Gradient elution capillary electrochromatography and hyphenation with nuclear magnetic resonance.

Coupling of gradient capillary electrochromatography (gradient CEC) and capillary zone electrophoresis (CZE) with nuclear magnetic resonance spectroscopy (NMR) was performed using a recently developed capillary NMR interface. This technique was applied for the analysis of pharmaceuticals and food. An analgesic was investigated using isocratic and gradient continuous-flow CEC-NMR. Comparison of the results demonstrated the superiority of gradient CEC over isocratic CEC. Aspartame and caffeine, both ingredients of soft beverages, were separated and analyzed by continuous flow CZE-NMR. The order of elution could be reversed by altering the pH. This reversal led to an increased sample concentration in the NMR detection cell, thus allowing the acquisition of a totally correlated spectroscopy (TOCSY) two-dimensional (2-D) spectrum of the synthetic peptide aspartame.

Peer Reviewed: On-Line Coupling of Capillary Separation Techniques with 1H NMR.

Improved sensitivity, deuterated solvents, and less sample make this approach feasible for many applications.

Directly coupled CZE-NMR and CEC-NMR spectroscopy for metabolite analysis: paracetamol metabolites in human urine.

Direct coupling of NMR spectroscopic detection with both capillary zone electrophoresis (CZE) and capillary electrochromatography (CEC) was applied to the separation of metabolites of the drug paracetamol in an extract of human urine. Continuous-flow CZE-NMR and CEC-NMR allowed the detection of the major metabolites, the glucuronide and sulfate conjugates of the drug and the endogenous material hippurate. Identification of these substances was achieved by examination of individual rows of the NMR chromatogram and this also gave estimates of the detection limits. For CEC-NMR, spectra were also obtained in the stopped-flow mode including a two-dimensional TOCSY NMR experiment which afforded confirmatory evidence for paracetamol glucuronide. Characterisation of drug metabolites using NMR spectroscopy is therefore possible with nanolitre sample volumes.

On-line coupling of capillary electrochromatography, capillary electrophoresis, and capillary HPLC with nuclear magnetic resonance spectroscopy.

A novel capillary NMR coupling configuration, which offers the possibility of combining capillary zone electrophoresis (CZE), capillary HPLC (CHPLC), and for the first time capillary electrochromatography (CEC) with nuclear magnetic resonance (NMR), has been developed. The hyphenated technique has a great potential for the analysis of chemical, pharmaceutical, biological, and environmental samples. The versatile system allows facile changes between these three different separation methods. A special NMR capillary containing an enlarged detection cell suitable for on-line NMR detection and measurements under high voltage has been designed. The acquisition of 1D and 2D NMR spectra in stopped-flow experiments is also possible. CHPLC NMR has been performed with samples of hop bitter acids. The identification and structure elucidation of humulones and isohumulones by on-line and stopped-flow spectra has been demonstrated. The suitability of the configuration for electrophoretic methods has been investigated by the application of CZE and CEC NMR to model systems.

Dihydrophencomycin methyl ester, a new phenazine derivative from a marine Streptomycete.

The novel 5,10-dihydrophencomycin methyl ester (4) and the known microbial metabolites (2-hydroxyphenyl)-acetamide (1), menaquinone MK9 (II, III, VIII, IX-H8) (2), and phencomycin (3a) were isolated from an unidentified marine Streptomyces sp. and the structures were elucidated by NMR methods. Compound 4 shows weak antibiotic activity against Escherichia coli and Bacillus subtilis.

Morulin Pm: a modified polypeptide containing TOPA and 6-bromotryptophan from the morula cells of the ascidian, Phallusia mammillata.

A novel polypeptide containing the unusual posttranslationally modified amino acids L-3,4,5-trihydroxyphenylalanine (TOPA) and L-6-bromotryptophan (6-BrW) has been isolated from the morula cells of the vanadium-accumulating ascidian, Phallusia mammillata. The polypeptide, designated Morulin Pm, has a molecular weight of 3825 +/- 0.6 and has a simple amino acid composition consisting mainly of TOPA and 6-BrW as well as Ser, Leu, Phe, and Ala. To our knowledge, this is the first reported example of multiple sites of brominated tryptophan in a polypeptide of this size. Edman degradation revealed the N-terminal sequence to be BrW-Leu-Phe-BrW before sequencing was blocked. While the N-terminal tripeptide could be isolated from chymotrypsin digests of Morulin Pm, the rest of the polypeptide resisted further cleavage by the proteases, a feature common among this class of peptides. However, unlike other ascidian blood cell peptides examined to date, microheterogeneity was minimal. For the first time a detailed NMR investigation could be undertaken on a member of this class of polypeptides. In addition to signals assignable to the constituent amino acids by extensive 2D experiments, resonances were present both in the 13C and 1H spectra not typical of a simple linear peptide. Two proton resonances were identified with a cross peak in the correlation spectrum strongly indicative of a C-terminal decarboxy-delta 2,3-unsaturated TOPA residue as observed in certain tunichromes and clionamide. Chemical degradation experiments were undertaken in an effort to produce identifiable fragments to which these signals could be assigned, including full and partial acid hydrolysis and tryptophan-targeted BNPS-skatole treatment. However, the nature of the modification remains unknown. Possible structures for the modification, which may represent the source of the difficulties encountered in the structural elucidation of this and related peptides, are assessed. Conjecture is made as to the biological relevance of Morulin Pm, based on its localization and chemical characteristics.