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PubMed1979

Application of a high-performance liquid chromatographic fluorescence method for the rapid determination of alpha-tocopherol in the plasma of cattle and pigs and its comparison with direct fluorescence and high-performance liquid chromatography-ultraviolet detection methods.

High-performance liquid chromatography is used to develop a sensitive method for the determination of tocopherol levels in the plasma of cattle and pigs. This method is compared with a similar method using UV detection and one using direct fluorescence determination of tocopherol. Finally a double injection technique used in conjunction with fluorescence detection is shown to enhance the rate of analysis of the tocopherol levels in bovine plasma extracts.

Animals↗

PubMed1987

Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography. V. Performance of non-porous monodisperse 1.5-microns bonded silicas in the separation of proteins by hydrophobic-interaction chromatography.

Non-porous monodisperse 1.5-microns silicas were allowed to react with (A) and (B) N-acetylaminopropyltriethoxysilane to generate bonded phases useful in high-performance hydrophobic-interaction chromatography (HIC). Differences in the selectivity were observed between the amide and the ether phase. Peak capacities between 10 and 30 were achieved for several proteins with the amide and ether phase packed into columns of 36 X 8 mm I.D. and elution of the proteins under chromatographic conditions in which the gradient volume, VG, was held constant by varying the gradient time between 20 and 2.5 min and the flow-rate between 0.5 and 4.0 ml/min. The S values derived from the dependences of log k' on the volume fraction of the low ionic strength buffer, phi b, were of the same magnitude as reported for porous HIC silicas and showed a dependence on the molecular weight of the protein. Using these HIC stationary phases based on non-porous 1.5-microns supports, fast separations (less than 5 min) could be carried out with high biological recoveries.

Ammonium Sulfate↗

PubMed1987

Size-exclusion high-performance liquid chromatography of peptides. Requirement for peptide standards to monitor column performance and non-ideal behaviour.

A series of five synthetic peptide polymers with the sequence Ac-(G-L-G-A-K-G-A-G-V-G)n-amide, where n = 1-5, was employed to assess the resolving power of high-performance size-exclusion columns in peptide separations. The peptide standards showed great versatility in monitoring both ideal (no interactions of solutes with the column material) and non-ideal (hydrophobic and/or ionic interactions of solutes with the column material) size-exclusion behaviour in volatile and non-volatile mobile phases. The effectiveness of adding salts or organic solvents to overcome non-specific interactions of solutes with the column materials was well illustrated by the standards. In addition, the advantageous use of non-ideal size-exclusion behaviour was highlighted. The ability to predict the position and/or elution order of peptides during size-exclusion chromatography (SEC) requires peptides to be separated by a pure size-exclusion process. Although the peptide standards demonstrated similar ideal size-exclusion profiles in non-denaturing medium on all the columns studied, this study suggested that, if the conformational character of a peptide-protein mixture in a particular mobile phase is uncertain and ideal size-exclusion behaviour is required, SEC should be carried out under highly denaturing conditions.

Acetonitriles↗

PubMed1987

High-performance liquid chromatography of amino acids, peptides and proteins. LXXIV. Separation of heparin-binding growth factors by reversed-phase high-performance liquid chromatography.

The separation of several immunologically related forms of the bovine brain basic heparin-binding growth factor by reversed-phase high-performance liquid chromatography is described. With Bakerbond C4 reversed-phase columns, it is possible to resolve the 0.8-1.0 M sodium chloride and the 1.0-1.3 M sodium chloride components from the preceding heparin-Sepharose affinity chromatographic step in the purification procedure for these polypeptide mitogens into multiple active forms, all of which exhibit similar molecular weights and immunoreactivity with specific polyclonal antisera. Structural characterisation suggests that it is likely that these forms represent different stages in the post-translational processing of these polypeptide mitogens.

Amino Acids↗

PubMed1984

High-performance liquid chromatography of amino acids, peptides and proteins. LVIII. Application of reversed-phase high-performance liquid chromatography to the separation of tyrosine-specific phosphorylated polypeptides related to human growth hormone.

Procedures for the purification of native and phosphorylated human growth hormone (hGH), S-carboxymethylated hGH, and hGH tryptic peptides, based exclusively on reversed-phase chromatography have been developed. Combinations of several volatile ion-pairing systems with small- and large-pore n-alkylsilicas were exploited in a general strategy, which allowed high recoveries of various hGH-related polypeptides from enzymatic incubations, as well as rapid desalting of samples following chemical modifications of the native protein, such as reductive alkylation in 6 M guanidine hydrochloride. The influence of the elution conditions on retention behaviour of phosphorylated hGH and its tryptic peptides in reversed-phase high-performance liquid chromatography is discussed.

Chromatography, High Pressure Liquid↗

PubMed1990

High-performance liquid chromatography of amino acids, peptides and proteins. XCVII. The influence of the gradient elution mode and displacer salt type on the retention properties of closely related protein variants separated by high-performance anion-exchange chromatography.

The influence of different elution modes, gradient times and flow-rates on the relative retention of closely related variants of carbonic anhydrase and ovalbumin has been investigated using high-performance ion-exchange chromatography. Three isoform species of carbonic anhydrase and four isoforms related to ovalbumin eluted by anion-exchange chromatography were characterised by isoelectric focusing and sodium dodecylsulphate-polyacrylamide electrophoresis. Gradient retention data were collected using several different alkali metal halides as the displacer salt, in order to systematically evaluate the effect on selectivity of different anions and cations in the series F-, Cl- and Br-, and Li+, Na+ and K+. While the selectivity between the different ovalbumin isoform species remained essentially constant with each displacer salt, solute Zc-values [J. Chromatogr., 458 (1988) 27] varied with the type of salt. In contrast, non-parallel retention plots were obtained for the carbonic anhydrase isoforms with the Zc values different for each isoform. Furthermore, significant differences in chromatographic behaviour for these proteins were observed between experiments carried out under gradient elution conditions with either varied gradient time and constant flow-rates or fixed gradient time and varied flow-rates. These results are discussed in terms of the influence of column residence time and protein-salt interactions of the solute's interactive ionotope and the concomitant effects these structural perturbations may have on chromatographic behaviour.

Amino Acids↗

PubMed1989

High-performance liquid chromatography of amino acids, peptides and proteins. XC. Investigations into the relationship between structure and reversed-phase high-performance liquid chromatography retention behaviour of peptides related to human growth hormone.

The gradient elution behaviour of eight synthetic peptides encompassing residues [6-13] of human growth hormone, i.e. Leu1-Ser-Arg-Leu-Phe-Asp-Asn-Ala8, has been investigated, by using an octadecylsilica, a butylsilica, and a polymeric fluorocarbon as stationary phases. Quantitative expressions, derived from the linear-solvent-strength theory and the general plate-height theory, were used to assess the influence of gradient time on the relative retention and bandwidths of these peptides. It was demonstrated that the chromatographic properties of the cyclised imide form involving Asp6 are consistent with the formation of a highly stabilised amphipathic helix, while the open-chain alpha- and beta-rearranged forms eluted as less rigid structures. The putative hydrophobic contact region consists of two leucine residues and one phenylalanine residue. From an analysis of the retention and bandwidth data obtained at pH 9, a surface-induced molecular reorientation of the beta-linked peptides was observed, in which the repulsion of the aspartyl carboxyl group from the hydrophobic stationary phase directs the C-terminal moiety away from the sorbent surface. Furthermore, the fluorocarbon sorbent exhibited characteristics favourable for use in preparative purification of these peptides. The present results demonstrate the sensitivity of reversed-phase high-performance liquid chromatography (RP-HPLC) to monitor small changes in the interactive behaviour of peptides with hydrocarbonaceous ligands and aquo-organic solvent combinations in reversed-phase systems. These observations further illustrate the general utility of HPLC for investigating the conformational behaviour of peptides at solid-liquid interfaces.

Amino Acids↗

PubMed1987

Predicting bandwidth in the high-performance liquid chromatographic separation of large biomolecules. II. A general model for the four common high-performance liquid chromatography methods.

A general model for describing gradient elution separations of peptides and proteins by reversed-phase high-performance liquid chromatography (HPLC) has been presented previously. This model has now been modified so that it can be applied to any of the four HPLC methods used for separating biological macromolecules: reversed-phase, ion-exchange, hydrophobic-interaction and size-exclusion chromatography, carried out in either an isocratic or gradient elution mode. The role of sample molecule structure and the particular column used has been further studied, so that previous empirical parameters for different column/sample choices can now be estimated from three physical properties of the sample and the column: sample molecular weight, native vs. denatured sample, column packing pore diameter. This eliminates much of the empiricism of our preceding model, and minimizes the number of experimental runs now required in order to apply the model in practice. The final model has been tested for several hundred runs involving peptides and proteins in the molecular weight range 600-162,000, all four of these HPLC methods, in both isocratic and gradient elution modes, and using data from several different laboratories (including our own). The model is able to predict bandwidth in HPLC separations of proteins and peptides with an accuracy of +/- 17% (1 standard deviation), for the case of "well-behaved" separations. Separations that are not "well-behaved" will give wider bands than predicted by the model.

Chemical Phenomena↗

PubMed1990

High-performance liquid chromatography of amino acids, peptides and proteins. C. Characterisation of coulombic interactive regions on hen lysozyme by high-performance liquid anion-exchange chromatography and computer graphic analysis.

The molecular characteristics of the dominant anion-exchange binding site of hen egg white lysozyme (HEWL) has been investigated using a combination of high-performance liquid chromatographic techniques and computer graphic analysis of the X-ray crystallographic structure. These studies have indicated that the site of highest electrostatic potential, in terms of the density of negatively charged amino acid side chains, is located around the catalytic cleft area. The four residues tentatively identified to be involved in the electrostatic binding domain were aspartic acid 48, 52, 101 and glutamic acid 35. The number of these charged groups correlated with the maximum value of the chromatographically determined retention parameter (Zc value). Variations in the range of experimental Zc values obtained under different elution conditions have been interpreted in terms of conformational flexibility of the structural domains of HEWL which result in the opening or closure of the catalytic cleft during the retention process.

Amino Acid Sequence↗

PubMed1984

High-performance liquid chromatography of amino acids, peptides and proteins. LVII. Analysis of radioiodinated thyrotropin polypeptides by reversed-phase high-performance liquid chromatography.

The chromatographic behaviour of bovine and human thyrotropin, radiolabelled stoichiometrically with lactoperoxidase, on octadecylsilicas and other reversed-phase n-alkylsilicas has been investigated. As part of this investigation the effects of a variety of elution systems on resolution and recovery have been examined. Analysis of the tryptic peptides of radioiodinated bovine thyrotropin (bTSH) preparations by reversed-phase high-performance liquid chromatographic mapping methods resulted in the separation of more than fourteen major radioactive peptide components. The data indicate that bTSH is radioiodinated with unequal incorporation into both the alpha- and beta-subunits. Further, assessment of the level of microheterogeneity of radiolabelled bTSH preparations can be achieved with these reversed-phase techniques.

Amino Acids↗

PubMed1997

On-line column-switching high-performance liquid chromatography analysis of cardiovascular drugs in serum with automated sample clean-up and zone-cutting technique to perform chiral separation.

A selective and highly reproducible, multi-column HPLC method is described for the analysis of the following cardiovascular drugs: lidocaine, pindolol, metoprolol, oxprenolol, diltiazem and verapamil, in serum. Column-switching devices are employed in combination with advanced separation media technologies for the automated analysis of samples containing complex matrices. The method consists of on-line sample clean-up using a restricted access sorbent, HPLC analysis of the drugs on a microsphere non-porous silica RP-18 column, and front-cutting to perform the chiral separation of pindolol enantiomers on a second HPLC system. Simultaneous control of the two HPLC systems and data analysis is achieved from a single centralized software. The R.S.D. values of the peak areas for spiked serum are less than 1% for metoprolol and oxprenolol, 2-5% for lidocaine, diltiazem and verapamil, and 1.2 and 2.4% for the two pindolol enantiomers. Recoveries, limits of detection and linearities are provided.

Cardiovascular Agents↗

PubMed1998

Determination of furanochromones and pyranocoumarins in drugs and Ammi visnaga fruits by combined solid-phase extraction-high-performance liquid chromatography and thin-layer chromatography-high-performance liquid chromatography.

A new, simple and rapid solid-phase extraction method for the determination of furanochromones and pyranocoumarins in Ammi visnaga L. fruits and pharmaceuticals by reversed-phase high-performance liquid chromatography (RP-HPLC) was developed. The isolation of compounds examined was carried out on octadecyl BakerBond SPE columns using various concentrations of methanol, acetonitrile and tetrahydrofuran in water. High and reproducible recoveries were obtained. To compare the results of quantitative analysis a preparative TLC procedure was also elaborated and carried out.

Antineoplastic Agents↗

PubMed1993

High-performance liquid chromatography of amino acids, peptides and proteins. CXXVIII. Effect of D-amino acid substitutions on the reversed-phase high-performance liquid chromatography retention behaviour of neuropeptide Y[18-36] analogues.

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. Further, these results indicate that, in hydrophobic environments, NPY[18-36] adopts a significant degree of secondary structure which is severely disrupted by the presence of the D-amino acids in the central portion of the molecule.

Acetonitriles↗

PubMed1993

Characterisation of TNF-alpha-related peptides by high-performance liquid chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry.

Ion-spray triple quadrupole mass spectrometry and high-performance liquid chromatography were used to investigate the products from the solid phase synthesis of (H)-Leu-Thr-Glu-Asn-(OH), a TNF-alpha active-site probe. The target sequence was assembled using tert.-butoxycarbonyl (Boc) chemistry in stepwise fashion from the C-terminal on an Boc-Asn-OCH2-Pam-copoly(styrene-divinylbenzene) resin [Pam = 4-(carboxamidomethyl)benzyl ester]. The crude product was deprotected and cleaved from the resin by HF-p-cresol treatment for 1 h at 0 degrees C. HPLC analysis at 214 nm indicated two late-eluting major products and an early-eluting product. Preparative HPLC demonstrated that the early-eluting product contained ca. 80% of the expected recovered sample mass. Each component was then directly analysed by mass spectrometry and tandem mass spectrometry. The early eluting peak was confirmed as the desired LTEN sequence. Synthesis of the same sequence using 9-fluorenyl methoxycarbonyl (Fmoc) chemistry gave an identical product and confirmed the above analysis. The most significant by-product was derived from arylation of the glutamyl group by the quencher p-cresol. The likely origins of the by-products are discussed.

Amino Acid Sequence↗

PubMed2000

Performance of a pentafluorophenylpropyl stationary phase for the electrospray ionization high-performance liquid chromatography-mass spectrometry-mass spectrometry assay of cocaine and its metabolite ecgonine methyl ester in human urine.

A pentafluorophenylpropyl (PFPP) bonded silica column has been used for the high-performance liquid chromatography-electrospray ionization-mass spectrometry-mass spectrometry assay (HPLC-ESI-MS-MS) of cocaine (COC) and its metabolite, ecgonine methyl ester (EME) in human urine. COC and EME were used as model basic solutes to demonstrate that a PFPP phase yields excellent results for the assay and validation of drugs in biological fluids. The assay was linear over three orders of magnitude (1.0-1000 ng/ml) and precision and accuracy of the assay was 4 and 15%, respectively. The limit of detection (LOD) for COC and EME was 1.6 and 2.8 pg on column, respectively. In addition, only a simple 1:10 dilution of the urine was necessary for the sample preparation procedure thus saving time on a laborious extraction step. The major advantage of the PFPP phase was the enhancement of the ESI-MS signal by providing good retention and good peak shape of COC and EME with a mobile phase of 90% acetonitrile. The MS signal for COC was a factor of 12 times greater on the PFPP phase than on the C18 phase.

Calibration↗

PubMed1980

Investigations of the performance of a high-performance liquid chromatography system with an electrochemical detector.

A procedure for the determination of norepinephrine and dopamine, based on high-performance liquid chromatography, is evaluated using an electrochemical detector system. The use of an inorganic mobile phase to provide resolution of low retention amines and extend column life is discussed. A high degree of correlation between estimations of endogenous catecholamine levels is reported using both electrochemical and fluorometric detector systems. Inter-assay reproducibility of the extraction method, and sensitivity and linearity of response of the electrochemical detector system are shown to be consistent across trials. The system described is determined to be accurate, sensitive, and reliable over time.

Animals↗

PubMed1997

Comparison of quantitative high performance thin layer chromatography and the high performance liquid chromatography of parabens.

A method is described for the densitometric determination of the p-hydroxybenzoic esters and p-hydroxybenzoic acid in mixtures or in drugs. This method is compared with the one used in high performance liquid chromatography (HPLC). The calibration curves were linear in interval 0.250-3.60 mumol ml-1 per 200 nl per spot. The limit of detection and the relative standard deviation (RSD) are higher than in HPLC (RSD is 6% in HPTLC. 3% in HPLC; limit of detection about 40 pmol in HPTLC and 25 pmol in HPLC) but HPTLC quantitative determination of parabens in drugs is faster.

Chromatography, High Pressure Liquid↗

PubMed2002

Metabolism of cymoxanil and analogs in strains of the fungus Botrytis cinerea using high-performance liquid chromatography and ion-pair high-performance thin-layer chromatography.

The metabolism of cyano-oxime fungicide 1-(2-cyano-2-methoxyiminoacetyl)-3-ethylurea (cymoxanil) and analogs was studied on several strains of the fungus Botrytis cinerea owing to their difference in sensitivity towards cymoxanil. Chromatographic analysis of the unextracted culture medium was simpler and more accurate, particularly for ionizable metabolites because it avoids problems associated with extraction. Reversed-phase high-performance liquid chromatography was applied to compare the decrease of cymoxanil and analogs caused by different strains of B. cinerea, by periodic injections of incubated culture medium aliquots, directly on a C4 wide-pore column. Furthermore, a thin-layer chromatographic monitoring on C18 bonded silica gel with ion-pairing allowed the monitoring of the ionizable metabolites for substrates that were demonstrated to decompose most rapidly. These complementary analyses showed that the sensitivity of the highly sensitive strain towards cymoxanil was related to the disappearance of cyano-oximes studied from culture medium, namely to the ability of the strain B. cinerea to metabolize them.

Acetamides↗