Journal of Chromatography

A method is described for measuring glutamine (GLN) and alpha-ketoglutarate (KG) concentration and specific activity (SA) using high-performance liquid chromatography (HPLC). Plasma GLN and KG are separated on miniature ion-exchange columns. KG is derivatized with O-phenylene diamine, the derivative is extracted in ethyl acetate, dried, and dissolved in pH 7 phosphate buffer. The isolated GLN is enzymatically converted to KG and analysed as such. Derivatized samples are stable for weeks at -20 degrees C. Samples are injected onto a reversed-phase HPLC column...

A method was developed for simultaneous determination of urinary hydroquinone, catechol and phenol using high-performance liquid chromatography (HPLC) with variable-wavelength fluorimetric detection. Urine samples, after acid hydrolysis, were saturated with sodium sulphate and extracted by diethyl ether. The two buffers used for gradient elution were (A) 10 mM sodium acetate containing 0.5% (v/v) acetic acid and (B) the same as buffer A but containing an additional 20% (v/v) acetonitrile. Hydroquinone, catechol and phenol were separated in a C18 column and detected at 2...

A gas chromatographic-mass spectrometric method has been developed for the identification of 1,2,3,4-tetrahydroisoquinoline and six metabolites extracted from urine in the picogram range. The derivatization procedure for the substances, formed by reaction of formaldehyde with biogenic amines, employs propionic anhydride and can take place in aqueous medium. In this way artificial formation of these compounds via condensation of biogenic amines with aldehydes or alpha-keto acids during the work-up procedure is eliminated...

A high-performance liquid chromatographic method for the determination of p-substituted N,N-dialkylaniline N-oxides [N,N-dimethylaniline N-oxide (DMANO), N,N-dimethyl-p-toluidine N-oxide, N,N-diethylaniline N-oxide, N-ethyl-N-methylaniline N-oxide (EMANO), p-cyano-N,N-dimethylaniline N-oxide (pCNDMANO), and N-phenylpyrrolidine N-oxide (PPNO)] has been developed. It uses an octadecylsilica column, a mobile phase of methanol-phosphate buffer (pH 7.0, adjusted by triethylamine) and ultraviolet detection. N-Oxides were eluted in the order of hydrophilicity except for PPNO, which eluted between DMANO and EMANO...

Multiple phosphoseryl-containing sequences of peptides and proteins stabilize amorphous calcium phosphate at neutral and alkaline pH and have been implicated in the nucleation/regulation of biomineralization. In an approach to analyze these peptides using capillary zone electrophoresis (CZE) we have attempted to relate the absolute electrophoretic mobility of various casein phosphopeptides to their physicochemical properties. Multiple phosphoseryl-containing peptides were selectively precipitated from enzymic digests of sodium caseinate and further purified using RP-HPLC and anion-exchange fast protein liquid chromatography...

In large-scale chromatography, process optimisation is one of the key elements for success. This paper presents a method for determining the optimum operating parameters for affinity and ion-exchange chromatographic columns when used for protein purification. Based on a mathematical model developed as part of our association investigations, computer programs have been developed to describe the dynamic relationships acting within the chromatographic system. Two basic operating parameters, the flow-rate and the effluent concentration at which the adsorption stage is terminated, can be optimised to give a maximum production rate...

The reversed-phase high-performance liquid chromatographic (RP-HPLC) gradient elution behaviour of a series of peptides related to Neuropeptide Y (NPY) has been investigated. The peptides studied included NPY, NPY[13-36], NPY[18-36] and a series of 16 analogues of NPY[18-36], each with a single D-amino acid substitution. Chromatographic parameters which relate to the interactive contact area and the binding affinity have been evaluated with two different stationary phase ligands and two organic modifiers. The results demonstrate that D-amino acid substitutions in the sequence region encompassing amino acid residues NPY[27-31] of these NPY[18-36] peptides significantly influence the interactive behaviour of these peptides relative to the unsubstituted NPY[18-36] molecule, while substitutions in the N- and C-terminal regions had little effect...

Peptides from a Staphylococcus aureus (V8) proteinase digest of human pepsin 3b have been identified by amino acid sequence analysis. Only 137 out of 326 expected residues were detected from the C and N terminal regions of the molecule. Comparison with amino acid sequences derived from nucleotide analysis of three different pepsinogen A genes, identified 2 out of 4 possible substitutions. The presence of valine at position 30 and leucine at 291 indicates that the major pepsin component of gastric juice, pepsin 3b, corresponds to pepsinogen genotype PGA-3...

A proline-rich peptide was isolated and purified to homogeneity from an extract of bovine neutrophil granules using semi-preparative RP-HPLC. The relative molecular mass of the peptide (called Bac-X) was determined by ionspray MS to be 5149 +/- 0.5. The amino acid composition of the peptide was characterized by its limited number of amino acid types, which included a high proline (43.3%) and arginine content (20.3%), and hydrophobic residues. Bac-X had similar characteristics to Bac-5, a previously characterised bactenecin of bovine neutrophil granules, with respect to its proline, arginine and hydrophobic amino acid content, molecular mass and antibacterial specificity...

We have examined the contribution of the alpha-amino group to retention behaviour for peptides in reversed-phase chromatography using two series of peptide analogues, one containing an N alpha-acetylated terminal and the other containing an alpha-amino group (non-acetylated). The effect of the alpha-amino group, at pH 2, on the hydrophobicity of the side-chain of the N-terminal residue was obtained by referencing the retention time of the acetylated or non-acetylated peptide to the retention time of a glycine analogue...

Porous zirconia based particles have been modified using different derivatisation procedures. The modified particles were characterised in terms of their accessible surface areas and degree of surface coverage of the bounded or physicoated phases utilising the strong and specific adsorption of phosphate ions to the zirconia surface. The hydroxyl group density was determined by a 1H NMR technique. The particles were modified by immobilising different silanes to introduce either hydrophobic ligands or reactive groups onto the zirconia surface...

Malto-oligosaccharides were derivatized via their reducing end with 8-aminonaphthalene-1,3,6-trisulfonic acid by reductive amination, and the separation and electrophoretic migration behavior of the labelled sugars were investigated by capillary zone electrophoresis. Series of linear malto-oligosaccharides were found particularly suitable for both the study of the effect of the operating conditions on the separation and the investigation of the relationship between the electrophoretic mobility and the molecular size of the homologues...

High-performance affinity chromatography was used to study the separation and binding of D- and L-tryptophan on an immobilized human serum albumin (HSA) column. Frontal analysis and zonal elution studies indicated that both D- and L-tryptophan were binding to single but distinct sites on HSA. L-Tryptophan bound to the indole site of HSA. D-Tryptophan had indirect interactions with the warfarin site of HSA but no interactions with the indole site. The association constants for the binding of D- and L-tryptophan at pH 7...

A rapid procedure for the preparation of short-chain (C s-C.) isoprenoid diphosphates is described . It is based on the method of Cornforth and Popjak [Methods Enzymol., 15 (1969) 359-390] which utilizes bis-triethylammonium phosphate in trichioroacetonitriie as the phosphorylating reagent . The reaction takes place in 15 min, and product isolation, previously requiring several steps, is done in a single step using either preparative thin-layer chromatography or flash chromatography on silica . From a single TLC plate, up to 50 μ mol of pure farnesyl diphosphate (i ...

The peculiarities of zone migration and band broadening in the reversed-phase gradient HPLC of proteins were investigated. In the isocratic mode a critical composition of the mobile phase was found at which all proteins regardless of their molecular mass migrate with equal velocity and have a capacity factor equal to the phase ratio (VP/V0), i.e., the same capacity factor as a marker of total accessible volume would have in steric exclusion chromatography. It is shown that steric exclusion conditions are never achieved in gradient HPLC...

The enantiomers of zopiclone and its two chiral N-desmethyl and N-oxide metabolites were determined in urine using a coupled achiral-chiral liquid chromatographic method. After liquid-liquid extraction, zopiclone and its two metabolites were quantified on a cyanopropyl column. After fluorimetric detection on the achiral system, the eluent was switched through a silica precolumn in order to trap and concentrate the analytes. Each fraction was then backflushed separately onto a carbamate cellulose chiral stationary phase in order to determine the enantiomeric ratios...

A rapid and sensitive reversed-phase ion-pair liquid chromatographic system with fluorimetric detection by post-column sodium nitrite oxidation was established for measuring six pterin compounds (6R-5,6,7,8-tetrahydrobiopterin, 6S,5,6,7,8-tetrahydrobiopterin, 7,8-dihydrobiopterin, biopterin, pterin and D-neopterin). The coefficients of variation for these pterins were 0.705-3.714%, and the minimum detectable amount was ca. 10-20 pg at a signal-to-noise ratio of 3. A linear detector response was also verified...

Poly(ethylene glycol) (PEG)-bound chelated metal ions partition preferentially into the top, PEG-rich, phase of a PEG-salt or PEG-dextran aqueous two-phase system. Extraction by this soluble affinity ligand of proteins is due to a selective interaction of the chelated metal ion with accessible histidine residues on the protein surface. Using Cu-iminodiacetate-PEG (Cu-IDA-PEG) the surface of lactate dehydrogenase (LDH) isoenzymes from different species was probed for the presence of metal chelate binding sites...

A copper-modified strong cation exchange stationary phase was used for the solid phase extraction of the antineoplastic drug doxifluridine (5'-deoxy-5-fluorouridine) and some of its main metabolites from human serum. Sorption of fluoropyrimidine derivatives due to complex formation with copper(II) was maximal at pH values around neutrality. Analytes were eluted by ligand exchange with ammonia. The average recoveries ranged from 70% to 108%. Reversed-phase chromatography with ultraviolet detection was used for the separation and quantitation of the analytes...

An overview is given of the methods that have been used in the study of polychlorodibenzo-p-dioxins and polychlorodibenzofurans in agriculture and the human food chain in a national survey and monitoring programme, including sampling strategies, sampling in the field and clean-up and analysis in various biological and environmental samples by high-resolution gas chromatography-high resolution mass spectrometry. The quality of data was evaluated as a result of internal quality control protocols and participation in interlaboratory comparison studies...