Gradient elution reversed-phase chromatographic isolation of individual glycerophospholipid molecular species

We describe a gradient elution reversed-phase high-performance liquid chromatographic approach for isolation of individual glycerophospholipid molecular species which greatly improves resolution and reduces run time compared to isocratic techniques. Separations were optimized and elution order and retention time data established by synthesizing 37 different homogeneous phospholipids comprising the major alkylacyl, diacyl and plasmalogen molecular species in samples derived from mammalian sources. Empirical equations which predict the elution order of individual species were derived. The method was validated with the use of complex mixtures of choline and ethanolamine glycerophospholipid species from isolated rabbit cardiomyocytes and porcine endothelial cells.     

Optimization of separation of purine and pyrimidine compounds using ion-pair reversed-phase high performance chromatography (HPLC)

Utilization of ion-air reagents in a reversed-phase chromatographic system allows solving a number of problems related to the separation of purine and pyrimidine derivatives. Simultaneous analysis of nucleotides, nucleosides and their bases was carried out by acetonitrile gradient elution using tetrabutyl ammonium phosphate as a counterion in the mobile phase. Besides, optimal conditions were selected for isocratic separation of adenine nucleotides and their metabolites. Furthermore, isocratic separation of certain purines and pyrimidines was achieved by modifying the stationary C18-phase with pentane- and heptane sulphonic acids.     

Separation of steroidal estrogens and their major unconjugated metabolites by high performance liquid chromatography

A high performance liquid chromatographic method is described for the rapid, non-destructive separation of a number of physiologically important steroidal estrogens, including the labile catechol estrogens. This procedures uses a "Diol" column and gradient elution to separate in a single run, estrogens ranging from 2-methoxy estrone, one of the least polar C18 steroids, to estriol, one of the most polar. Simpler, isocratic conditions, are provided for the separation of estrogens of similar polarity. A semi-preparative column of similar composition was used for the purification of samples containing 25 to 50 mg of individual steroids.     

Investigation, identification and dosage of local anesthetics and antihistaminics in cosmetic products

A rapid and accurate reversed-phase HPLC method for separation and simultaneous quantitation of some local anesthetics, antihistamines and preservatives in skin cosmetics is described. The investigated compounds (procaine, lidocaine, mepivacaine, bupivacaine, benzocaine, brompheniramine, benzoic acid, methyl, ethyl and propyl p-hydroxybenzoate, o-phenylphenol) are extracted by ultrasonic treatment in methanol from cosmetic form. Methanol-aqueous orthophosphoric acid (pH 2.8) containing 0.9% dibutylamine has been used as eluent. The influence of different percentages of methanol on chromatographic behaviour has been studied using both isocratic conditions and gradient elution program. The gradient program allows a rapid resolution and quantitation also for acidic preservatives.     

High-pressure liquid chromatography: I. Quantitative separation of purine and pyrimidine nucleosides and bases

A high-pressure liquid column chromatographic method has been developed for the separation and identification of ribonucleosides, deoxyribonucleosides, and bases. This method is capable of detecting these components at 1.0 to 5.0 ng applied; is reproducible under conditions of constant pressure, temperature, and pH; and is rapid, requiring about 30 min for a complete chromatographic separation of ribonucleosides from deoxyribonucleosides and from bases. These separations were carried out under different pH values and buffers, namely, phosphate buffer containing 2.5% methanol at pH 6.9 and 3.0 or 50 mm sodium borate buffer, pH 9.0. These different conditions were utilized to obtain more definitive identification and quantitation of normal metabolites and their counterparts, the antimetabolites. The advantage of this method is that the 20 naturally occurring components are separated from each other by an isocratic elution method, alleviating the need for a gradient elution system, which produces a drift in the baseline with increasing concentration of the eluting buffer, especially when the instrument is operating at maximum sensitivity, thus hindering the quantitation of the separated components. The potential application of this method for the quantitation of plasma metabolites and antimetabolites such as Ara-C², Ara-U, and F-pyrimidine is describe.     

Apolipoprotein A-I(Milano) anion exchange chromatography: Self association and adsorption equilibrium

The self-associative properties of apolipoprotein A-I(Milano) (apoA-I(M)) were investigated in relationship to its anion exchange behavior on Q-Sepharose-HP with and without the addition of urea as a denaturant. Self-association was dependent on protein and urea concentration and both influenced interactions of the protein with the chromatographic surface. In the absence of urea, apoA-I(M) was highly associated and existed primarily as a mixture of homodimer, tetramer and hexamer forms. Under these conditions, since the binding strength was greater for the oligomer forms, broad, asymmetrical peaks were obtained in both isocratic and gradient elution. Adding urea depressed self-association and caused unfolding. This resulted in sharper peaks but also decreased the binding strength. Thus, under these conditions chromatographic elution occurred at lower salt concentrations. The adsorption isotherms obtained at high protein loadings were also influenced by self-association and by the varying binding strength of the differently associated and unfolded forms. The isotherms were thus dependent on protein, urea, and salt concentration. Maximum binding capacity was obtained in the absence of urea, where adsorption of oligomers was shown to be dominant. Adding urea reduced the apparent binding capacity and weakened the apparent binding strength. A steric mass action model accounting for competitive binding of the multiple associated forms was used to successfully describe the equilibrium binding behavior using parameters determined from isocratic elution and isotherm experiments.     

A simple and inexpensive sample-handling method for the semi-preparative RP-HPLC of polypeptides and non-polar peptide derivatives: pre-adsorption of samples

A simple method is described for the application onto HPLC columns of very crude or alternatively poorly soluble polypeptide samples prior to their chromatographic purification. The procedure involves the batch pre-adsorption of the crude polypeptide mixture from a dilute solution onto an appropriate preparative-grade chromatographic adsorbent, removal of the solvent by rotary evaporation or lyophilisation and then dry-packing the pre-adsorbed chromatographic material into guard column cartridges of suitable dimensions. The polypeptide products can then be eluted either by isocratic or gradient elution methods through the cartridge coupled in tandem with prepacked semi-preparative HPLC columns. This method has been successfully utilised for the routine RP-HPLC purification of polar and hydrophobic polypeptides prepared by solid phase peptide synthesis (SPPS) methods as well as peptide derivatives and intermediates used as part of SPPS procedures.     

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity.

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity is described. The method uses the small synthetic peptide benzyloxycarbonyl-L-glutaminylglycine and the fluorescent amine monodansylcadaverine as substrates. Very small amounts of substrates and enzyme are required for this assay. The reaction product is separated from substrates on a reversed-phase, C-18 column, using an isocratic elution solvent consisting of 50% methanol in water, and is detected fluorometrically with didansylcadaverine as standard. A detection limit of 31 pmol of product per injection was measured. An apparent Km of 34.7 +/- 2.4 mM was determined for the peptide substrate with purified guinea pig liver enzyme. Using this assay, a series of alkyl aldehydes was shown to inhibit transglutaminase. Modification of this assay using either gradient or isocratic elution with various proportions of acetonitrile (0.1% trifluoroacetic acid)/water (0.1% trifluoroacetic acid) afforded assays for a series of glutamine-containing peptides including substance P, alpha-endorphin, and two small, synthetic peptides. The assay is suitable for measurement of transglutaminase activity with purified enzyme or with crude preparations. This method provides a sensitive, quantitative assay for the determination of substrate and inhibitor properties of small peptides toward transglutaminases.     

High-performance liquid chromatographic determination of ibuprofen and its major metabolites in biological fluids

A sensitive and selective high-performance liquid-chromatographic assay for ibuprofen and its major metabolites in biological fluids is described. To ensure good chromatographic separation the drug and metabolites were run on a gradient elution system and detected with a variable wavelength detector set at 220 nm. A second, more rapid, isocratic system is also described for the detection of only ibuprofen.     

High-performance liquid chromatography of methylated phospholipids

A rapid high-performance liquid chromatographic method for the separation of methylated phospholipids is described. The separation is accomplished on an amine column using acetonitrile—methanol—water as the eluting solvent and UV detection at 203 nm. The choice between gradient and isocratic elution for the separation depends upon the condition of column. The method is suitable for the isolation of phosphatidylcholine, sphingomyelin, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine and lysophosphatidylethanolamine from tissues. It is applicable to the study of reaction products in phosphatide methyltransferase assay mixtures. Choline and ethanolamine plasmalogens can be determined indirectly by converting them into lysophosphatidylcholine and lysophosphatidylethanolamine with exposure to hydrochloric acid fumes.     

Sensitive and reproducible method for neutral monosaccharides and uronic acid determination in sugar beet pulp enzymatic treatment media and press juices by High Performance Anion Exchange Chromatography with pulsed amperometric detection

The resolution and the elution of arabinose, rhamnose, xylose, glucose, galactose, fructose, sucrose and galacturonic acid by an High Performance Anion Exchange Chromatography method were studied. The best results were obtained with a two step elution composed of a 25mM NaOH isocratic phase for 20 min followed by a 300mM sodium acetate gradient for 10 min. The reliability, repeatability and sensitivity of these analytical conditions were set.     

Ternary gradient elution markedly improves silver-ion high performance liquid chromatography of unsaturated sterols

A wide variety of unsaturated sterols can accumulate in eukaryotic cells as a consequence of normal metabolism, genetic disorders, and actions of enzyme inhibitors. Resolving these sterol mixtures into individual components by conventional chromatographic methods is inefficient because unsaturated sterols differ little in polarity, hydrophobicity, and volatility. Although sterol mixtures are well-resolved by silver-ion high performance liquid chromatography (Ag(+)-HPLC), existing methods require derivatization to acetates for best results, and the isocratic mobile phases lead to long analysis times and low sensitivity for late-eluting sterols. We show that these problems can be overcome with ternary gradient elution using acetone, hexanes, and acetonitrile. Separation of a mixture of 20 underivatized sterols gave dramatically shortened analysis times, with good peak shapes for both early- and late-eluting components. In a similar separation of blood sterols from a patient with Smith-Lemli-Opitz syndrome, the band for 7-dehydrocholesterol was much narrower than with isocratic elution. Column re-equilibration was rapid, and the separations could be monitored with ultraviolet spectroscopy at 210 nm, which affords universal, non-destructive detection of unsaturated sterols. Also discussed are retention mechanisms and reproducibility of Ag(+)-HPLC separations. The overall results represent a major advance in chromatographic methods for resolving mixtures of unsaturated sterols differing in the number and position of olefinic bonds.     

Enantioresolution of Chiral Derivatives of Xanthones on (S,S)‐Whelk‐O1 and l‐Phenylglycine Stationary Phases and Chiral Recognition Mechanism by Docking Approach for (S,S)‐Whelk‐O1

The resolution of seven enantiomeric pairs of chiral derivatives of xanthones (CDXs) on (S,S)‐Whelk‐O1 and l ‐phenylglycine chiral stationary phases (CSPs) was systematically investigated using multimodal elution conditions (normal‐phase, polar‐organic, and reversed‐phase). The (S,S)‐Whelk‐O1 CSP, under polar‐organic conditions, demonstrated a very good power of resolution for the CDXs possessing an aromatic moiety linked to the stereogenic center with separation factor and resolution factor ranging from 1.91 to 7.55 and from 6.71 to 24.16, respectively. The chiral recognition mechanisms were also investigated for (S,S)‐Whelk‐O1 CSP by molecular docking technique. Data regarding the CSP–CDX molecular conformations and interactions were retrieved. These results were in accordance with the experimental chromatographic parameters regarding enantioselectivity and enantiomer elution order. The results of the present study fulfilled the initial objectives of enantioselective studies of CDXs and elucidation of intermolecular CSP–CDX interactions. Chirality 25:89–100, 2013. © 2012 Wiley Periodicals, Inc.     

Enantioselective biocatalysis optimized by directed evolution

Directed evolution methods are now widely used for the optimization of diverse enzyme properties, which include biotechnologically relevant characteristics like stability, regioselectivity and, in particular, enantioselectivity. In principle, three different approaches are followed to optimize enantioselective reactions: the development of whole-cell biocatalysts through the creation of designer organisms; the optimization of enzymes with existing enantioselectivity for process conditions; and the evolution of novel enantioselective biocatalysts starting from non-selective wild-type enzymes.     

Membrane chromatographic method for the rapid purification of vitellogenin from fish plasma

A membrane chromatographic method was developed for the rapid purification of vitellogenin (Vtg) from the plasma of 17beta-estradiol induced loach (Misgurnus angaillicaud atus) and carp (Cyprinus carpio). The time required for the proposed procedure is less then 10 min at a flow-rate of 5 ml/min of the mobile phase, and 0.5 ml of fish plasma could be separated in one cycle. Multistep gradient elution was more suitable for the separation than linear gradient elution. Under optimized conditions, a single Vtg peak can be obtained and its identity was confirmed by SDS-PAGE and gel-permeation chromatography assessment. This method is rapid and easy to operate compared to conventional HPLC and FPLC columns for Vtg separation.     

Isocratic separation of estrogen conjugates on DEAE-sephadex.

Mixtures of estrogen conjugates containing estrone-3-glucosiduronate, 17β-estradiol-3-glucosiduronate, 17β-estradiol-17-glucosiduronate, estrone-3-sulfate and 17β-estradiol-3-sulfate have been separated on DEAE-Sephadex resin by isocratic elution using NaCI concentrations ranging from 0.05M to 0.4M. The results indicate that an NaCI gradient is not necessary for the Chromatographic separation of these estrogen conjugates. An NaCl concentration of 0.05M was adequate to separate the various monoglucosiduronates and sulfates. Isocratic elution of the columns with or without a possible stepup in the salt concentration was shown to give a higher resolution of estrogen conjugates in a more convenient volume than a gradient elution. For ideal chromatography of estrogen conjugates on a DEAE-Sephadex column, isocratic elution with 0.05 or 0. IM NaCl is preferred for the separation of monoglucosiduronates and 0.2 or 0.25M NaCl for the separation of sulfate conjugates. Contrary to current expectations, the molarity at which a particular conjugate elutes in a gradient mode does not bear a consistent relationship to the structure of the conjugate. However, the holdback volume in the isocratic mode may be used for identification purposes. When holdback volume was plotted against molarity, separate curves were obtained for each of the above mentioned conjugates. Tests of fit were carried out using a number of models. The best fit was obtained using the simple model $\text{y}\text{=}\text{a}\text{+}\text{b}\text{1}\text{x}$ where the independent variable, x, is the molarity; the dependent variable, y, is the volume and a and b are the intercept and slope respectively. Each curve fitted the model, but the values for a and b were significantly different. Using this model, a simple and predictable relationship between molarity and holdback volume can be demonstrated for each of the estrogen conjugates. The advantages of the isocratic mode of elution over gradient elution are discussed.     

Analysis of glycosphingolipid-derived oligosaccharides by high pH anion exchange chromatography

As an adjunct to existing thin layer and column chromatographic methods for the identification of glycolipids a method that utilizes the high pH anion chromatographic (HPAEC) analysis of the oligosaccharides released from the glycolipids by endoglycoceramidase has been developed. Using a Dionex Carbo Pak PA1 column and elution with a linear gradient of sodium acetate in 0.2M NaOH, the elution times of eight neutral and fourteen acidic oligosaccharides derived from glycolipids were determined. Under these conditions the neutral oligosaccharides were well separated from each other but some of the acidic oligosaccharides had overlapping elution times. The ganglioside-derived oligosaccharides could be further identified by treating them with sialidase or by mild acid hydrolysis and reanalysing the products by HPAEC. The method was applied to the analysis of mixed bovine brain gangliosides. The procedure provides an additional approach for the initial identification of glycolipids by analysing the component oligosaccharides rather than the intact glycolipids.     

Monosaccharide determination of glycoconjugates by reverse-phase high-performance liquid chromatography of their phenylthiocarbamyl derivatives.

A method for the determination of neutral sugars and hexosamines present in glycoconjugates by reverse-phase high-performance liquid chromatography (HPLC) of their phenylthiocarbamyl (PTC) derivatives has been developed. After acid hydrolysis, neutral sugars are converted to glycamines by reaction with ammonium acetate in the presence of sodium cyanoborohydride and are subsequently derivatized with phenylisothiocyanate, while the hexosamines present in the same hydrolysate, after separation on Dowex 50, are treated directly with this reagent. HPLC of the PTC-glycamines of the neutral sugars is performed on Microsorb C18 in an isocratic manner while chromatography of the PTC-hexosamines employs a Pico-Tag column with gradient elution to achieve separation from the PTC-amino acids. The procedure has proven to be highly sensitive, requiring as little as picomole amounts for the chromatographic step; monosaccharide compositions determined on glycoproteins and glycopeptides by this method were found to compare favorably to those previously obtained by other techniques.     

Comparative study of coated and immobilized polysaccharide-based chiral stationary phases and their applicability in the resolution of enantiomers

The enantioselective and chromatographic properties of Chiralpak AD and Chiralpak IA as well as those of Chiralcel OD and Chiralpak IB have been evaluated using a set of 48 compounds that differ in their physical and chemical properties. The impact of the different immobilisation methodologies of the chiral polysaccharide, i.e., coated or immobilized on retention and enantioselectivity was studied. The study on immobilized chiral stationary phases (CSPs) was expanded to also include mobile phases containing mixtures of alkanes and more non-conventional solvents such as ethyl acetate, ethers, acetone and dichloromethane. In this paper we report some of the general trends observed for the 48 racemic compounds with respect to retention, alpha and Rs. Further, the impact of the immobilisation methodology and the choice of the mobile phase on the elution order of the enantiomers is also discussed.     

Determination of tryptophan and its kynurenine pathway metabolites in human serum by high-performance liquid chromatography with simultaneous ultraviolet and fluorimetric detection

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of tryptophan and four metabolites of the kynurenine pathway (kynurenine, 3-hydroxykynurenine, kynurenic acid and 3-hydroxyanthranilic acid) in human serum is described. This new method, which uses both isocratic elution and two on-line connected programmable ultraviolet and spectrofluorimetric detectors, allows the determination of these metabolites, in the physiological ranges, with satisfying specificity and sensitivity within 30 min.