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Countercurrent chromatography: liquid-liquid partition chromatography without solid support.

The liquid-liquid partition chromatographic system reported here involves a long helix of narrow-bore tubing. When the coiled tube is filled with one phase of a two-phase system and fed with the other phase, phase-interchange takes place in each turn of the coil, leaving a segment of the former phase as the stationary phase. Consequently, solutes present in either phase are subjected to a multistep partition process. The column efficiency, estimated on a separation of dinitrophenyl amino acids, is comparable to that of gas chromatography.

Amino Acids↗

Liquid chromatography-mass spectrometry and liquid chromatography-NMR characterization of in vitro metabolites of a potent and irreversible peptidomimetic inhibitor of rhinovirus 3C protease.

In vitro metabolism of AG7088 [trans-(4S,2'R,5'S,3"'S)-4-[2'-4-(4-fluorobenzyl)-6'-methyl-5'-[(5"-methylisoxazole-3"-carbonylamino]-4-oxoheptanoylamino]-5-(2"'-oxopyrrolidin-3-"'-yl)pent-2-enoic acid ethyl ester] was studied in liver microsomes isolated from mice, rats, rabbits, dogs, monkeys, and humans. The structures of the metabolites were characterized by liquid chromatography (LC)-tandem mass spectrometry and LC-NMR methods. Hydrolysis of the ethyl ester to produce metabolite M4 (AG7185) is the predominant pathway in all species, with the greatest activity observed in rodents and rabbits, followed by monkeys, dogs, and humans. Several hydroxylation products were identified as minor metabolites, including diastereomers M1 and M2, with a hydroxy group at the P1-lactam moiety, and M3, with a hydroxy group at the methyl position of the methylisoxazole ring. Rodent and rabbit liver microsomes formed almost exclusively the acid metabolite M4 (AG7185), with very little hydroxylated metabolites, whereas monkey liver microsomes formed more secondary metabolites (i.e., acid analogs of the hydroxylated metabolites). The overall metabolic profile of AG7088 formed in dog liver microsomes closely resembled that of human liver microsomes; therefore, this species may be the most appropriate animal model relative to humans for exposure to AG7088 and its metabolites.

3C Viral Proteases↗

Quantitative gas chromatography and gas chromatography-mass spectrometry of Cephalotaxus alkaloids.

Plants of the genus Cephalotaxus contaim many alkaloids, some of which have demonstrated antitumor activity. Analysis of crude alkaloid mixtures by gas chromatography provides quantitation of the active principles and other, non-active, alkaloids. Mass spectrometry is used to identify known alkaloids in extracts and to confirm the presence of previously unknown ones. Such data provide a means for predicting the biological activity of new plant accessions.

Alkaloids↗

Chelating peptide-immobilized metal ion affinity chromatography. A new concept in affinity chromatography for recombinant proteins.

We report our experimental results supporting the hypothesis that a specific metal-chelating peptide (CP) on the NH2 terminus of a protein can be used to purify that protein using immobilized metal ion affinity chromatography (IMAC). The potential utility of this approach resides with recombinant proteins since the nucleotide sequence that codes for the protein can be extended to include codons for the chelating peptide and thereby generate the gene for a chimeric CP-protein that can be cloned, expressed, and affinity-purified with immobilized metal ions. The chelating peptide purification handle could then be removed chemically or enzymatically after purification has been achieved to generate a protein with the natural amino acid sequence. The feasibility of using a chelating peptide as a purification handle has been demonstrated using a leuteinizing hormone-releasing hormone (LHRH) analog, 2-10 LHRH, which contains the previously identified chelating peptide, His-Trp, on the NH2 terminus. 2-10 LHRH had a high affinity for a Ni(II) IMAC column due to the NH2-terminal dipeptide sequence His-Trp, forming a coordination complex with Ni(II), whereas the controls, 3-10 LHRH and 4-10 LHRH, lacking the CP sequence, did not bind. Furthermore, 2-10 LHRH could be purified from a mixture of histidine-containing peptides on a Ni(II) IMAC column in one step. His-Trp proinsulin was used as a model of a recombinant CP-protein. The S-sulfonates of His-Trp-proinsulin and proinsulin were isolated from Escherichia coli engineered to overproduce these proteins as trpLE' fusion proteins. His-Trp-proinsulin(SSO3-)6 had a higher affinity for immobilized Ni(II) than proinsulin (SSO3-)6. Both proteins were eluted by decreasing the pH or by introducing a displacing ligand into the buffer. Ni(II) eluted from the column with much higher concentrations of displacing ligand than the proteins.

Amino Acid Sequence↗

Chemotaxonomy of selected species of the Actinobacillus-Haemophilus-Pasteurella group by means of gas chromatography, gas chromatography-mass spectrometry and bioenzymatic methods.

Instrumental analytical and bioenzymatic methods were used to differentiate between species of the Actinobacillus-Haemophilus-Pasteurella group. Long-chain fatty acids were analysed directly with gas chromatography (GC) without derivatization. GC of trifluoroacetylated whole-cell methanolysates was a rapid method for differentiation. Cellular sugars were more suitable for differentiation than fatty acids. D-Glycero-D-mannoheptose, the major localization of which was lipopolysaccharide, distinguished H. aphrophilus from A. actinomycetemcomitans, H. paraphrophilus, H. influenzae type b, P. haemolytica, P. multocida, and P. ureae. GC of single colonies, which is a new chemotaxonomic method, was preferable to GC of liquid-grown cells. Lysozyme-and EDTA-induced bacteriolysis and reduction of methylene blue by cellular hydrogenase served as additional criteria for differentiation.

Actinobacillus↗

Determination of desaminosulfamethazine, sulfamethazine, and N4-acetylsulfamethazine by gas chromatography with electron capture detection and confirmation by gas chromatography-chemical ionization mass spectrometry.

An electron capture gas chromatographic method for the determination of sulfamethazine was modified to separate and quantitate simultaneously sulfamethazine and 2 of its metabolites, N4-acetylsulfamethazine and desaminosulfamethazine. The modified method was applied to incurred residues in a veal calf depletion study and to incurred residues in swine tissues. With capillary column gas chromatography-positive ion chemical ionization mass spectrometry, confirmation of the identities of incurred desaminosulfamethazine, N4-acetylsulfamethazine, and sulfamethazine in tissues was obtained from a single injection.

Animals↗

Paired-ion chromatography and high performance liquid chromatography of labetalol in feeds.

A high performance liquid chromatographic (HPLC) method using reverse phase paired-ion chromatography and ultraviolet detection at 280 nm has been developed to determine labetalol, an alpha and beta adrenoceptor blocking agent, in Purina No. 5001 rodent chow. The method is simple and rapid, and demonstrates a separation technique applicable to other acidic and basic drugs. It requires only extraction of the drug with methanol--water--acetic acid (66 + 33 + 1) and separation of insoluble material by filtration before HPLC. Labetalol, is chromatographically separated from soluble feed components by means of a microBondapak C18 column and methanol--water--acetic acid (66 + 33 + 1) mobile phase, 0.005M with respect to sodium dioctylsulfosuccinate paired-ion reagent. Average recovery is 98.7% with a relative standard deviation of +/- 2.3% for the equipment described.

Animal Feed↗

[The automated "SynChrom" system for solid-phase synthesis of peptides and liquid column chromatography. II. Use in the solid-phase synthesis of peptides and liquid column chromatography].

Automatic solid phase peptide synthesis using the SynChrom system is described. Problems of swellographic monitoring are discussed. Combined monitoring (swellographic, spectrophotometric, and manometric) of all steps of the synthetic cycle are suggested. Potential applications of the system to liquid column chromatography were demonstrated.

Amino Acid Sequence↗

[Gel-permeating chromatography of interacting multi-component systems. I. Study of protein isomerization by the method of gel-permeation chromatography. II. Method of determining the kinetic constant of isomerization].

A technique is suggested of determining the isomerization constants by means of gel-permeation chromatography. Chromatograms of proteins and expressions for statistical moments of their distribution along the chromatographic column obtained in the authors previous work [1], are used. A necessary condition for optimal setup of the GPCh experiments is found. Fulfilment of this condition provides a maximal accuracy of determination of the isomerization constants.

Chromatography, Gel↗

Application of gas-liquid chromatography to the analysis of essential oils. Part XVII. Fingerprinting of essential oils by temperature-programmed gas-liquid chromatography using capillary columns with non-polar stationary phases. Analytical methods committee.

Problems in obtaining reproducible results when 'fingerprinting' essential oils by temperature-programmed gas-liquid chromatography have been reported on in Parts VII and VIII of this series. Those reports were concerned with the general problems and the use of packed columns. This report is concerned with the use of capillary columns and non-polar stationary phases. A collaborative study using capillary columns with non-polar stationary phases has resulted in a method which specifies the 'g-pack value' of a column and gives reproducible relative retention indices for the test compounds limonene, acetophenone, linalol, naphthalene, linalyl acetate and cinnamyl alcohol. The method has been applied successfully to the examination of oil of rosemary. A recommended method is given for the reproducible temperature-programmed gas-liquid chromatographic fingerprinting of essential oils using capillary columns with non-polar stationary phases.

Chromatography, Gas↗

Determination of phenylurea herbicide residues in vegetables by liquid chromatography after gel permeation chromatography and Florisil cartridge cleanup.

A liquid chromatographic method for quantitative determination of 9 phenylurea herbicide residues (metoxuron, metobromuron, monolinuron, chlortoluron, isoproturon, diuron, linuron, chloroxuron, and neburon) in potatoes, carrots, and mixed vegetables is described. Samples are extracted with acetone, partitioned with ethyl acetate-cyclohexane (50 + 50, v/v) and cleaned up by gel permeation chromatography with ethyl acetate-cyclohexane (50 + 50, v/v) as eluant. A small column (1 cm id) packed with Biobeads SX3 resin is used to reduce solvent consumption and analytical time. After solid-phase extraction on a Florisil cartridge, herbicide residues are successfully separated on a C18 column by gradient elution and determined by UV detection at 242 nm. Average recoveries of 9 compounds from different samples range from 70 to 98% at 0.010 and 0.100 mg/kg fortification levels. Quantitation limits are 0.010 mg/kg.

Chromatography, Gel↗