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At least 865 records · Page 48Linked to original sources
PubMed1993

Identification of gonadorelin (LHRH) derivatives: comparison of reversed-phase high-performance liquid chromatography and micellar electrokinetic chromatography.

A number of reversed-phase (RP) HPLC systems for the separation of gonadorelin (gonadoliberin, LHRH) and five therapeutically important analogues have been systematically examined. The selectivity of RP-HPLC has been compared with several micellar electrokinetic chromatographic (MEKC) systems and free solution capillary electrophoresis. RP-HPLC exhibits greater selectivity towards structural differences, but complete separation of the peptides in one isocratic analytical run is tedious due to the large differences in retention. Gradient elution gives satisfactory separation in an acceptable time span. Of the micellar systems examined (sodium dodecyl sulphate, cetrimide, 3-[(cholamidopropyl)dimethylamino]-1-propanesulphonate and Triton X-100) only MEKC with cetrimide micelles gave a complete separation showing selectivity similar, but not identical, to RP-HPLC, and providing a complete separation of all six compounds as rapidly as gradient RP-HPLC.

Amino Acid Sequence↗
PubMed1993

Protein sorting by high-performance liquid chromatography. I. Biomimetic interaction chromatography of recombinant human deoxyribonuclease I on polyionic stationary phases.

Chromatographic separations can be tailored to exploit specific interactions between a stationary phase ligand and a protein structural feature of interest. Variations in this feature then form the basis for sorting a mixture of closely related proteins into defined subpopulations. This report describes the sorting of variants of recombinant human deoxyribonuclease I (rhDNase) that differ in the occurrence of deamidation at a single residue. rhDNase, an enzyme that non-specifically hydrolyzes DNA, is glycosylated and exhibits considerable charge heterogeneity owing to the sialylation and phosphorylation of its N-linked oligosaccharides. This heterogeneity obscures the relatively subtle differences between deamidated and intact rhDNase, preventing separation on this basis in conventional ion-exchange HPLC. Published structural information on bovine DNase reveals that the analogous labile asparagine residue is involved in DNA binding, so stationary phases containing polyanionic ligands mimicking nucleic acids were employed to separate the deamidation variants of rhDNase. Electrostatically immobilized DNA, a "tentacle" cation exchanger (TCX) and immobilized heparin columns all resolved the deamidated and intact forms of rhDNase when operated at pH 4.5. The ligands of the TCX and heparin columns are sufficiently long, flexible and polyanionic to interact with rhDNase in a manner similar to DNA and to sort rhDNase variants on the basis of the charge difference of a single residue involved in that interaction. A non-hydrolyzable double-stranded oligonucleotide analogue of DNA was also synthesized and immobilized to an HPLC support. This column, operated at pH 6, where rhDNase is active, resolved the two isomeric products of deamidation of rhDNase, i.e., variants of the enzyme containing either aspartate or isoaspartate in lieu of asparagine at the deamidation site in rhDNase. This is the first reported separation of intact variants of a glycoprotein differing on the basis of these isomeric products of deamidation through the common cyclic imide mechanism.

Anions↗
PubMed1994

Analysis of amino acids by gas chromatography-flame ionization detection and gas chromatography-mass spectrometry: simultaneous derivatization of functional groups by an aqueous-phase chloroformate-mediated reaction.

The one-step ethyl chloroformate derivatization of amino acids in an aqueous medium is extended with the use of a variety of alkyl chloroformate reagents. This provides a new and convenient procedure for preparing esters with different alkoxy groups. A new mechanism for esterification during chloroformate derivatization is proposed based on the formation of an intermediate mixed carboxylic-carbonic acid anhydride followed by the exchange with an alcohol. Among the different reagents investigated, isobutyl chloroformate derivatized amino acids were found to provide more sensitivity for analyses by GC-flame ionization detection and GC-MS relative to derivatives prepared by other alkyl chloroformates.

Amino Acids↗
PubMed1987

Determination of diazepam and its metabolites by high-performance liquid chromatography and thin-layer chromatography.

A sensitive, simple high-performance liquid chromatographic assay, capable of simultaneously measuring diazepam, its active metabolites oxazepam, temazepam and N-desmethyldiazepam and two phenyl hydroxylated metabolites, 4'-hydroxy-N-desmethyldiazepam and 4'-hydroxydiazepam, is described. The assay is easily modified to include separation of additional metabolite(s), e.g. oxazepam glucuronide(s). A thin-layer chromatographic assay, which resolves diazepam, the active metabolites and the two phenyl hydroxylated derivatives in one solvent system, is also reported. Application of these procedures to the quantitation of diazepam and its metabolites was shown, after delivery of diazepam (5 micrograms/ml or 16 microM) at a constant flow-rate (10 ml/min per liver) through the single-pass perfused rat liver preparation. Blood perfusion medium and bile were analysed for parent drug and metabolites before and after enzyme hydrolysis. These assay methods are found to be particularly pertinent and useful in providing a more comprehensive metabolic profile of diazepam metabolism, especially when aromatic hydroxylation pathways predominate.

Animals↗
PubMed1991

Simultaneous analysis of flunixin, naproxen, ethacrynic acid, indomethacin, phenylbutazone, mefenamic acid and thiosalicylic acid in plasma and urine by high-performance liquid chromatography and gas chromatography-mass spectrometry.

Simple and reproducible high-performance liquid chromatographic (HPLC) and gas chromatographic-mass spectrometric (GC-MS) methods have been developed for the simultaneous analysis of several acidic drugs in horse plasma and urine. Although the capillary GC-MS column provided better separation of the drugs than the reversed-phase C8 (3 microns, 75 mm) HPLC column, the total analysis time with HPLC was shorter than the total analysis time with GC-MS. The HPLC system equipped with a diode-array detector provided simultaneous screening (limit of detection 100-500 ng/ml) and confirmation (limit 1.0 micrograms/ml) of the drugs. The HPLC system equipped with fixed-wavelength ultraviolet and fluorescence detectors provided a relatively sensitive screening [limit of detection 50-150 ng/ml for ultraviolet and 10 ng/ml for fluorescence (naproxen only) detectors] of the drugs. However, the positive samples had to be confirmed by using either the diode-array detector or the GC-MS system. The GC-MS system provided simultaneous screening and confirmation of the drugs at very low concentrations (20-50 ng/ml).

Analgesics↗
PubMed1992

Urinary testosterone measurement by gas chromatography after solid-phase extraction and high-performance liquid chromatography.

A method was developed for the rapid determination of testosterone in urine. The procedure consists of solid-phase extraction (SPE) followed by high-performance liquid chromatographic (HPLC) clean-up before gas chromatographic determination. Recovery was evaluated by adding [3H]testosterone (10(4) cpm) to urine samples; the mean recovery of radioactivity after SPE and HPLC was 82%. Precision was estimated by repeated measurement of testosterone in four different urine samples; the coefficient of variation was 7.9% (95% confidence limits 6.1-11.4%). Accuracy was evaluated by standard addition and dilution assays; a linear relationship was found between the expected and observed values (r2 = 0.982). The method is rapid, effective and suitable for routine analysis.

Chromatography, Gas↗
PubMed1992

Determination and identification of amiloride in human urine by high-performance liquid chromatography and gas chromatography-mass spectrometry.

A simple and sensitive high-performance liquid chromatographic method was developed to screen and determine amiloride (I) in human urine. The detection limit of the method is 0.12 micrograms/ml and the recovery of amiloride from urine was 80.4-85.5% at different concentrations. The coefficients of variation were less than 2.8 and 4.4% for intra- and inter-assays, respectively. Total urinary excretion of I in 24 h after oral administration of 5 mg or 15 mg of I ranged from 22.0 to 33.3% of the total dose for three different subjects. I could be detected in urine up to at least 44 h after a 5-mg dose and 72 h after a 15-mg dose. A gas chromatographic-mass spectrometric (GC-MS) confirmatory method was established based on the methanolysis of I to methyl 3,5-diamino-6-chloropyrazine-carboxylate (II). The di-N-trimethylsilyl derivative of II showed very good GC-MS properties and provided reliable structure information for confirmation analysis of I. This is the first time that a reliable GC-MS method has been reported for the detection of urinary I.

Adult↗