An efficient method for the determination of enantiomeric purity of racemic amino acids using micellar chromatography, a green approach.

A simple, sensitive and green micellar liquid chromatographic method (RP-HPLC) was developed for enantioseparation of four racemic amino acids, namely, (RS)-selenomethionine, (RS)-methionine, (RS)-cysteine and (RS)-penicillamine. An aqueous solution of sodium dodecyl sulfate and Brij-35 was prepared and used as mobile phase for HPLC analysis. Activated esters of (S)-ibuprofen, (S)-ketoprofen and (S)-levofloxacin were synthesized by reacting them with N-hydroxybenzotriazole. These esters were characterized by UV, IR, 1 HNMR, HRMS and elemental analysis. These chiral reagents (activated esters) were used for the synthesis of diastereomeric derivatives of the chosen amino acids. The diastereomeric derivatives were separated on a C18 column by micellar liquid chromatography. Chromatographic conditions were optimized by varying concentration of surfactant in aqueous solution, and by varying the concentration and pH of the buffer. The green assessment score was calculated for the developed method (78, an excellent green method score). In addition, density functional theory calculations were performed, using Gaussian 09 rev. A.02 and hybrid density functional B3LYP with a 6-31G* basis set program, in order to develop lowest energy optimized structures of diastereomeric derivatives. The method was validated according to International Conference on Harmonization guidelines and the retention factor (k), selectivity factor (α), resolution factor (RS ) and limit of detection (0.295 ng ml-1 ) and limit of quantification (0.896 ng ml-1 ) were calculated.

Simultaneous determination of ascorbic acid, aminothiols, and methionine in biological matrices using ion-pairing RP-HPLC coupled with electrochemical detector.

A novel highly sensitive ion-pairing reversed-phase high performance liquid-chromatography/electrochemical detection method for simultaneous determination of l-ascorbic acid, aminothiols, and methionine in biological matrices was developed, optimized, and validated. Reduced forms of the analytes were extracted from the sample matrices with 10% meta-phosphoric acid solution((aqueous)). To determine the total vitamin C, the total aminothiols, and the total methionine, samples were treated with tris(2-carboxyethyl)phosphine solution in 0.05% trifluoroacetic acid solution((aqueous)) subsequent to deproteination to reduce the oxidized forms of these compounds. Various analytes were separated on a C(18) (250 × 4.6 mm, 5 µm) analytical column using methanol-0.05% trifluoroacetic acid solution((aqueous)) (05/95, v/v), containing 0.1mM 1-octane sulphonic acid as the ion-pairing agent) as the isocratic mobile phase pumped at a flow rate of 1.5 mL min(-1) at room temperature. The column eluents were monitored at a voltage of 0.85 V. These analytes were efficiently resolved in less than 20 min using n-acetyl cysteine as the internal standard. The present method was specific for the analysis of these analytes and demonstrated acceptable values for linearity (r(2)>0.999 in the range of 0.2-10,000 ng mL(-1) for all the analytes), recovery (>96%), precision (%RSD ≤ 2.0), and sensitivity (on column limit of detection: 250-400 fg and limit of quantification: 0.8-1.25 pg), indicating that the proposed method could be efficiently used for determination of these analytes in the context of clinical research.

Isolation and characterization of new Metschnikowia pulcherrima strains as producers of the antimicrobial pigment pulcherrimin.

Metschnikowia pulcherrima is a highly effective biocontrol yeast due to its pigment pulcherrimin that accumulates in the cells and in the growth medium. Three different strains of M. pulcherrima were isolated from local grapes. The yeast isolates were characterized on the basis of their biochemical, physiological and ITS1-5.8 s rDNA-ITS2 region. Based on the obtained results, the M. pulcherrima isolates were identified as new strains of M. pulcherrima. Strong antagonistic activities of the M. pulcherrima strains on the human pathogens Proteus vulgaris, Escherichia coli, Candida albicans, Candida parapsilosis, Candida krusei, and Trichosporon mucoides were determined. In addition, antagonistic effects of these M. pulcherrima strains were also tested against Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Trichoderma spp., Paecilomyces spp., and Bipolaris spp. and it was shown that the three different strains of M. pulcherrima also have an antagonistic effect on the growth of these fungal species at different extents. This study showed that all three strains of M. pulcherrima produce the same amount of the pigment pulcherrimin, but their antimicrobial activities on different microorganisms show important variations.

Chiral separation of natural and unnatural amino acid derivatives by micro-HPLC on a Ristocetin A stationary phase.

This paper deals with the chiral separation of Fmoc- and Z-derivatives of natural and unnatural sulfur containing amino acids by micro-HPLC. The separations were carried out in microbore columns packed with a new material based on Ristocetin A bonded to 3.5 microm silica gel. The columns were run in the normal phase, polar organic mode as well as in the reversed phase mode, whereby best results were obtained with the reversed-phase mode using mixtures of triethylamine acetate (TEAA) buffer and methanol as mobile phases.

Novel enantioselective strong cation exchangers based on sulfodipeptide selectors: evaluation for enantiomer separation of chiral bases by nonaqueous capillary electrochromatography.

Strong cation exchange (SCX)-type chiral stationary phases (CSPs) based on beta-amino sulfonic acid-terminated dipeptide derivatives as chiral selectors, immobilized on thiol-modified silica particles (3.5 microm), were synthesized and applied to enantiomer separations of chiral bases by nonaqueous capillary electrochromatography (CEC). The effect of structural variations of the sulfodipeptide selectors on the separation factors alpha was investigated. These studies included variation of the acid-terminal amino sulfonic acid residue, variation of the configurations, i.e., comparison of the diastereomeric (S,S)- and (R,S)-configurations of the sulfodipeptides, and finally comparison of sulfodipeptide selectors with corresponding beta-amino sulfonic acid analogs. In general, the capillary columns (100 microm ID) packed with the new SCX-type CSPs showed enantioselectivity for an elaborated set of chiral basic drugs in CEC acting by an enantioselective cation-exchange retention mechanism. N-[N-(4-Allyloxy-3,5-dichlorobenzoyl)-leucyl]-2-amino-3,3-dimethylbutane sulfonic acid, in particular with (R,S)-configuration, turned out to be a more effective SCX-type selector than a more rigid analog based on N-[N-(4-Allyloxy-3,5-dichlorobenzoyl)-leucyl]-2-pyrrolidinemethane sulfonic acid. Both of the former diastereomers were capable to baseline-resolve the enantiomers of ca. 40% of the tested basic chiral solutes including sympathomimetics and beta-blockers, while for the latter SCX-type CSPs only 10-20% of the selected solutes afforded resolutions > 1.5.

High-throughput capillary electrophoretic method for determination of total aminothiols in plasma and urine.

Increased interest in the analysis of aminothiols in body fluids during the last years results in a request for high-throughput analytical methods for their determination. We report here a novel, high-throughput method for the determination of total concentrations of biogenous aminothiols - homocysteine, cysteine, glutathione, cysteinylglycine, gamma-glutamylcysteine, and of penicilamine, mercaptopropionylglycine, and cysteamine, three compounds used to treat disorders of aminothiol metabolism in plasma and urine. Samples were reduced with tris(carboxyethyl)phosphine and labeled with 5-(bromomethyl)fluorescein. Capillary electrophoretic separations were performed in 60 mmol/L borate - 15 mmol/L sodium dodecyl sulfate - 2-amino-2-methyl-1-propanol, pH 10.0, with laser-induced fluorescence detection. Analysis time was less than 2 min. The assay is linear (r > 0.999) up to 500 micromol/L. Reproducibilities of migration times (coefficient of variation, CV) were < 0.5%. Interassay repeatabilities (CV, n = 10) were 5.08% and 6.09% for 5 micromol/L addition of homocysteine and 0.60% and 3.78% for 100 micromol/L addition of cysteine in plasma and urine, respectively. Recovery values were within 94-106% and sensitivity was better than 0.19 micromol/L for all analyzed compounds. Results agreed well with a standard high-performance liquid chromatography (HPLC) method. The diagnostic usefulness of the method has been proven on 79 samples of cystinuric patients and 12 samples of homocystinuric patients. We report here a novel method for the determination of aminothiols in body fluids by capillary electrophoresis (CE). Determination is fast and sensitive enough for diagnostic purposes.

Isolation and structure of pulcherrimine, a novel bitter-tasting amino acid, from the sea urchin (Hemicentrotus pulcherrimus) ovaries.

A novel sulfur-containing amino acid, pulcherrimine, has been isolated as a bitter principle from ovaries of the sea urchin Hemicentrotus pulcherrimus. The structure was elucidated as 4-(2'-carboxy-2'-hydroxy-ethylthio)-2-piperidinecarboxylic acid by spectroscopic and chemical methods. Absolute stereochemistry was determined by NOE experiments and chiral HPLC analysis. Pulcherrimine exhibited bitterness with a threshold value of 0.306 mM.

Identification of an oxidation product of aminoethylcysteine ketimine dimer.

In continuation of our previous work dedicated to the detection of the oxidation products of aminoethylcysteine ketimine dimer by oxygen reactive species, we give here data for the identification of the alpha, beta unsaturated sulfoxide as the main product of interaction of the dimer with H2O2. Identification has been done on the basis of mass spectrometry and NMR analyses of the product isolated by preparative chromatography.

Separation of selenium analogues of sulphur-containing amino acids by high-performance liquid chromatography and high-resolution gas chromatography.

The historically conditioned adaptation of living organisms to chemically corresponding elements is influenced in nature by anthropogenic activities in many regions, the selenium-sulphur pair being one example of such a case. The separation of selenomethionine, selenoethionine and selenocystine was studied by HPLC and high-resolution GC. Ion-exchange chromatography followed by temperature-programmed GC gives the possibility of the analytical separation of trace amounts of selenomethionine in a complex mixture of common amino acids. Diastereoisomers of selenocystine were identified by HPLC in the AccQ-Tag mode.

L-1-N-methyl-4-mercaptohistidine disulfide, a potential endogenous regulator in the redox control of chloroplast coupling factor 1 in Dunaliella.

A water-soluble, highly polar, heat-stable, small molecule has been isolated from cell-free extracts of the halotolerant green alga Dunaliella salina. This compound, soluble inhibitory factor (SIF), when added to in vivo light-activated, thylakoid membrane-bound preparations of the D. salina coupling factor 1 (CF1), causes a rapid inactivation of the ATPase activity. SIF must be in its oxidized form to inactivate the CF1 ATPase and probably functions by oxidizing the reduced form of the light-activated enzyme. SIF has been purified to homogeneity and characterized by UV-visible and IR absorption spectroscopy, 1H and 13C NMR spectroscopy, and mass spectrometry. SIF has five different kinds of nonexchangeable protons and seven different kinds of carbon atoms. Three of the carbon atoms and one proton are part of a heterocyclic (imidazole) ring. One carbon atom is a carbonyl (carboxylic acid). One carbon atom and three protons form a methyl group attached to the aromatic ring. One carbon atom and two protons are a methylene group, and one carbon atom (an alpha-amino carbon) is attached to a single proton. In addition, in its reduced form, SIF contains a thiol group attached to the heterocyclic ring. From high resolution mass spectrometry, the molecular weight of SIF was determined to be 401 (M + H+) and is consistent with the composition being C14H21N6O4S2. The UV absorption of SIF shows a large increase at 240 nm upon reduction. An effective difference extinction coefficient for this absorbance change has been calculated to be 6.84 meq/cm. A comparison of SIF with the oxidized form of ovothiol A (1-N-methyl-4-mercaptohistidine disulfide) shows the two compounds to be identical in all respects. In addition, ovothiol A disulfide is as effective as SIF in inhibiting the light-triggered, CF1 ATPase activity. It is concluded, therefore, that SIF and L-1-N-methyl-4-mercaptohistidine disulfide are identical.

High-performance liquid chromatography of 2-mercaptopropionylglycine and its metabolite 2-mercaptopropionic acid in plasma and urine after treatment with thiopronine.

2-Mercaptopropionic acid has been identified as a normal metabolite of 2-mercaptopropionylglycine (thiopronine) when this drug was given to humans and dogs. A high-performance liquid chromatographic method was developed to resolve the derivatives of these two thiols and thus enable simultaneous determination of the two compounds in plasma and urine.

Ovothiols, a family of redox-active mercaptohistidine compounds from marine invertebrate eggs.

We have previously reported a novel thiol compound, 1-methyl-N alpha,N alpha-dimethyl-4-mercaptohistidine, or ovothiol, present at high concentration in the eggs of the sea urchin Strongylocentrotus purpuratus [Turner, E., Klevit, R., Hopkins, P. B., & Shapiro, B. M. (1986) J. Biol. Chem. 261, 13056-13063]. Here we report two related compounds, 1-methyl-N alpha-methyl-4-mercaptohistidine, or ovothiol B, from the scallop Chlamys hastata, and 1-methyl-4-mercaptohistidine, or ovothiol A, from the starfish Evasterias troschelii. These two compounds, as well as the S. purpuratus compound now designated ovothiol C, were isolated from eggs or ovarian tissue by S-carboxymethylation with [3H]iodoacetic acid, ion-exchange chromatography and ion-pairing high-pressure liquid chromatography. The structures of S-(carboxymethyl)ovothiols A and B were determined by 1H NMR, and that of ovothiol A was confirmed by comparison with authentic methylhistidine samples after desulfuration with Raney nickel. In the ovary of each species, the predominant methylation form of ovothiol accounts for at least 80% of the total 4-mercaptohistidine. The ovothiol concentration of the ovary far exceeds that of the testis or somatic tissues. The ovothiol C content of unfertilized S. purpuratus eggs is 1.14 mumol/10(6) eggs, equivalent to approximately 4.3 mM average concentration; the glutathione (GSH + GSSG) content is 0.9 mumol/10(6) eggs. In this species, high ovothiol levels persisted for the first 2 weeks of embryonic development. Ovothiol and glutathione account for virtually all of the trichloroacetic acid soluble-SH groups in the egg; these results are compared to several previous studies.(ABSTRACT TRUNCATED AT 250 WORDS)