An efficient and stereodivergent synthesis of threo- and erythro-β-methylphenylalanine. Resolution of each racemic pair by semipreparative HPLC

threo and erythro diastereoisomers of the constrained amino acid (βMe)Phe can be obtained separately on a multigram scale through a three-step synthesis from the corresponding Z and E isomers of 2-phenyl-4(α-phenylethylidene)-5(4H)-oxazolone. The 5(4H)-oxazolones are readily available from acetophenone and hippuric acid. The four enantiomerically pure isomers of β-methylphenylalanine, (2R,3R)-(βMe)Phe, (2S,3S)-(βMe)Phe, (2R,3S)-(βMe)Phe and (2S,3R)-(βMe)Phe, have been prepared by HPLC resolution of the racemic precursors methyl threo (or erythro)-2-benzamide-3-phenylbutanoates.     

HPLC and electrophoresis — Competitors or partners

Summary Protein structure analysis is an indispensible tool in modern biological research. However, the isolation of a protein or the separation of peptides in a form suitable for sequence analysis is a considerable technical challenge. The two predominant separation methods in protein biochemistry are chromatography and electrophoresis. In this paper the position, advantaged and disadvantages of both HPLC and electrophoresis for the separation of amino acids, peptides and proteins in protein structure analysis are discussed.     

Investigation of surface structure and composition of in situ annealed Fe–Mn binary alloy samples using RHEED and electron spectroscopy

The formation of oxides at the surface of Fe–1.5%Mn and Fe–0.6%Mn binary alloys was investigated as a function of the conditions of the heat treatments. Both the influence of temperature and the atmosphere under which the experiments were performed were studied. The range of annealing temperatures was adjusted to 800°C. The atmosphere consisted of a mixture of N₂–5%H₂ and traces of water vapour, with different fixed dew points ranging from −10°C to −30°C. The state of the annealed surfaces was determined using in situ analytical devices attached to the annealing reactor in order to avoid surface contamination or the formation of native oxides after the experiments due to contact with air. The structure and composition of the surfaces were determined by reflection high-energy electron diffraction (RHEED) and electron spectroscopy (XPS, AES). Copyright © 2002 John Wiley & Sons, Ltd.     

Aspects of investigating scrambling in the synthesis of porphyrins: different analytical methods

Herein, we discuss the analyses and quantification of the different components in porphyrin mixtures, prepared from p-anisaldehyde, p-tolualdehyde, and 5-(4-bromophenyl)-dipyrromethane with acid catalysis, using NMR and HPLC. The advantages and disadvantages of these analytical methods are emphasized. Due to the similar size of a bromine atom and a methyl group it was possible to grow crystals suitable for X-ray crystallographic studies from a mixture of porphyrins, where the 4-position of the meso-phenyl rings was either substituted with methyl groups or bromine atoms. We also show that X-ray studies are inferior to NMR analysis for determining the components in a porphyrin mixture.     

Separation and characterisation of sphingoceramides by high-performance liquid chromatography-electrospray ionisation mass spectrometry

We developed a simple and reliable analytical method for the quantification and the characterization of ceramides extracted from biological samples by high-performance liquid chromatography (HPLC) coupled to electrospray ionisation tandem mass spectrometry (ESI/MS/MS). The chromatographic separation of analytes was carried out in a RP8 column, eluting with a methanol-water mixture in gradient elution mode. The separated lipids were detected by total ion monitoring and characterised by MS/MS spectra; quantitative analysis was performed by integrating the extracted ion peaks obtained in the negative ion mode. Good repeatability was obtained for retention time (0.3-2%), peak area ratio (A(S)/A(IS), 2-8%), as well as limit of detection (LOD, 5-26 pg) and quantification (LOQ, 13-53 pg). The method was validated for the analysis of N-palmitoyl-D-erythro-sphingosine (Cer16), N-stearoyl-D-erythro-sphingosine (Cer18), N-tetracosanoyl-D-erythro-sphingosine (N24:0, lignoceric ceramide, Cer24:0), and N-tetracos-15'-enoyl-D-erythro-sphingosine (N24:1, nervonic ceramide, Cer24:1), giving good results. Lipid mixtures, extracted from skin and epidermal cells, were analysed for their content of the studied ceramides.     

First synthesis of enantio-uracil dinucleotide, comparison of physicochemical properties of their enantiomers, and separation by chiral column chromatography

Enantio-uracil dinucleotide 5, which consists of two l-uridylic acids and one pyrophosphate, was synthesized for the first time in our laboratory. Benzolyated l-uridine was prepared by a stereoselective glycosylation of silylated uracil with l-1-O-acetyl-2,3,5-tri-O-benzoylribose (l-ABR 7). After deprotection, l-uridine 9 was converted to P¹,P⁴-di(l-uridine 5′-) tetraphosphate tetrasodium salt (l-UP₄U 5) by treatment of l-UMP morpholidate 10c with triethylammonium pyrophosphate (TEA-PPi 11b). Spectral data of synthesized l-UP₄U 5 are given in the references. All spectral data were identical with those of UP₄U 3 except the specific rotation, which showed a positive value compared to UP₄U 3 having a negative value. Furthermore, the separation by chiral column chromatography was investigated.     

Analysis of selected ionic liquid cations by ion exchange chromatography and reversed-phase high performance liquid chromatography

The chromatographic behavior of 8 ionic liquids - 7 homologues of 1-alkyl-3-methylimidazolium and 4-methyl-N-butylpyridinium - has been investigated with a strong cation exchange adsorbent. In particular, the dependence of the retention properties of these solutes on mobile phase composition, pH, and buffer concentration was evaluated with the aim of optimizing and improving the selectivity and retention of solute separation. While using the SCX stationary phase, several interactions occurred with varying strengths, depending on the mobile phase composition. Cation exchange, nonspecific hydrophobic interactions, and adsorption chromatography behavior were observed. Reversed phase chromatography occurred at low concentrations of acetonitrile, electrostatic and adsorption interactions at higher organic modifier concentrations. Elevated buffer concentrations lowered the retention factors without affecting the selectivity of ionic liquids. Obtained results were further compared to the chromatographic behaviour of ionic liquids in the reversed phase system. All analyzed ionic liquids follow reversed-phase behavior while being separated. Much lower selectivity in the range of highly hydrophilic compounds is obtained. This suggests preferred use of ion chromatography for separation and analysis of compounds below 4 carbon atoms in the alkyl side chain.     

High performance liquid chromatographic determination of metoclopramide in plasma and urine and its application to biopharmaceutical investigations

An optimized HPLC method for the quantification of metoclopramide (MCP) in human plasma and urine is described. MCP and internal standard are extracted from alkalinized substrate into diethyl ether and back-extracted into dilute acid. The analytes are separated with a ternary mobile phase at cyanopropyl-silica and detected at 312 nm (UV detection). The lower limit of quantification is 0.5 ng/ml in plasma and 50 ng/ml in urine. Optimization of extraction, chromatography, and detection is discussed. The method is selective to numerous common drug substances with excellent accuracy and precision data. After validation, the method is applied to the samples of a pharmacokinetic study. Pharmacokinetic parameters indicate the need for a sophisticated method as tool for optimization of metoclopramide formulations.     

Electrochemical vapor generation of selenium species after online photolysis and reduction by UV-irradiation under nano TiO<Subscript>2</Subscript> photocatalysis and its application to selenium speciation by HPLC coupled with atomic fluorescence spectrometry

An online UV photolysis and UV/TiO₂ photocatalysis reduction device (UV–UV/TiO₂ PCRD) and an electrochemical vapor generation (ECVG) cell have been used for the first time as an interface between high-performance liquid chromatography (HPLC) and atomic fluorescence spectrometry (AFS) for selenium speciation. The newly designed ECVG cell of approximately 115 L dead volume consists of a carbon fiber cathode and a platinum loop anode; the atomic hydrogen generated on the cathode was used to reduce selenium to vapor species for AFS determination. The noise was greatly reduced compared with that obtained by use of the UV–UV/TiO₂ PCRD–KBH₄–acid interface. The detection limits obtained for seleno-DL-cystine (SeCys), selenite (Se^IV), seleno-DL-methionine (SeMet), and selenate (Se^VI) were 2.1, 2.9, 4.3, and 3.5 ng mL^–1, respectively. The proposed method was successfully applied to the speciation of selenium in water-soluble extracts of garlic shoots cultured with different selenium species. The results obtained suggested that UV–UV/TiO₂ PCRD–ECVG should be an effective interface between HPLC and AFS for the speciation of elements amenable to vapor generation, and is superior to methods involving KBH₄.     

Quantitative determination of sotolon, maltol and free furaneol in wine by solid-phase extraction and gas chromatography-ion-trap mass spectrometry

A method for the analytical determination of sotolon [4,5-dimethyl-3-hydroxy-2(5H)-furanone], maltol [3-hydroxy-2-methyl-4H-pyran-4-one] and free furaneol [2,5-dimethyl-4-hydroxy-3(2H)-furanone] in wine has been developed. The analytes are extracted from 50 ml of wine in a solid-phase extraction cartridge filled with 800 mg of LiChrolut EN resins. Interferences are removed with 15 ml of a pentane-dichloromethane (20:1) solution, and analytes are recovered with 6 ml of dichloromethane. The extract is concentrated up to 0.1 ml and analyzed by GC-ion trap MS. Maltol and sotolon were determined by selected ion storage of ions in the m/z ranges 120-153 and 79-95, using the ions m/z 126 and 83 for quantitation, respectively. Furaneol was determined by non-resonant fragmentation of the m/z 128 mother ion and subsequent analysis of the m/z 81 ion. The detection limits of the method are in all cases between 0.5 and 1 microg l(-1), well below the olfactory thresholds of the compounds. The precision of the method is in the 4-5% range for levels in wine around 20 microg l(-1). Linearity holds at least up to 400 microg l(-1), and is satisfactory in all cases. The recoveries of maltol and sotolon are constant (70 and 64%, respectively) and do not depend on the type of wine. On the contrary, in the case of furaneol, red wines show constant and high recoveries (97%), while the recoveries on white wines range between 30 and 80%. Different experiments showed that this behavior is probably due to the existence of complexes formed between furaneol and sulphur dioxide or catechols. Sensory experiments confirmed that the complexed forms found in white wines are not perceived by orthonasal olfaction, and that the furaneol determined by the method can be considered as the free and odor-active fraction.