Glass capillary columns applied to prostaglandins measurement: a useful tool for gas-chromatography mass spectrometry analysis

The difficulties to analyse prostaglandins (PG) by gas-liquid chromatography are mainly due to the lack of sensitivity of the gas-chromatograph itself (higher than 200 ng) and to the poor resolution of the packed columns. Therefore the use of glass capillary columns which has been applied with success for other biological compounds was tempting. We describe a comparison of the preparation of the columns and their use for PG analysis of standards and of human semen. A complete resolution of PG-1 from PG-2 series was achieved. The sensitivity was multiplied 100 fold with a flame ionisation detector when compared to packed columns and was equal to the one obtained with electron capture detectors without the inconveniences of this technique. The successful coupling of glass capillary columns to a mass spectrometer leads to promising results and allows profile studies of primary PG and their metabolites as seen with human semen.     

Affinity chromatography of microsomal enzymes on immobilized detergent-solubilized cytochrome b5

Highly selective chromatography of microsomal enzymes has been carried out on columns of immobilized cytochrome b5 that was obtained by detergent solubilization (d-b5) of the complete amphipathic molecule. Several partially purified isozymes of cytochrome P-450 are resolved on d-b5 columns, and one high-affinity isozyme has been readily purified to homogeneity. Chromatographic selectivity and correlation of elution order of isozymes of cytochrome P-450 with direct spectral measurements of affinity constants suggests affinity chromatography on d-b5 columns. Substantial one-step enrichments of NADH-cytochrome-b5 reductase and an unstable cytochrome b5-dependent oxidase of cholesterol synthesis, 4-methyl sterol oxidase, have been obtained on d-b5 columns which further supports this conclusion. Comparison of chromatographic behavior on columns of immobilized cytochrome b5 that was obtained by trypsin solubilization (t-b5) with d-b5 columns shows marked differences which must be attributed to the absence of the hydrophobic domain of the t-b5 molecule. NADH-cytochrome-b5 reductase and the high affinity isozyme of cytochrome P-450 purified by d-b5 affinity chromatography are poorly retained on t-b5 columns. A different cytochrome P-450 isozyme with lower affinity for cytochrome b5 is only retained on d-b5 columns. Cytochrome-P-450 reductase is not retained on either column. Because affinity chromatography is suggested on d-b5 columns, the procedure may be generally applicable for predicting protein-protein interactions of microsomal electron transport components that either donate electrons to, or receive electrons from, cytochrome b5. In addition, the procedure should have considerable utilitarian application for enzyme enrichment.     

TopCount coupled to ultra-performance liquid chromatography for the profiling of radiolabeled drug metabolites in complex biological samples

The recent commercial availability of small particle packed columns (<2microm) and associated instrumentation capable of withstanding the high pressures of such columns, has lead to an increase in the application of so called ultra-performance liquid chromatography (UPLC). It has recently been shown that the improved efficiency, resolution and peak capacity of these columns, when coupled to mass spectrometry, provides particular benefit for the identification of drug metabolites in complex biological samples. In this work, the ability of TopCount, a microplate scintillation counter, to act as a suitable radiodetection system for ultra-performance liquid chromatography methods is tested. TopCount, has innumerable benefits over more traditional on-line radioactivity flow detection methods, when dealing with the narrow peak widths and small peak volumes associated with the enhanced efficiency of sub-2microm columns. The system is tested for robustness and sensitivity, and then used to undertake successful metabolite profiling of actual samples, and the data compared to traditional HPLC with on-line radioactivity flow detector.     

Use of generic fast gradient liquid chromatography–tandem mass spectroscopy in quantitative bioanalysis

Short narrow analytical HPLC columns have been used successfully with high linear flow-rates and combined with mass spectrometric detection to produce a generic approach to quantitative bioanalysis. The approach has been used to validate several assays in the low ng/ml region and an example is given in this paper. When combined with a simple solid-phase extraction process the need for complicated, time consuming method development has been removed for the majority of pharmaceutical compounds. The approach takes advantage of not only the extra selectivity of the MS–MS detector but the excellent resolution and peak shape produced by gradient elution.     

A system for coupled multiple-column separation of proteins

Purification of proteins is commonly a multiple-step process involving size exclusion, ion exchange, affinity, hydrophobic, and other modes of chromatography. In an effort to circumvent the laborious process of collecting the solutes from each column and reintroducing them onto a second column, a valving system is described that directs the samples eluted from a high-performance liquid chromatographic column through a detector with a high-pressure cell into either a second column or into storage loops of a multiloop value. This multiloop value is referred to as a high-pressure fraction collector. After development of the first column is complete, a second solvent can be directed to the second column or high-pressure fraction collector to elute the solutes back through the detector and onto any other column in the system. The process of eluting a sample from a column through a single detector and directing it to the high-pressure fraction collector or any other column in the system may be repeated a number of times. Such valving systems make it possible to chromatograph a single protein component on two or three columns in a short time.     

Rapid separation of urinary acids by high-performance liquid chromatography

Over a hundred acidic urinary constituents were separated within 30 min by using 5-μm octadecyl-silica columns and gradient elution with increasing acetonitrile concentration in dilute aqueous phosphoric acid solution at 70°. The column effluent was monitored with a UV detector at 280 nm or with a fluorescence detector at 260 nm excitation and 340 nm emission wavelengths. The high sensitivity and speed of analysis, the excellent reproducibility and adequate resolution obtained suggest that this technique may be useful to obtain metabolic profiles in routine clinical work.     

Determination of tryptophan and several of its metabolites in physiological samples by reversed-phase liquid chromatography with electrochemical detection

A new method for the concurrent assay of three tryptophan metabolites at the picomole level is described. The method has been developed for blood, urine, cerebrospinal fluid, and tissue samples such as whole brain, brain parts, and endocrine glands. Tryptophan itself, serotonin, and 5-hydroxyindoleacetic acid are isolated initially on extraction columns, eluted with a suitable solvent, and injected onto a liquid chromatograph with an amperometric detector. This general approach may be applicable to a variety of other tryptophan metabolites and should be useful in both research and clinical investigations.     

Development and validation of a column-switching high-performance liquid chromatographic method for the determination of sanfetrinem in rat and dog plasma by direct injection

A direct injection column-switching HPLC method was developed and validated for quantification of sanfetrinem in rat and dog plasma. Following dilution with buffer, samples were directly injected onto the system. The analyte was retained in an enrichment column while endogenous plasma components were eluted to waste. Sanfetrinem was then back-flushed to the analytical column for separation and quantification with an ultraviolet detector. Sample batch size was increased by adding a washing phase of the enrichment column and by alternating the injections between two enrichment columns. The method is very simple and sample preparation is minimal. The method has been fully validated and shown to be specific, accurate and reproducible.     

High performance liquid chromatographic determination of N-butyryl glucosamine in rat plasma

PURPOSE: A high performance liquid chromatography (HPLC) method for determination in plasma of N-butyryl glucosamine (GLBU), a highly water-soluble compound with no chromophore was developed. METHOD: To 100 muL of plasma containing GLBU was added fucose as internal standard. GLBU and fucose were derivatized using 1-phenyl-3-methyl-5-pyrazolone in the presence of sodium hydroxide at 70 degrees C for 30 min. The solution was neutralized with hydrochloric acid and the excess derivatizing reagent was extracted with chloroform. The aqueous layer was injected into an isocratic HPLC system consisting of an autoinjector, a single pump and a UV detector set at 245 nm. Two different 25 cm reversed phase columns were used, a 4 and a 10 microm C(18) columns. The mobile phase was a mixture of phosphate buffer (pH 7) and acetonitrile (80:20), which was run through a pump at a flow rate of 1.0 mL/min at ambient temperature. RESULTS: Derivatized fucose and GLBU appeared 24 and 28 min, and at 34 and 37 min using 4 and 10 microm columns, respectively. The assay was linear over the range of 0.2-200 microg/mL with a limit of quantification of 0.2 and 1 microg/mL for the 4 and 10 microm columns, respectively. The method was applied to the determination of GLBU in rat plasma after oral administration of 233 mg/kg of GLBU. CONCLUSION: The present assay is precise, and accurate with sufficient sensitivity for pharmacokinetic studies following therapeutically relevant doses.     

Determination of urinary amino acids by means of glass capillary gas—liquid chromatography with alkali-flame ionisation detection and flame ionisation detection

The determination of amino acids as their N(O)-heptafluorobutyryl-isobutyl ester derivatives by glass capillary gas—liquid chromatography has been studied. Separations of amino acids obtained from insulin hydrolysate and human urine analysed with a flame ionisation detector, an alkali-flame ionisation detector and an electron-capture detector are shown. The quantitative results of urinary amino acids of one deep-sea diver are presented.     

Sorbitol determination by liquid chromatography: application to red blood cells of diabetic rats

This report describes an application of liquid chromatography to the determination of sorbitol in red blood cells. The chromatograph employed in the present study was made up of sub- and main-separation systems and a detector portion. The sub-separation system was for concentration of polyols and involved two small columns, each containing the same anion exchange resin. The first was a tiny column which, in borate form, served as the concentrator of polyols and sugars charged in a large volume, while the second, in acetate form, separated the carbohydrates from the borate. The main system was for the fine separation of each carbohydrate and employed cation exchange columns. The detector part utilized a flow fluorometric method comprising two successive reactions: periodate oxidation followed by the Hantzsch reaction. The resulting whole chromatographic system was applied to the determination of sorbitol in red blood cells obtained from normal rats and rats made diabetic by the administration of streptozotocin; a part of the latter group had also received an aldose reductase inhibitor. Our results supported the concepts that a prolonged duration of high blood glucose level induces an elevated level of sorbitol inside red blood cells and that aldose reductase inhibitors are effective in reducing this level.     

High-performance liquid chromatographic method for guanylhydrazone compounds

A high-performance liquid chromatographic method has been developed for a series of aromatic guanylhydrazones that have demonstrated therapeutic potential as anti-inflammatory agents. The compounds were separated using octadecyl or diisopropyloctyl reversed-phase columns, with an acetonitrile gradient in water containing heptane sulfonate, tetramethylammonium chloride, and phosphoric acid. The method was used to reliably quantify levels of analyte as low as 785 ng/ml, and the detector response was linear to at least 50 μg/ml, using a 100 μl injection volume. The assay system was used to determine the basic pharmacokinetics of a lead compound, CNI-1493, from serum concentrations following a single intravenous injection in rats.     

Heterogeneity of tropocollagen as investigated by chromatography on hydroxyapatite columns

The chromatography of native acid-soluble tropocollagen from calf skin on hydroxyapatite columns has been investigated. Resolution of a number of chromatographic peaks has been obtained by using shallow slopes of the eluting phosphate gradient. The results obtained suggest a heterogeneity of tropocollagen molecules which might be due to different distributions of absorbing sites on the molecules. The results obtained have also been used to study the mechanism of chromatography of proteins on hydroxyapatite and the resolving power of the columns.     

Enzymatic synthesis of 3H-labeled Ring-A reduced metabolites of aldosterone and their separation by high pressure liquid chromatography

Specific methods are described for the enzymatic synthesis of each of the six possible 3H-labeled Ring-A reduced metabolites of aldosterone (5 alpha- and 5 beta-DHAldo; 3 alpha,5 alpha-THAldo; 3 beta,5 alpha-THAldo; 3 alpha,5 beta-THAldo; and 3 beta,5 beta-THAldo; see footnote 1 for full names). Use of heated jacketed columns (C8-reverse phase) and two HPLC solvent systems, with isocratic aqueous methanol or acetonitrile, respectively, have been developed which resolve all six Ring-A reduced metabolites of aldosterone. The relative retention times and elution order of each reduced metabolite are different with each solvent system and hence help confirm the identities of Ring-A reduced metabolites made in vivo from physiological quantities of [3H]aldosterone. The use of an on-line beta-radioactivity detector (Berthold LB-504) enhanced the sensitivity of detection and markedly improved the resolution of these metabolites, compared with that obtained by off-line scintillation counting. Thus, the use of increased temperature with these two solvent systems, together with an on-line radioactivity detector, provide a useful and efficient analytical tool for the separation and identification of each reduced metabolite of aldosterone.     

The reaction of iodobenzene-p-sulphonyl chloride (pipsyl chloride) with certain amino acids and peptides, and with insulin

1. A system of separation using buffered Celite columns is described that enables the pipsyl derivatives of most of the common amino acids to be separated. 2. The reaction of pipsyl chloride with several amino acids not included in previous studies has been investigated. In particular, knowledge of the acid-soluble pipsyl derivatives of arginine, histidine, lysine, tyrosine and cysteic acid has been extended. 3. Reproducible factors have been obtained that enable corrections to be applied for the breakdown of pipsylamino acids on acid hydrolysis. 4. The reaction of pipsyl chloride with peptides has been studied under various conditions. 5. The extent of the reaction between pipsyl chloride and insulin depends on the nature of the solvent–buffer system, and under the best conditions so far found is about 75% complete. 6. In an Appendix, the separation of pipsylamino acids by thin-layer chromatography is described.     

Lysozyme-agarose interaction.

It has been observed that the elution volumes of hen egg-white lysozyme on agarose columns exceeded by far the total volumes of the corresponding columns. Similar results have been obtained also on polyacrylamide-agarose gel columns. This anomalous behaviour of lysozyme on agarose columns is interpreted in terms of an interaction between the enzyme and agarose which resembles in some way the physiological substrates of lysozyme.     

Synchronized oscillation in a modular neural network composed of columns

The columnar organization is a ubiquitous feature in the cerebral cortex. In this study, a neural network model simulating the cortical columns has been constructed. When fed with random pulse input with constant rate, a column generates synchronized oscillations, with a frequency varying from 3 to 43 Hz depending on parameter values. The behavior of the model under periodic stimulation was studied and the input-output relationship was non-linear. When identical columns were sparsely interconnected, the column oscillator could be locked in synchrony. In a network composed of heterogeneous columns, the columns were organized by intrinsic properties and formed partially synchronized assemblies.     

A comparison of coulometric detectors for catecholamine analysis by LC-EC

An ion-pairing chromatographic method which uses a controlled potential coulometric detector is described. Two coulometric detectors with different electrolytic cell designs have been investigated. The resulting sensitivity can be comparable to the conventional amperometric detector. This technique has been applied to the analysis of catecholamines.     

Isoelectric focusing of brain adenylate cyclase

Mouse brain adenylate cyclase has been solubilized with Lubrol PX and separated by isoelectric focusing on polyacrylamide gels. The enzyme activity has been measured with a sensitive assay isolating cyclic AMP from Dowex and alumina columns. The technique allows a one-step analysis of this membrane enzyme from a heterogeneous sample within 6 hr.     

High-performance liquid chromatographic determinations of disaccharides resulting from enzymatic degradation of isomeric chondroitin sulfates.

The use of Whatman Partisil-10 PAC and Altex LiChrosorb NH₂ bonded phase columns for the high-performance liquid chromatographic separation of the disaccharides obtained from digestion of isomeric chondroitin sulfates with chondroitinases has been investigated. The substituted unsaturated disaccharides in the enzymatic degradation mixtures undergo rapid isocratic separations on both bonded stationary phases. A complete separation can be established within 10 min. The development of these procedures has expedited enzymatic studies of isomeric chondroitin sulfates.The rate of depolymerization of chondroitin sulfates by the action of chondroitinases based on quantitative high-performance liquid chromatographic separations and detection of the disaccharide products was studied.