Crystalline degradation products of vancomycin as chiral stationary phase in microcolumn liquid chromatography.

The ability of crystalline degradation products (CDPs) of vancomycin as a chiral stationary phase was reported in a previous study for enantioselective separation of drugs, amino acids and agrochemical toxins by conventional LC column (250 x 4.6 mm). In this work, the potential of CDP of vancomycin for the enantiomeric separation in micro-LC (200 x 1 mm) has been studied. The obtained separation results are better than in our previous study with conventional LC columns. The enantiomers of D,L-phenylalanine, D,L-alanine, methyldopa, atropine and propranolol were used for this evaluation. Experiments have been carried out in a stainless steel tube that was packed with chiral silica particles of 3 and 12 microm diameters. Also, three different ratios of 3 and 12 microm silica particles were used for packing material of chiral columns and the effect on aspect ratio and resolving powers was compared.

Fluorescence and mass spectrometry studies of meta-tetra(hydroxyphenyl)chlorin photoproducts.

The meta-tetra(hydroxyphenyl)chlorin (m-THPC), a second-generation sensitizer used in photodynamic therapy (PDT), is currently under clinical trial. In vivo fluorometry provides direct evidence that photobleaching processes are induced at the tumor site during PDT. Photoproduct formation has thus to be taken into account to fully understand PDT treatment. A preliminary step is to determine the fluorescence characteristics of photoproducts formed in solution. Solutions of m-THPC irradiated at 514 nm have been separated by HPLC using absorption and fluorescence detection. Six main photoproducts have been isolated. According to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) results, five fluorescent photoproducts emitting at 652 nm have been attributed to three mono-, one di- and one tri-hydroxy derivatives (m/z 697, 713 and 729, respectively). Fluorescence characteristics of mono-hydroxy forms were found to be similar to those of m-THPC, whereas fluorescence yields in di- and tri-hydroxy derivatives were very low. Another product, corresponding to a MALDI-TOF MS main signal at m/z 542, showed an absorption spectrum maximum at 522 nm while a weak fluorescence was detected at 480 nm. The loss of the Soret band suggests that this photoproduct results from the opening of the reduced pyrrole ring. The part played by each of these products in the photobleaching phenomenon of m-THPC is discussed.

Direct high-performance liquid chromatographic determination of the enantiomeric purity of levodopa and methyldopa: comparison with pharmacopoeial polarimetric methods.

Chiral high-performance liquid chromatography was employed for determination of the enantiomeric purity of levodopa and methyldopa. The determination of D-DOPA in levodopa was accomplished using a chiral ligand-exchange chromatograpy with an ordinary C18 column and a chiral mobile phase containing N,N-dimethyl-L-phenylalanine and Cu(II) acetate or by means of LC on a teicoplanin column in conjunction with ethanol-water (65:35, v/v). Both methods gave good performance, however, the latter was faster and more convenient and suitable for routine analyses. For the determination of D-methyldopa a LC method based on the use of a teicoplanin column in polar organic mode with methanol-acetic acid-triethylamine (1,000:0.05:0.05, v/v/v) mobile phase was developed. The precision, accuracy, linearity and selectivity were satisfactory. In comparison with pharmacopoeial polarimetric methods (according to the European Pharmacopoeia and the Pharmacopoea Bohemoslovaca), the LC methods proved to be much more sensitive giving detection limits 0.04% of D-DOPA and 0.3% of D-methyldopa.

Separation of aromatic acids, DOPA, and methyl-DOPA by capillary electrophoresis with dendrimers as buffer additives.

Polyamidoamine starburst dendrimers with terminal carboxylate groups are used as a pseudo-stationary phase in electrokinetic chromatography for separating the selected aromatic amino acids phenylalanine, phenylglycine, homophenylalanine, and tyrosine and the catecholamines 3-(3,4-dihydroxyphenyl)alanine and 3-(3,4-dihydroxyphenyl)-2-methylalanine at different pH levels. A significant difference in analyte selectivity is observed between dendrimers of different generations and at different pH levels. At pH 7.0, the utility of the dendrimers for separating these analytes is limited by relatively low selectivity and a noisy baseline. However, good separation and selectivity are obtained with low generation dendrimers (G0.5 and G1.5) at low pH. Strong association of the solute with dendrimers is observed for high generations (G2.5, G3.5, and G5.5).

[Mechanism and kinetic of methyldopa removal by Fenton's reagent].

Methyldopa was selected to be degraded by Fenton's reagent in the experiment. The experimental results showed that it was feasible to the removal of Methyldopa and COD by Fenton's reagent. The mechanism of Methyldopa removal by Fenton's reagent was significantly different according to the Fe2+ :H2O2 ratio. With high ratio of Fe2+ :H2O2 (> or = 2), the Fenton reaction was coagulation enhanced by H2O2. With medium ratio of Fe2+ :H2O2 (= 1), the Fenton reaction could be characterized into two specific systems : oxidation and coagulation. With low ratio of Fe2+ :H2O2 (< or = 0.2), the Fenton reaction was oxidation, including H2O2 oxidation catalyzed by Fe2+ and degradation by a hydrated ferryl-complex Fe(aq)4+. With the analysis of the mechanism according to low ratio of Fe2+ :H2O2 (< or = 0.2), a kinetics model was adopted to describe the reaction, and the rate equation could provide the evidence for the main reaction pathway, which was fitted very well with the experiment data for the relative error below 10%. It was showed that the kinetic models could primary describe the process of the removal of Methyldopa and COD by Fenton's reagent.

Liquid chromatographic determination of methyldopa and methyldopa-thiazide combinations in dosage forms.

A liquid chromatographic (LC) method, using a reverse phase C18 column, an acetic acid-methanol-water mobile phase, and detection at 280 nm, was developed for the determination of methyldopa in tablets and oral suspensions and combinations of methyldopa with hydrochlorothiazide or chlorothiazide in tablets. A mixture of these 3 drugs was resolved in less than 8 min. Detector responses were linear for the following amounts (mg/mL) of drug injected: methyldopa 0.031-0.393, chlorothiazide 0.019-0.114, and hydrochlorothiazide 0.004-0.083. Recoveries from commercial dosage forms ranged from 99.1 to 100.9% for methyldopa, 99.2-100.4% for chlorothiazide, and 100.0-101.2% for hydrochlorothiazide. Replicate injections of methyldopa, chlorothiazide, and hydrochlorothiazide standard preparations alone or in combination gave overall relative standard deviations of less than 1.6% (n = 10). The results for methyldopa tablets by the proposed method were in agreement with those obtained by the USP XX method. The LC method detected as little as 0.6 micrograms 3-O-methylmethyldopa/mL and 0.5 micrograms 4-amino-6-chloro-1,3-benzenedisulfonamide/mL, which are sometimes found as contaminants of methyldopa and thiazides, respectively, and resolved methyldopa from its methyldopa glucose adduct, a substance found in methyldopa oral suspensions.

New procedure for isolation of amino acids based on selective hydrolysis of trimethylsilyl derivatives.

A rapid procedure for the isolation of amino acids from physiological fluids by class separation suitable for gas chromatographic and gas chromatographic-mass spectrometric analysis is described. A physiological fluid such as plasma is adjusted to pH 2 and extracted with diethyl ether to remove organic acids and neutrals. After precipitation of proteins with trichloroacetic acid, the aqueous plasma is dried and derivatized by trimethylsilylation. Organic compounds like sugars and amino acids are rendered soluble in petroleum ether leaving inorganic salts when the soluble layer is transferred. Separation of sugars from amino acids is achieved by taking advantage of the different rates of aqueous hydrolysis of the trimethylsilyl (TMS) derivatives. Mixing the petroleum ether extract with a small volume of water results in two phases. The petroleum ether layer contains TMS-Sugar constituents of plasma and the aqueous layer contains free amino acids and amines. This procedure was used to isolate L-dopa, 3-O-methyldopa and tyrosine from human plasma in a quantitation assay using 18O-labelled amino acids and gas chromatography-mass spectrometry.

Chiral separation of alpha-amino acids by ligand-exchange capillary electrophoresis using N-(2-hydroxy-octyl)-L-4-hydroxyproline as a selector.

The direct chiral resolution of underivatized alpha-amino acids by capillary zone electrophoresis (CZE) based on the principle of ligand exchange is described. An N-(2-hydroxyoctyl)-L-4-hydroxyproline/Cu(II) complex was used as a chiral selector. Besides amino acids containing aromatic residues, the basic amino acid histidine was resolved. Baseline separations were obtained for all amino acids investigated. The influence of selector concentration, electrolyte composition and pH on the resolution was investigated. It was found that there is a correlation between pI of the amino acids and the optimal pH.

Enantiomeric separation of dihydroxyphenylalanine (DOPA), methyldihydroxyphenylalanine (MDOPA) and hydrazinomethyldihydroxyphenylalanine (CDOPA) by using capillary electrophoresis with sulfobutyl ether-beta-cyclodextrin as a chiral selector.

Capillary electrophoresis (CE) was successfully applied to the enantiomer resolution of racemic structurally related compounds, namely dihydroxyphenylalanine (DOPA), methyldihydroxyphenylalanine (MDOPA) and hydrazinomethyldihydroxyphenylalanine (CDOPA). The chiral resolution was performed in an untreated fused-silica capillary by using a phosphate buffer at pH 2.5 or 3.0 supplemented with sulfobutylated beta-cyclodextrin (SBE-CD). Resolution was strongly influenced by the concentration of the chiral selector added to the background electrolyte. In fact, 2-5 mM of SBE-CD enabled the resolution of DOPA and MDOPA enantiomers, while CDOPA optical isomers were resolved by using either 0.5 mM or 6-20 mM of SBE-CD. The latter separation conditions (reversed polarity mode) made it possible to obtain inversion of migration order.