slovak grape wines with subsequent capillary gas chromatography-flame ionization detection, -electron-capture detection (ECD) and -mass spectrometry-ion-trap detection (MS-ITD) analysis. Recovery was tested at various concentration levels of vinclozolin and iprodione in standard solutions (R = 80-97%, R.S.D. < or = 5). The value of recovery in spiked wines is dependent on concentration level (studied in the range of 5.9 micrograms/1-1.96 mg/l) and on the variety of wine (R = 80-96%; R.S.D. = 3-5%). Limits of quantitation (for sample volume 50 ml) were determined to be with GC-ECD for both fungicides in ppt range and with GC-MS-ITD in the multiple ion detection mode monitoring in ptt range for vinclozolin and ppb range for iprodione. Concentration levels of vinclozolin residues were determined in treated wines (with 0.1% Ronilan 50 WP) as well as iprodione residues (with 0.15% Rovral 50 WP) and a strong dependence on the protective term before the harvest is shown.

Aminoimidazole Carboxamide↗

Screening and analysis of polar pesticides in environmental monitoring programmes by coupled-column liquid chromatography and gas chromatography-mass spectrometry.

Screening and analysis of polar pesticides based on coupled-column reversed-phase liquid chromatography (LC-LC) and GC- or LC-MS is a powerful tool in the execution of environmental monitoring programmes. This paper presents a unified approach utilising LC-LC screening followed by GC-MS confirmation. As polar pesticides are not generally amenable to GC a widely applicable derivation technique is used. The results demonstrate that the proposed LC and MS techniques are capable of analysing a wide range of polar pesticides down to levels of 0.1 microgram/l (EU limit for drinking water). LC switching techniques for group analysis or individual compounds rely on the reversed-phase retention and the UV detectability of the pesticides in combination with the choice of the LC columns. Fast miniaturised derivatization prior to GC-MS forms an integral part in the proposed strategy. In order to avoid extraction losses, derivation in the aqueous sample, preferably with electrophoric reagents with enhanced sensitivity in GC-NICI-MS are employed where possible. In this communication, method development and validation fitting in the strategy are evaluated and the results of the combined approach are discussed.

Chromatography, Liquid↗

Probing protein structure using biochemical and biophysical methods. Proteolysis, matrix-assisted laser desorption/ionization mass spectrometry, high-performance liquid chromatography and size-exclusion chromatography of p21Waf1/Cip1/Sdi1.

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53 tumor suppressor-dependent cell growth control in humans and other organisms, mediates G1/S-phase arrest through inhibition of cyclin-dependent kinases (Cdks). The enzymatic activity of these kinases is essential for progress through the cell division cycle and one level of cell cycle regulation is exerted through inhibition of Cdks by a family of small proteins, including p21. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2-terminus. Using proteolytic mapping, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, HPLC and size-exclusion chromatography, we show that p21, active as a Cdk inhibitor, exists in an extended, non-globular conformation in the absence of its biological target and that p21 lacks the hallmarks of stable secondary and tertiary structure. We have developed an efficient approach to obtain detailed proteolytic maps that takes advantage of the high accuracy and sensitivity of MALDI mass spectrometry. Our method allows a proteolytic map to be obtained from a single mass spectrum for fragments produced from a single proteolytic reaction.

Chromatography, Gel↗

Determination of chlorpyrifos in air, leaves and soil from a greenhouse by gas-chromatography with nitrogen-phosphorus detection, high-performance liquid chromatography and capillary electrophoresis.

Chlorpyrifos was determined in air, leaves and soil in a greenhouse in order to establish performance differences between gas-chromatography with nitrogen-phosphorus detection (GC-NPD) and high-performance liquid chromatographic and capillary electrophoretic methods and to assess the farm workers' risk of overexposure due to air exposure and/or skin contact with this compound. Results obtained indicate that the three analytical techniques, with the specific procedures described, can be used, although only GC-NPD provides an operative limit of detection in air. Chlorpyrifos levels in air are dependent on time and greenhouse ventilation, whereas it remains for a long time on leaf surfaces and soil. As a consequence, specific instructions can be established for farm workers in order to avoid skin and respiratory exposure to chlorpyrifos.

Agriculture↗

Determination of acids and volatile compounds in red Txakoli wine by high-performance liquid chromatography and gas chromatography.

The Txakoli is a wine from the north of Spain with acid characteristics and medium ethanol content. We report the comparative results obtained from red Txakoli elaborated with different grape varieties. We have used chromatographic methods for the characterization of this wine. The volatile compounds were analyzed the means of gas chromatography and the organic acid content was determined by a newly validated liquid chromatographic procedure. The aim of this study is to characterize the red Txakoli and to know the major differences between Hondarrabi Beltza grape variety and the other varieties. Hondarrabi Beltza grape variety is the one which was awarded the Generic Label. The major differences observed in the samples obtained from Hondarrabi Beltza variety are their higher contents in ethanol, propanol, ethyl acetate and tartaric and malic acids.

1-Propanol↗

Clinical applications of gas chromatography and gas chromatography-mass spectrometry of steroids.

This review article underlines the importance of gas chromatography-mass spectrometry (GC-MS) for determination of steroids in man. The use of steroids labelled with stable isotopes as internal standard and subsequent analysis by GC-MS yields up to now the only reliable measurement of steroids in serum. Isotope dilution GC-MS is the reference method for evaluation of routine analysis of serum steroid hormones. GC-MS is an important tool for detection of steroid hormone doping and combined with a combustion furnace and an isotope ratio mass spectrometer the misuse of testosterone by athletes can be discovered. Finally the so called urinary steroid profile by GC and GC-MS is the method of choice for detection of steroid metabolites in health and disease.

Anabolic Agents↗

Electrokinetically-driven cation-exchange chromatography of proteins and its comparison with pressure-driven high-performance liquid chromatography.

This paper examines protein ion-exchange behavior in electrokinetically-driven open-tubular chromatography with columns produced by immobilization of poly(aspartic acid) on capillary walls. Retention and selectivity are similar in the electrokinetic elution mode to that observed in HPLC. The separation mechanism was found to depend on the relationship of mobile phase pH to that of protein pI and ionic strength. Column efficiency in the electrokinetic elution mode was found to be 10-100-times higher than in HPLC. The best separations were achieved at intermediate ionic strength and high pH. The great advantage of these low-phase-ratio, high-efficiency open tubular columns is that isocratic separations in the electrokinetic elution mode were equivalent to gradient elution in the HPLC mode. Low phase ratio has the net effect of collapsing the chromatogram into a narrow elution window while the very high efficiency produces the requisite resolution.

Animals↗

High-performance liquid chromatography of amino acids, peptides and proteins. CXXXI. O-phosphoserine as a new chelating ligand for use with hard Lewis metal ions in the immobilized-metal affinity chromatography of proteins.

Conditions for the immobilization of O-phosphoserine (OPS) to epoxy-activated Sepharose CL-4B are described. The binding behaviour of OPS and iminodiacetic acid (IDA) immobilized onto Sepharose CL-4B, toward the hard Lewis metal ions Al3+, Fe3+, Ca2+ and Yb3+, and Cu2+ ion as a borderline metal ion control, over the pH range pH 4.0 to pH 8.0, was examined. Immobilized OPS shows a stronger affinity for Fe3+ and Al3+ ions but a lower affinity for Cu2+ and Yb3+ ions, compared to immobilized iminodiacetic acid (IDA), over the equilibrating range examined. Immobilized OPS-Mn+ was screened for protein binding using as model proteins tuna heart cytochrome c (THCC), horse myoglobin (HMYO) and hen egg while lysozyme (HEWL) over the pH range 5.5 to 8.0. Immobilized OPS-Fe3+ bound THCC under all the examined equilibrating conditions, bound HMYO between pH 5.5 and pH 7.0 and did not bind HEWL under any condition examined. Immobilized OPS thus presents an additional mode of metal ion and protein selectivity in immobilized-metal affinity chromatography.

Amino Acids↗

Determination of therapeutic and toxic concentrations of doxepin and loxapine using gas-liquid chromatography with a nitrogen-sensitive detector, and gas chromatography-mass spectrometry of loxapine.

A gas-liquid chromatographic procedure is presented for the determination of therapeutic and toxic serum levels of doxepin and loxapine, using a nitrogen-phosphorus-sensitive detector. Amitriptyline is used as the internal standard. The method is accurate, sensitive and specific with no derivatization required prior to analysis. An advantage of the procedure is the small serum sample size needed for analysis and the selectivity and sensitivity of the detector, with the limit of detection being 3 and 2 microgram/l for doxepin and loxapine, respectively. Nine cases of doxenin and loxapine misuse are presented. Serum doxepin concentrations ranged from 113 to 439 microgram/l, with a loxapine concentration of 192 microgram/l observed in one patient. The presence of the tricyclics was identified and confirmed by gas chromatography-mass spectrometry and the mass spectrum of loxapine is reported.

Adult↗

Determination of dexamethasone in bovine tissues by coupled-column normal-phase high-performance liquid chromatography and capillary gas chromatography-mass spectrometry.

A new method for the determination of dexamethasone in bovine liver and muscle tissues has been developed. Crude tissue extracts were obtained by means of a three-phase liquid-liquid extraction scheme. The resulting residue was subjected to coupled-column normal-phase high-performance liquid chromatography which served to isolate the drug for the purpose of screening and quantification. Sample was injected onto the first column of the system, a phenyl column, from which a heart-cut was diverted to a short silica column which retained dexamethasone. The contents of this column were backflushed onto a cyanopropyl column which isolated dexamethasone. Mobile phases consisted of hexane modified with 2-propanol, acetic acid, and water. Analysis of each sample was completed in 15 min. Quantitation was performed by external standard calibration of ultraviolet response at 239 nm. Limits of detection were estimated to be 4 and 6 ppb in muscle and liver, respectively. In addition to screening and quantitation, the coupled-column system purified tissue extracts for gas chromatographic-mass spectrometric analysis which, in the selected-ion monitoring mode, confirmed the identity of the trimethylsilyl-enol-trimethylsilyl derivative of dexamethasone.

Animals↗

Identification of a pyrovalerone metabolite in the rat by gas chromatography-mass spectrometry and determination of pyrovalerone by gas chromatography-nitrogen-phosphorus detection.

Pyrovalerone and its hydroxylated metabolite have been identified by gas chromatography-mass spectrometry in rat urine and plasma. A sensitive gas chromatographic method for the quantitative analysis of pyrovalerone in rat urine and plasma is described. The method also permits the quantitative monitoring of the urinary excretion of the drug and its metabolite. Pyrovalerone and its hydroxylated metabolite are detected up to 18 h after a single oral administration to the rat at a dose of 20 mg/kg.

Animals↗

Use of high-performance liquid chromatography as an extraction procedure for analysis of triazolam in decomposed human muscle by gas chromatography-mass spectrometry.

A reliable and sensitive method for the determination of triazolam in human muscle using gas chromatography-mass spectrometry (GC-MS) is described. The drug was extracted from decomposed human muscle using three-step liquid-liquid extraction and HPLC which was performed isocratically on a conventional ODS column with a mobile phase of 0.01 M phosphate buffer (pH 6.5)-acetonitrile (7:3). Estazolam was used as an internal standard. GC-MS analysis was performed on a DB-5 capillary column. Excellent linearity was obtained over the concentration range 1-200 ng/g. The lower limit of detection was approximately 0.5 ng/g. Forensic application of the present method was also described.

Chromatography, High Pressure Liquid↗

Gas chromatography-mass spectrometry with high-performance liquid chromatography prepurification for monitoring the endonuclease III-mediated excision of 5-hydroxy-5,6-dihydrothymine and 5,6-dihydrothymine from gamma-irradiated DNA.

The endonuclease III from Escherichia coli is a repair enzyme which exhibits both a glycosylase and an endonuclease function. The activity of the enzyme can be assayed by measuring the released targeted bases in solution from a sample of modified DNA. In the present study, gas chromatography-mass spectrometry was used together with an HPLC prepurification step in order to single out the released bases. The prepurification was found to enhance the specificity and the sensitivity of the assay. Thus, the overall method allowed us to analyze separately 5-hydroxy-5,6-dihydrothymine from the cis and trans isomers of 6-hydroxy-5,6-dihydrothymine. Examples of application of the assay are provided with the measurement of the E. coli endonuclease III-mediated excision of 5-hydroxy-5,6-dihydrothymine and 5,6-dihydrothymine from samples of gamma-irradiated DNA in the presence of cysteine.

Chromatography, High Pressure Liquid↗

Analysis of nifedipine-acebutolol hydrochloride binary combination in tablets using UV-derivative spectroscopy, capillary gas chromatography and high performance liquid chromatography.

Three methods are described for the simultaneous determination of nifedipine and acebutolol hydrochloride in combined pharmaceutical tablets. The first method depends on first-derivative ultraviolet spectrophotometry, with peak-to-base and zero-crossing measurements methods. The first derivative amplitudes at 400 and 352 nm were selected for the assay of nifedipine and acebutolol hydrochloride, respectively. Calibration graphs follow Beer's law in the range of 4-12 and 44-132 micrograms ml-1 and the linearity was satisfactory (r = 0.999) for nifedipine and acebutolol hydrochloride, respectively. The second method was based on the separation of nifedipine from acebutolol hydrochloride, with an internal standard thymolphthalein, using capillary gas-liquid chromatography with a programmable temperature change. The third method was based on high performance liquid chromatographic separation of the two drugs on a reversed-phase, C18, column using a mobile phase of methanol-water (55:45, pH 4.5) with a programmable flow rate of 1 ml min-1 for 4 min which changed to 2 ml min-1 for the rest of the run. The detection was done at 260 nm using oxprenolol hydrochloride as an internal standard. Both chromatographic methods showed good linearity, precision and reproducibility. No spectral or chromatographic interference from the tablet excipients were found. The proposed methods were successfully applied to the assay of commercial tablets and a content uniformity test. The procedures were rapid, simple nondestructive and suitable for quality control application.

Acebutolol↗

Metabolism of cymoxanil and analogs in strains of the fungus Botrytis cinerea using high-performance liquid chromatography and ion-pair high-performance thin-layer chromatography.

The metabolism of cyano-oxime fungicide 1-(2-cyano-2-methoxyiminoacetyl)-3-ethylurea (cymoxanil) and analogs was studied on several strains of the fungus Botrytis cinerea owing to their difference in sensitivity towards cymoxanil. Chromatographic analysis of the unextracted culture medium was simpler and more accurate, particularly for ionizable metabolites because it avoids problems associated with extraction. Reversed-phase high-performance liquid chromatography was applied to compare the decrease of cymoxanil and analogs caused by different strains of B. cinerea, by periodic injections of incubated culture medium aliquots, directly on a C4 wide-pore column. Furthermore, a thin-layer chromatographic monitoring on C18 bonded silica gel with ion-pairing allowed the monitoring of the ionizable metabolites for substrates that were demonstrated to decompose most rapidly. These complementary analyses showed that the sensitivity of the highly sensitive strain towards cymoxanil was related to the disappearance of cyano-oximes studied from culture medium, namely to the ability of the strain B. cinerea to metabolize them.

Acetamides↗