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At least 505 records · Page 28Linked to original sources
PubMed2001

Effect of liquid chromatography separation of complex matrices on liquid chromatography-tandem mass spectrometry signal suppression.

The effect of liquid chromatography separation on liquid chromatography-tandem mass spectrometry (LC-MS-MS) signal response for the characterization of low-molecular-mass compounds in a complex matrix was investigated. Matrix induced signal suppression appears throughout the entire LC-MS-MS analysis of wheat forage extract, with greatest suppression occurring at early retention times. Experimental results show that co-elution of matrix components and analytes from the LC column may be most strongly attributed to column overloading rather than similar analyte and matrix retention behavior. As a result, two-dimensional (LC-LC) separation can be a highly effective approach to address signal suppression effects for the quantitative LC-MS-MS analysis of complex matrix samples.

Chromatography, Liquid↗
PubMed2001

Determination of capsaicin, dihydrocapsaicin, and nonivamide in self-defense weapons by liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry.

Sensitive and selective liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods for the analysis of capsaicin, dihydrocapsaicin, and nonivamide in pepper spray products have been developed. Chromatographic separation of the capsaicinoid analogues was achieved using a reversed-phase HPLC column and a stepwise gradient of methanol and distilled water containing 0.1% (v/v) formic acid. Identification and quantification of the capsaicinoids was achieved by electrospray ionization single-stage mass spectrometry monitoring the protonated molecules of the internal standard (m/z 280), capsaicin (m/z 306), dihydrocapsaicin (m/z 308), and nonivamide (m/z 294) or by tandem mass spectrometry monitoring the appropriate precursor-to-product-ion transitions. The plot of concentration versus peak area ratio was linear over the range of 10-750 ng/ml using LC-MS and 10-500 ng/ml using LC-MS-MS. However, to accurately quantify the capsaicinoids in the pepper spray products calibration curves between 10 and 1000 ng were constructed and fit using a weighted quadratic equation. Using the quadratic curve, the accuracy of the assay ranged from 91 to 102% for all analytes. The intra-assay precision (RSD) for capsaicin was 2% at 25 ng/ml, 10% at 500 ng/ml, and 3% at 800 ng/ml. The inter-assay precision (RSD) for capsaicin was 6% at 25 ng/ml, 6% at 500 ng/ml, and 9% at 800 ng/ml. Similar values for inter- and intra-assay precision were experimentally obtained for both dihydrocapsaicin and nonivamide. The analysis of selected pepper spray products demonstrated that the capsaicinoid concentration in the products ranged from 0.7 to 40.5 microg/microl.

Capsaicin↗
PubMed2001

Multiresidue analysis of insecticides in soil by gas chromatography with electron-capture detection and confirmation by gas chromatography-mass spectrometry.

A rapid multiresidue method has been developed for the analysis of nine insecticides (organochlorines, pyrethroids and organophosphorus) in soil. The method is based on the sonication extraction of residues from a certain amount of soil placed in a small column, using ethyl acetate. The effect of the residence time of insecticides in soil, the material of the columns used (glass or plastic columns) and the soil moisture content on the recovery of these compounds was also studied. Residues were determined by gas chromatography with electron-capture detection. The average recovery through the method obtained for these compounds varied from 90 to 108% with a relative standard deviation between 1 and 11%. The results of this study pointed out that the recoveries of insecticide residues obtained with plastic or glass columns at different soil moisture content were similar and that the residence of these compounds in soil during several days did not affect their recovery from soil. Confirmation of residue identity was performed by gas chromatography coupled with mass spectrometry.

Chromatography, Gas↗
PubMed1979

Celite column chromatography followed by reversed-phase high-performance liquid chromatography: a simple, two-step method for separating 14 testicular steroids.

The separation of 14 major testicular steroids has been effected by a simple, two-step procedure involving Celite column chromatography followed by reversed-phase high-performance liquid chromatography. The technique does not alter the resolved steroids, which are then available for subsequent analysis.

Chromatography, High Pressure Liquid↗
PubMed1991

Liposome chromatography: liposomes immobilized in gel beads as a stationary phase for aqueous column chromatography.

Liposomes have been used as a stationary phase for column chromatography with an aqueous mobile phase. They were immobilized in the pores of carrier gel beads by two methods: (A) hydrophobic ligands were coupled to the matrix of gel beads, which then were packed into a column and liposomes were applied and became associated with the ligands by hydrophobic interaction; and (B) phospholipids and detergent were dialysed in the presence of gel beads; many of the liposomes that formed in the pores of the beads were sterically immobilized by the gel matrix. Proteoliposomes containing red cell glucose transport protein in the lipid bilayers were immobilized in a column by method A. This column retained D-glucose longer than L-glucose. In contrast to L-glucose, D-glucose was transported into and out of the immobilized liposomes, causing an increased retention. Liposomes with (stearylamine)+ or (phosphatidylserine)- in their lipid bilayers were immobilized by method B and the gel beads were packed into a column. A protein of opposite charge was applied in excess. Under suitable conditions, the protein molecules became close-packed on the liposome surfaces. Ion-exchange chromatographic experiments with proteins showed that these sterically immobilized liposomes were also stable enough to be used as a stationary phase. The loss of lipids was 5-23% in the first run at high protein load and with sodium chloride gradient elution but was lower in subsequent runs. It is proposed that water-soluble molecules can be separated and their interactions with liposome surfaces studied by chromatography on immobilized liposomes in detergent-free aqueous solution. Membrane proteins can be inserted and ligands can be anchored in the lipid bilayers for chromatographic purposes.

Chemical Phenomena↗
PubMed1987

Isolation of bovine cardiodilatin by fast protein liquid chromatography and reversed-phase high-performance liquid chromatography.

Cardiodilatin (CDD), a polypeptide exhibiting vasorelaxant and diuretic natriuretic bioactivity, was isolated from bovine atria. The isolation procedure reported here is different from that originally used for the purification of porcine and bovine CDD. Instead of cation-exchange chromatography on Fractogel TSK-CM 650 S and several purification steps on different high-performance liquid chromatographic (HPLC) columns, it is now possible to obtain CDD-88 by an automated fast-protein liquid chromatography system for repeated injections and a motor valve as fraction collector and only one final step of reversed-phase HPLC on a TSK-ODS-120T column.

Amino Acid Sequence↗
PubMed1984

High-performance liquid chromatography of amino acids, peptides and proteins. LVIII. Application of reversed-phase high-performance liquid chromatography to the separation of tyrosine-specific phosphorylated polypeptides related to human growth hormone.

Procedures for the purification of native and phosphorylated human growth hormone (hGH), S-carboxymethylated hGH, and hGH tryptic peptides, based exclusively on reversed-phase chromatography have been developed. Combinations of several volatile ion-pairing systems with small- and large-pore n-alkylsilicas were exploited in a general strategy, which allowed high recoveries of various hGH-related polypeptides from enzymatic incubations, as well as rapid desalting of samples following chemical modifications of the native protein, such as reductive alkylation in 6 M guanidine hydrochloride. The influence of the elution conditions on retention behaviour of phosphorylated hGH and its tryptic peptides in reversed-phase high-performance liquid chromatography is discussed.

Chromatography, High Pressure Liquid↗
PubMed2002

Organic analysis by ion chromatography. 1. Determination of aromatic amines and aromatic diisocyanates by cation-exchange chromatography with amperometric detection.

A method has been developed for the simultaneous determination of a range of aromatic amines using cation-exchange chromatography performed on a standard ion chromatography column using d.c. amperometric detection. The analytes separated were 2,4- and 2,6-toluenediamine (2,4- and 2,6-TDA), aniline, o-toluidine, benzidine, p-chloroaniline, 4,4'-diaminodiphenyl (4,4'-DDP), m-nitroaniline and 1-naphthylamine. A Dionex CS12 column was used with gradient elution from an initial eluent of 5% CH3CN+35 mM H2SO4 to 27% CH3CN+35 mM H2SO4 (at 35 min). Detection limits in the range 2.6-22.6 microg/l were observed for all analytes except m-nitroaniline, for which the detection limit was 201 microg/l. Linear calibrations and good precision were observed and the method was applied to the determination of benzidine, p-chloroaniline and 1-naphthylamine in wastewater samples. Further, the separation was also used (after some modification of the eluent conditions) for the determination of 2,4- and 2,6-toluene diisocyanate (2,4- and 2,6-TDI) and 4,4'-methylenediphenyl diisocyanate (4,4'-MDI) after their hydrolysis to 2,4-TDA, 2,6-TDA and 4,4'-DDP. Detection limits for 2,6- and 2,4-TDI and 4,4'-MDI were 3.8, 8.2, and 11.2 microg/l, respectively. The method was applied to the determination of diisocyanates in air.

Amines↗
PubMed2002

Determining the molar mass of a plasma substitute succinylated gelatin by size exclusion chromatography-multi-angle laser light scattering, sedimentation equilibrium and conventional size exclusion chromatography.

The clinical effectiveness of succinylated gelatin as a plasma substitute depends strongly on its molar mass, determined conventionally by size exclusion chromatography (SEC). This study evaluates different SEC calibration standards in comparison with two independent "absolute" methods for determining the weight average molar mass (M(w)) of a succinylated gelatin sample. SEC calibrated using succinylated gelatin fractions correlated well with size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) and sedimentation equilibrium whereas SEC calibrated with unmodified gelatin, sodium polystyrene sulfonates or pullulans overestimated M(w) by over 20%. Universal calibration was equivocal. The problems associated with the preparation of succinylated gelatin fractions suggest that an absolute method such as SEC-MALLS may be a more suitable choice for determining the M(w) in succinylated gelatins.

Calibration↗
PubMed2003

Reduction of analysis time in gas chromatography. Application of low-pressure gas chromatography-tandem mass spectrometry to the determination of pesticide residues in vegetables.

An alternative to conventional capillary gas chromatography (GC) is evaluated as a new approach to determine pesticide residues in vegetables. Low-pressure gas chromatography-tandem mass spectrometry (LP-GC-MS-MS) is proposed after a fast and simple extraction of the vegetable samples with dichloromethane and without clean up. The use of the above-mentioned GC technique reduced the total time required to determine 72 pesticides to less than half the present time (31 min), increasing the capability of a monitoring routine laboratory. The use of guard column and plug of carbofrit into the glass liner in combination with LP-GC was evaluated. The method was validated with limits of quantitation low enough to determine the pesticide residues at concentrations below the maximum residue levels stated by legislation. In order to assess its applicability to the analysis of real samples, 25 vegetable samples previously determined using conventional-capillary GC-MS-MS were analysed by LP-GC-MS-MS. The results obtained with the compared techniques showed differences lower than 0.01 mg kg(-1).

Gas Chromatography-Mass Spectrometry↗
PubMed1998

Comparison of the retention behavior of beta-blockers using immobilized artificial membrane chromatography and lysophospholipid micellar electrokinetic chromatography.

We have demonstrated that significant differences exist between the retention of eight beta-blockers analyzed with immobilized artificial membrane (IAM) and lysophospholipid micellar electrokinetic capillary (LMEKC) chromatographic methods. The general retention trends are maintained with highly hydrophilic compounds such as atenolol eluting first and more hydrophobic compounds such as propranolol eluting last. The retention order, however, is different and would result in major ranking differences. LMEKC demonstrates a better correlation with liposomal partitioning (R2 = 0.95) than does IAM chromatography (R2 = 0.60). LMEKC, with its higher efficiency, can allow a more specific evaluation of lipophilicity than IAM chromatography and is useful in the analysis of pharmaceutical candidates, particularly for ranking purposes.

Adrenergic beta-Antagonists↗
PubMed1999

Carbohydrate profiling of bacteria by gas chromatography-mass spectrometry and their trace detection in complex matrices by gas chromatography-tandem mass spectrometry.

Bacterial cellular polysaccharides are composed of a variety of sugar monomers. These sugars serve as chemical markers to identify specific species or genera or to determine their physiological status. Some of these markers can also be used for trace detection of bacteria or their constituents in complex clinical or environmental matrices. Analyses are performed, in our hands, employing hydrolysis followed by the alditol acetate derivatization procedure. Substantial improvements have been made to sample preparation including simplification and computer-controlled automation. For characterization of whole cell bacterial hydrolysates, sugars are analyzed by gas chromatography-mass spectrometry (GC-MS). Simple chromatograms are generated using selected ion monitoring (SIM). Using total ion GC-MS, sugars can be readily identified. In more complex clinical and environmental samples, markers for bacteria are present at sufficiently low concentrations that more advanced instrumentation, gas chromatography-tandem mass spectrometry (GC-MS-MS), is preferred for optimal analysis. Using multiple reaction monitoring, MS-MS is used (replacing more conventional SIM) to ignore extraneous chromatographic peaks. Triple quadrupole and ion trap GC-MS-MS instruments have both been used successfully. Absolute chemical identification of sugar markers at trace levels is achieved, using MS-MS, by the product spectrum.

Acetates↗
PubMed1999

Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography.

A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification. This result was confirmed by isoelectric focusing. These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.

Animals↗
PubMed1999

Chiral high-performance liquid chromatography of N-octyl bicycloheptene dicarboximide and confirmatory studies using liquid chromatography-tandem mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy.

N-Octyl bicycloheptene dicarboximide (MGK 264) has exo and endo diastereomers. Each structure has a chiral center at the nitrogen side chain. Enantioselective separation of MGK 264 was achieved by normal-phase high-performance liquid chromatography (HPLC) using cellulose-based Chiralcel OD column with diode-array and optical rotation detectors. Peaks were isolated with the purpose of identifying their stereochemical structures. Molecular mass of the HPLC peaks and their structural information was determined by liquid chromatography-electrospray tandem mass spectrometry (LC-ES-MS-MS). A two-dimensional nuclear magnetic resonance (NMR) spectroscopic technique was used to establish the structural features. Correlation of the data obtained from chiral separation and NMR facilitated in unambiguous assignment of the HPLC peaks.

Chromatography, High Pressure Liquid↗
PubMed1978

Determination of cytosine arabinoside in human plasma by gas chromatography with a nitrogen-sensitive detector and by gas chromatography--mass spectrometry.

A method for the determination of cytosine arabinoside in the plasma of leukemic patients being treated with this drug is described using either gas--liquid chromatography with a nitrogen-sensitive flame ionization detector or gas chromatography--mass spectrometry (GC-MS). To increase volatility, a double derivative of cytosine arabinoside was used, prepared by acetylation and subsequent methylation. Cytidine was used as internal standard for the GC procedure. GC--MS was performed with either cytidine as internal standard and detection by single-ion monitoring or by the use of [2H3] acetate-methyl derivative of cytosine arabinoside as internal standard and subsequent multiple-ion monitoring. Attempted extraction of cytosine arabinoside from plasma with various organic solvents was unsuccessful, but protein precipitation with ethanol or trichloroacetic acid followed by washing of the aqueous residue with organic solvents to remove as many of the interfering substances as possible gave satisfactory results. The minimum detectable quantity of pure cytosine arabinoside was similar for both techniques (approximately 500 pg). However, with GC using a nitrogen-sensitive detector, the lower limit of detection from plasma was found to be approximately 40--70 ng per ml plasma whilst GC--MS showed greater analytical selectivity with a detection limit in some cases as low as 1 ng per ml plasma.

Chromatography, Gas↗
PubMed1984

A quantitative densitometric method for the rapid separation and quantitation of the major tissue and lipoprotein lipids by high-performance thin-layer chromatography. I. Sample preparation, chromatography, and densitometry.

A rapid method for the separation and quantitation of the major lipids of tissues and lipoproteins by automated high-performance thin-layer chromatography is presented. Solvent systems for one-dimensional separation of neutral lipids, of cholesteryl esters, and of phospholipids are described. Separated lipids are measured following treatment with methanolic sulphuric acid containing manganese chloride and scanned in fluorescence or absorption mode. Absolute quantitation is obtained by the use of an internal standard and by references to standards for each lipid run on the same plates as samples. The method described here is particularly suitable for the rapid quantitation of small amounts of lipid (0.01-0.02 nmol per sample), for example in tissue culture studies; 100 micrograms of fibroblast or macrophage protein are sufficient for complete lipid analysis. The coefficients of variation due to the sample preparation, application to the plates and densitometry are in the range 7.2-9.1%. The method was compared with enzymatic determinations for cholesterol and gave correlation coefficients of 0.95 for total cholesterol and 0.91 for unesterified cholesterol. Phospholipid estimation was compared with large-plate thin-layer chromatography and phosphorus analysis and gave correlation coefficients of 0.90 for phosphatidylcholine and 0.89 for sphingomyelin.

Animals↗
PubMed1984

Simultaneous determination of norepinephrine, dopamine, 5-hydroxytryptamine and their main metabolites in rat brain using high-performance liquid chromatography with electrochemical detection. Enzymatic hydrolysis of metabolites prior to chromatography.

In order to measure turnover rates of the noradrenergic, dopaminergic, and serotonergic transmitter systems in rat brain, a method was developed by which norepinephrine, dopamine, and 5-hydroxytryptamine, and their main metabolites 3-methoxy-4-hydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid, could be measured simultaneously. High-performance liquid chromatography in the reversed-phase mode, including ion pairing, separated the transmitters and their metabolites well. By means of enzymatic hydrolysis of the sample prior to chromatography, it was also possible to measure the conjugated forms of the metabolites. Since there was no prepurification step, the hydrolysed supernatants of tissue homogenates were injected directly into the chromatographic system; additional selectivity tests were necessary. Peak identification was confirmed by comparison of hydrodynamic voltagrams and capacity factors at different pH values of the mobile phase of the components in the sample and the standard solution. The method is demonstrated by analysing mediobasal hypothalamic tissues of probenecid-treated rats.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1997

Profiling degradants of paclitaxel using liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry substructural techniques.

A rapid and systematic strategy based on liquid chromatography-mass spectrometry (LC-MS) profiling and liquid chromatography-tandem mass spectrometry (LC-MS-MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS-MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC-MS and LC-MS-MS methodologies resulting in an LC-MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS-MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity ligh produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3-C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.

Antineoplastic Agents, Phytogenic↗