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PubMed1984

Analysis and quantitation of gangliosides as p-bromophenacyl derivatives by high-performance liquid chromatography.

A highly sensitive method for the analysis and quantitation of gangliosides by high-performance liquid chromatography is described. The method involves simple, quantitative and specific derivatization of gangliosides, involving their carboxylic acid groups, to UV-absorbing p-bromophenacyl derivatives. The molar extinction coefficient of the derivatives at maximum absorption at 261 nm was about 23,000. The acidic fraction separated by DEAE-Sephadex column chromatography was directly derivatized and the reaction mixture was directly injected into a high-performance liquid chromatograph without any purification of the derivatives. Monosialogangliosides, GM4, GM3, GM2, and GM1, were well separated by normal phase high-performance liquid chromatography. The standard curve for quantitation of gangliosides was linear up to 100 micrograms of ganglioside-sialic acid and the lower limit of detection was about 10 ng. Also, by reverse phase high performance liquid chromatography, the molecular species of gangliosides were successfully analyzed. The method was shown to be applicable to the analysis of gangliosides in biological materials.

Acetophenones↗
PubMed1993

Evaluation of cation exchange chromatography for the isolation of M glycoprotein from histoplasmin.

Cation exchange chromatography was evaluated to purify the M antigen from histoplasmin (HMIN). Two H and M antigen-containing fractions, soluble (S) and precipitate (PP), resulted from the initial 0.025 M, pH 3.5 citrate buffer dialysis step. The PP fraction contained 62% of the M antigen activity and was resolubilized. Both fractions were chromatographed on CM Sepharose CL-6B. Polysaccharide C antigen was abundant in the S fraction and most of it did not bind to CM Sepharose. M antigen-enriched fractions were eluted with 0.5 M NaCl. Re-chromatography of the relevant S fraction (S-II) and PP fraction (PP-II) by linear gradient fast protein liquid chromatography (FPLC) removed protein and C impurities. M antigen purified by FPLC from the PP-II fraction was depleted of other antigens when Western blots were probed with anti-M, anti-H and anti-C monoclonal antibodies (Mabs). M antigen was identified as a 94 kDa glycoprotein containing a specific-protein epitope and an epitope that reacted with a Mab against the polysaccharide C antigen. M antigen can be purified from HMIN by tandem cation exchange chromatography of the precipitable fraction on an open CM Sepharose CL-6B column followed by linear gradient FPLC.

Blotting, Western↗
PubMed1975

Isolation of bacterial and phage proteins by homopolymer RNA-cellulose chromatography.

Nucleic acid-free extracts of Escherichia coli have been analyzed by chromatography on columns of cellulose, to which poly(A), poly(U), or poly(C) have been attached by ultraviolet irradiation. Proteins are released from the columns by stepwise elution with increasingly higher concentrations of salt, followed by washing with urea to remove very tightly bound molecules. The pattern of protein elution is reproducibly different for each of the homopolymer RNA-cellulose columns used: some proteins bind very tightly to one column, but poorly to others. Analysis by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, by immunological cross-reactivity in double diffusion tests, and by enzymological assays, has allowed the identification of a number of these proteins. The RNA polymerase core enzyme binds to poly(C)- and to poly(U)-cellulose columns, and can be purified to 20 to 30 percent homogeneity in a single step. Ribosomal protein S1 and the termination factor rho bind very tightly to poly(C)-cellulose, and both can be purified to homogeneity rapidly, in much higher yields than previously reported. Poly(A)-cellulose chromatography allows the isolation of large amounts of an 80,000 molecular weight protein having an as yet unassigned cellular function. The host factor required for RNA phage Qbeta RNA replication in vitro can also be obtained from poly(A)-cellulose, and chromatography of extracts of phage Qbeta-infected E. coli on RNA-cellulose columns results in very rapid isolation of the Qbeta replicase enzyme. Homopolymer RNA-cellulose chromatography thus appears to be a simple, general technique, useful for the efficient isolation of a variety of RNA-binding proteins.

Bacterial Proteins↗
PubMed1998

Purification of alpha 1 proteinase inhibitor from human plasma fraction IV-1 by ion exchange chromatography.

BACKGROUND AND OBJECTIVES: Alpha-proteinase inhibitor (PI) protects the lungs from proteolytic damage caused by elastase and can be used to treat congenital emphysema. We describe an improved method of purification of alpha 1 PI from redissolved fraction IV-1 paste. MATERIALS AND METHODS: The process used dimethylaminoethyl anion exchange chromatography, sulfopropyl cation exchange chromatography, virus inactivation by dry heat, and tri-n-butyl-phosphate/cholate treatment, followed by a second strong cation exchange chromatography. Optimizations of loading conditions for ion exchange chromatography at small scale (20-60 ml of suspension) are described. Virus inactivation was adjusted to provide the best yield of alpha 1 PI consistent with effective inactivation. The process has been effectively scaled up. RESULTS: The final product was approximately 90% pure by SDS-PAGE, with a 60-70% yield from starting fraction IV-1 paste. The process has been characterized by methods including nonreduced SDS-PAGE, alpha 1 PI inhibition assay, and biuret protein assay. CONCLUSION: The method described is an effective way of preparing large quantities of alpha 1 PI from fractionated plasma.

Anions↗
PubMedDate not supplied

Partitioning of flunitrazepam into model membranes studied by temperature controlled gel filtration chromatography.

The use of gel filtration chromatography with Sephadex as a separation medium was used in order to study flunitrazepam (FNTZ) partitioning into artificial model membranes consisting of dipalmitoyl-phosphatidylcholine (dpPC) vesicles, under controlled temperature conditions. In this system two phenomena are taking place simultaneously: the ligand-liposome interaction and the lipid self-aggregation to form the liposome. The liposome-FNTZ interaction was evidenced by the non-enantiography of the first derivative of FNTZ elution peak in frontal chromatography through Sephadex G-75. On the other hand, the presence of FNTZ reduced liposomes mean size and increased their size dispersion as evidenced by molecular filtration through Sephadex G-200. The dpPC-buffer FNTZ partition coefficient determined in zonal chromatography through Sephadex G-10 increased about 33% when the temperature rose above the temperature of dpPC transition from the liquid crystalline to the fluid phase. Gel filtration chromatography seems a suitable technique to study lipid liposome-FNTZ interactions at a qualitative level. In addition, this technique has the advantage over other methods of giving the possibility of observing the mutual effects exerted between the drug and the self-aggregating structure.

Chromatography, Gel↗
PubMed2001

Use of atmospheric pressure ionization mass spectrometry in enantioselective liquid chromatography.

Recent advances in mass spectrometry have rendered it an attractive and versatile tool in industrial and academic research laboratories. As a part of this rapid growth, a considerable body of literature has been devoted to the application of mass spectrometry in studies involving enantioselectivity, molecular recognition, and supramolecular chemistry. In concert with separation techniques such as capillary electrophoresis and liquid chromatography, mass spectrometry allows rapid characterization of a large array of molecules in complex mixtures. A majority of these findings have been made possible by the introduction of 'soft-ionization' techniques such as electrospray ionization interface. Other techniques such as atmospheric pressure chemical ionization mass spectrometry have been widely used as a rugged interface for quantitative liquid chromatography-mass spectrometry. Herein, we present a brief overview of the above techniques accompanied with several examples of enantioselective capillary electrophoresis- and liquid chromatography-mass spectrometry in drug discovery and development. Although the emphasis of this article is on quantitative enantiomeric chromatography-mass spectrometry, we envisage that similar strategies are adaptable in qualitative studies.

Atmospheric Pressure↗
PubMed1995

Simultaneous concentration and purification through gradient deformation chromatography.

Mobile-phase additives, commonly used to modulate absorbate retention in gradient elution chromatography, are usually assumed to be either linearly retained or unretained. Previous theoretical work from our laboratory has shown that these modulators, such as salts in ion-exchange and hydrophobic interaction chromatography and organic modifiers in reversed-phase chromatography, can absorb nonlinearly, giving rise to gradient deformation. Consequently, adsorbate peaks that elute in the vicinity of the head of the deformed gradient may exhibit unusual shapes, form shoulders, and/or be concentrated. These effects for a reversed-phase sorbent with aqueous acetonitrile (ACN) as the modulator are verified experimentally. Gradient deformation is demonstrated experimentally and agrees with simulations based on ACN isotherm parameters that are independently determined from batch equilibrium studies using the layer model. Unusual absorbate peak shapes were found experimentally for single-component injections of phenylalanine, similar to those calculated by the simulations. A binary mixture of tryptophan and phenylalanine is used to demonstrate simultaneous concentration and separation, again in agreement with simulations. The possibility of gradient deformation in ion-exchange and hydrophobic interaction chromatography is discussed.

Acetonitriles↗
PubMed2005

Analysis of dichloroacetic acid in rat blood and tissues by hydrophilic interaction liquid chromatography with tandem mass spectrometry.

Dichloroacetic acid (DCA) is a compound found in chlorinated drinking water. In addition, the compound is a metabolite of several halogenated solvents, including trichloroethylene (TCE) and perchloroethylene (PCE). Exposure to DCA is of concern because high doses of the compound have been shown to cause cancer in laboratory animals. Dosages of TCE administered to animals in cancer studies are designed to elicit maximal DCA formation in vivo, whereas levels of DCA to which individuals are exposed in drinking water are very low. Analysis of DCA in biological samples has been quite challenging. Derivatizing reagents commonly used to convert DCA into a more volatile form for analysis by gas chromatography (GC) have been found to convert trichloroacetic acid (TCA), a major metabolite of TCE and PCE, into DCA. High-performance liquid chromatography (HPLC) analysis does not require derivatization of DCA and can thus avoid this problem. However, the most popular stationary phases in HPLC columns do not retain small, polar compounds such as DCA well. The liquid chromatography/tandem mass spectrometry (LC/MS/MS) method described in this paper uses hydrophilic interaction liquid chromatography (HILIC), a type of chromatography that is able to retain these small, polar compounds. Method validation was performed using the United States Food and Drug Administration (USFDA) and International Conference on Harmonziation (ICH) Guidance for Industry: Bioanalytical Method Validation as a guide. Levels of DCA found in rats dosed with 2 g/kg TCE were 17.2 ng/mL (liver), 262.4 ng/mL (kidney), 175.1 ng/mL (lung), and 39.5 ng/mL (blood).

Animals↗
PubMed1993

Quantification of diacylglycerols by capillary gas chromatography-negative ion chemical ionization-mass spectrometry.

We describe a method for quantifying diacylglycerols as their 1-pentafluorobenzoyl-2-acyl-3-acetyl-glycerol derivatives by capillary gas chromatography-negative ion chemical ionization-mass spectrometry. The basis of the method resides in the sequential treatment of diacylglycerols with acetic anhydride, pancreatic lipase, and pentafluorobenzoyl chloride. Cultured rat mesenteric artery vascular smooth muscle cells (VSMC) were incubated for 20 min in the presence of vehicle or vasopressin (10(-7) M). The incubations were stopped by aspirating the medium and adding 2 ml of methanol containing 790 pmol of internal standard 1-stearoyl-2-(10,13)-nonadecadienoyl- glycerol. After extraction, diacylglycerols were isolated by thin-layer chromatography, acetylated, and treated with pancreatic lipase. The resulting 2-acyl-3-acetylglycerols were then purified by thin-layer chromatography, transformed into their 1-pentafluorobenzoyl-derivatives, and monitored by capillary gas chromatography-negative ion chemical ionization-mass spectrometry on the selected ion-monitoring mode (m/z 614 and 604 for 2-arachidonoyl and 2-nonadecadienoyl species, respectively). The levels of diacylglycerols bearing an arachidonoyl moiety were 128 +/- 26 pmol/100 nmol lipid phosphorus in resting cells and 333 +/- 28 in stimulated cells (mean +/- SD, n = 3, P < 0.01). The presence of diacylglycerol species bearing an oleoyl or a linoleoyl group at the second position could also be detected in VSMC preparations by this approach. This new method can be applied to quantitate various diacylglycerol species bearing distinct acyl moieties at the second position of the glycerol molecule.

Acetic Anhydrides↗
PubMed1993

Separation of N-acetylglucosamine and N-acetylmannosamine by chromatography on Sephadex in borate buffer.

Several procedures have been used previously for the separation of N-acetylglucosamine and N-acetylmannosamine, which are based on the difference in strength of the borate complexes of the two N-acetylhexosamines and include paper chromatography on borate-treated paper, paper electrophoresis in borate buffer, and anion-exchange chromatography of the borate complexes. In the present study, we have observed that the two sugars, despite their identical size in noncomplexed form, may also be separated by gel chromatography in borate buffer. Nearly complete resolution was obtained by chromatography on a column (1.5 x 117 cm) of Sephadex G-15, which was eluted at room temperature with 0.27 M sodium borate, pH 7.8 (prepared from H3BO3 by addition of NaOH), at a flow rate of 10 ml/h. This procedure complements existing methods for the separation of N-acetylmannosamine and N-acetylglucosamine and has the advantage that it can be carried out on a relatively large preparative scale.

Acetylglucosamine↗
PubMed1994

Reversed-phase high-performance liquid chromatography and capillary electrophoresis in the stability study of the neuropeptide growth factor antagonist [Arg6,D-Trp7,9,MePhe8]-substance P (6-11): a comparative study.

Reversed-phase high-performance liquid chromatography and capillary zone electrophoresis are widely used in protein and peptide analysis. Degradation of the basic peptide [Arg6,D-Trp7,9,MePhe8]-substance P (6-11) (antagonist G) was monitored with reversed-phase high-performance liquid chromatography, free capillary zone electrophoresis, and capillary zone electrophoresis with a capillary cationic coating. Capillary zone electrophoresis with a dynamically coated capillary provided better separation between antagonist G and its degradation products (formed at pH/Hv 13) than high-performance liquid chromatography and free zone capillary electrophoresis. Rate constants of the alkaline degradation of antagonist G measured with reversed-phase high-performance liquid chromatography and capillary zone electrophoresis with a dynamic coated capillary wall are similar whereas the values measured with free zone capillary electrophoresis are lower. Rate constants for the degradation of antagonist G in acidic media are comparable for the three techniques. It is concluded that capillary zone electrophoresis using a dynamic coating with Fluorad is the most suited of the above-mentioned techniques in analyzing antagonist G and its degradation products.

Amino Acid Sequence↗
PubMed1994

Purification of a recombinant human respiratory syncytial virus chimeric glycoprotein using reversed-phase chromatography and protein refolding in guanidine hydrochloride.

FG glycoprotein is a recombinant chimeric protein consisting of the extracellular portions of human respiratory syncytial virus (RSV) F and G glycoproteins. In theory, highly purified FG glycoprotein may be effective as a RSV vaccine. Recombinant FG glycoprotein was expressed using the baculovirus/insect cell system. FG glycoprotein was isolated from cell culture supernatants using S Sepharose ion-exchange chromatography, Cu(2+)-immobilized metal affinity chromatography, preparative reversed-phase high-performance liquid chromatography, denaturation with 6 M guanidine hydrochloride, and protein refolding in Tween 80 detergent. The purified FG glycoprotein was concentrated on a S Sepharose column and exchanged into an appropriate buffer for vaccine formulation. Five batches of FG glycoprotein with protein purity of 92-99% were produced using this purification process. FG glycoprotein produced using reversed-phase chromatography and protein refolding was compared with nondenatured FG glycoprotein using a panel of 14 monoclonal antibodies directed against conformational and linear epitopes on RSV F and G glycoproteins. The results of these studies indicated that refolded FG glycoprotein had the same three-dimensional structure as nondenatured FG glycoprotein.

Amino Acid Sequence↗
PubMed1979

Bovine dihydropteridine reductase: purification by affinity chromatography and comparison of enzymes from liver and adrenal medulla.

Dihydropteridine reductase has been purified to homogeneity from bovine liver and bovine adrenal medulla by precipitation with polyethylene glycol, ion exchange chromatography, gel filtration, and affinity chromatography on 5'-AMP-Sepharose 4B. The enzymes from the two tissues seem identical by the criteria of gel filtration chromatography, affinity chromatography, polyacrylamide gel electrophoresis in the presence and absence of dodecyl sulfate, isoelectric focusing, amino acid analysis, and binding of NADH. Fluorescence studies show two independent binding sites for NADH and a dissociation constant of 10 nM at pH 6.8. Isoelectric focusing of the enzyme as purified in the presence of NADH revealed three different bands, which by removal of this coenzyme were converted into a single band, corresponding to pI 5.7. The enzyme contains no carbohydrate or zinc.

Adrenal Medulla↗
PubMed1987

Gas chromatographic comparison of peptone yeast glucose and gas liquid chromatography growth media for anaerobic bacteria.

The growth media peptone yeast extract glucose broth and gas liquid chromatography broth were compared with respect to their suitability for anaerobic bacteria, using 63 strains belonging to 12 species. After incubation for 24 and 48 h the broth cultures were subjected to viable counts and gas chromatographic analysis of short-chain alcohols and fatty acids in head-space vapours and ether extracts. Head-space analysis was superior to the ether extraction method for detecting the rapidly eluting short-chain alcohols. The gas liquid chromatography medium provided superior growth and contained fewer compounds that interfered with the chromatographic analyses. Whereas incubation in gas liquid chromatography medium for 24 h gave characteristic chromatograms for all but one of the studied species, most species required 48 h of incubation in peptone yeast extract glucose medium. Gas liquid chromatography broth is the preferred substrate for use in gas chromatographic identification of anaerobic bacteria.

Alcohols↗
PubMedDate not supplied

Colorimetric versus radioimmunological measurement of glycated and non-glycated serum albumin after affinity chromatography.

Affinity chromatography by m-aminophenylboronic acid has been proposed for routine measurement of glycated albumin. We assayed glycated and non-glycated fractions of serum albumin (HSA) eluted by affinity chromatography columns by both a specific RIA method for the human serum albumin (HSA) and by a colorimetric method. Sixteen diabetic patients presented a significantly higher percentage of glycated-HSA than 7 control subjects with both methods, and a strong correlation was found between the values obtained with the two methods. RIA was able to detect a significant concentration of glycated-HSA in all normal subjects, while the colorimetric method was not. The accuracy of separation between the glycated and non-glycated fractions of albumin was tested using [14C]glucose as tracer. When [14C]glycated-HSA purified by Sephadex G25 filtration was chromatographed using the m-aminophenylboronic acid, only 5.3% of the total 14C-radioactivity present in the solution was recovered in the bound fraction, while 44.0% was eluted in non-protein-bound fraction and 54.7% was retained in the column. Our findings confirm that affinity chromatography by m-aminophenylboronic acid can be a useful tool in the monitoring of short glycemic control of diabetic patients. Our data also indicate that the affinity chromatography with m-aminophenylboronic acid does not accurately discriminate between glycated and non-glycated fraction of HSA.

Adult↗
PubMed1993

Protein purification by dye-ligand chromatography.

Dye-ligand chromatography has developed into an important method for large-scale purification of proteins. The utility of the reactive dyes as affinity ligands results from their unique chemistry, which confers both the ability to interact with a large number of proteins as well as easy immobilization on typical adsorbent matrices. Reactive dyes can bind proteins either by specific interactions at the protein's active site or by a range of non-specific interactions. Divalent metals participate in yet another type of protein-reactive dye interactions which involve the formation of a ternary complex. All of these types of interactions have been exploited in schemes for protein purification. Many factors contribute to the successful operation of a dye-ligand chromatography process. These include adsorbent properties, such as matrix type and ligand concentration, the buffer conditions employed in the adsorption and elution stages, and contacting parameters like flowrate and column geometry. Dye-ligand chromatography has been demonstrated to be suitable for large-scale protein purification due to their high selectivity, stability, and economy. Also, the issue of dye leakage and process validation of large-scale dye-ligand chromatography has been discussed. Reactive dyes have also been applied in high performance liquid affinity chromatographic techniques for protein purification, as well as non-chromatographic techniques including affinity partition, affinity membrane separations, affinity cross-flow filtration, and affinity precipitation.

Adsorption↗
PubMed2002

Determination of thiopurine S-methyltransferase phenotype using thin-layer chromatography and quantitative scanning.

OBJECTIVE: To develop a non-high-performance liquid chromatography method for the determination of thiopurine- S-methyltransferase (TPMT) phenotype using thin-layer chromatography and quantitative scanning. METHODS: TPMT reaction was performed using a radiochemical assay. The reaction product [(14)C]-6-methylmercaptopurine was separated using thin-layer chromatography and quantified by means of radioactive scanning. Day-to-day variance was determined to validate results. RESULTS: Determination of TPMT phenotype using thin-layer chromatography and quantitative scanning is reliable (day-to-day variance 8.5+/-1.7%, mean+/-SEM). Mean TPMT activity in 314 randomly selected patient samples was 11.8+/-3.3 units/ml red blood cells (mean+/-SD, range 3.5-25 units/ml). CONCLUSION: We developed a new assay variant for the determination of TPMT phenotype that is easy to perform, reliable and reduces production of radioactive waste. This may lead to more frequent pretreatment determination of TPMT phenotype and increase drug safety and efficacy by individualising thiopurine doses.

Carbon Radioisotopes↗
PubMed1985

Assay of diadenosine tetraphosphate hydrolytic enzymes by boronate chromatography.

A new procedure was described for assay of diadenosine tetraphosphate (Ap4A) hydrolases based on boronate chromatography. Potential reaction products, AMP, ADP, and ATP, of the hydrolysis of Ap4A were separated from residual substrate by chromatography on a boronate-derivatized cation-exchange resin, Bio-Rex 70. Separation was achieved by changing the concentrations of ethanol and ammonium acetate in the elution buffers. Picomole masses of products were detectable, blank dpm values were less than 0.5% of the total dpm, and auxiliary enzymes were not required. The procedure was specifically described for Ap4A pyrophosphohydrolase from Physarum polycephalum. The assay is generally applicable for dinucleoside polyphosphate hydrolases which hydrolyze other substrates such as Ap3A, Ap5A, Ap6A, and Gp4G. Dinucleotide polyphosphates are readily purified by chromatography on this boronate resin in a volatile buffer. Tes, Tricine, and Tris buffers significantly interfered with the chromatography of ATP.

Acid Anhydride Hydrolases↗