High-performance liquid chromatographic determination of hippuric acid in human urine

A method is described for the determination of urinary hippuric acid by high-performance liquid chromatography. The method used ethyl acetate extraction for partial clean up of the urine. The separation was carried out on a reversed-phase column using 20% methanol in 0.01 M aqueous potassium phosphate containing 0.5% acetic acid as a mobile phase. The column effluent was monitored with a UV detector at 254 nm. Hippuric acid was separated from other normal urine constituents in less than 10 min. Metabolites of xylene and styrene did not interfere with the assay. Analytical recoveries from urine were excellent and peak height and concentration were linearly related.     

High-performance liquid chromatographic separation of cobalamins

Physiological cobalamins were separated by means of high-performance liquid chromatography (HPLC). Optimal conditions for elution of methylcobalamin, adenosylcobalamin, hydroxycobalamin and cyanocobalamin were determined. Excellent separation and resolution of these physiological cobalamins by HPLC were achieved. In addition, several cobalamin analogues were also studied and shown to be separable from the physiological forms. HPLC provides a rapid, sensitive, reproducible means of characterizing physiological cobalamins.     

High-performance liquid chromatographic separation of stereoisomers of N-phthaloyl-protected amino acids and dipeptidomimetics

The stereoisomers of N-phthaloyl-protected amino acids and dipeptidomimetics were separated on macrocyclic glycopeptide and cellulose-based chiral stationary phases (CSPs) in the RP and polar-ionic modes. The effects of the organic modifier, the mobile phase composition, and the pH on the separations were investigated. Optimization of these separations was achieved through variation of the mobile-phase additive combinations. The elution sequence was determined for some of the samples. A practical application for the monitoring of the reaction conditions for N-phthaloylation of (S)-Phe was demonstrated.     

High-performance liquid chromatographic analysis of hippuric acid in human blood plasma

A method has been developed for the isocratic high-performance liquid chromatographic analysis of hippuric acid in human blood plasma. After the addition of an internal standard (3-methoxysalicylic acid), plasma samples (1 ml) were made alkaline and extracted stepwise with methylene chloride and ethyl acetate. The detection limit was 50 pmol of hippuric acid per ml of plasma. The concentrations of hippuric acid in plasma from house painters (n = 8), with long-term exposure to solvent vapours from alkyd paints, were in the range 1-21 nmol/mol (median 11 nmol/ml). These values were statistically significantly higher than those for controls (n = 9): 2-8 nmol/ml (median 3 nmol/ml).     

High-performance liquid chromatographic separation of inositol phosphate isomers employing a reversed-phase column and a micellar mobile phase

Surfactants have been employed in high-performance liquid chromatography (HPLC) for the separation of ionic and non-ionic compounds. We have developed a method employing a reversed-phase column and a mobile phase containing a surfactant, hexadecyltrimethylammonium hydroxide (HDTMA+OH-), for the separation of several inositol phosphate positional isomers. Various parameters were studied for their effect on the chromatographic capacity factor (k'). They included the concentration of HDTMA+OH-, the pH of the bulk micellar suspension and the addition of inorganic salts to the mobile phase. Resolution of the inositol monophosphates was controlled by a mixed mechanism, where the predominant elements were electrostatic forces and the formation of micelles. The elution of the inositol polyphosphate isomers was obtained by increasing the amount of a non-polar solvent, in agreement with an ion-pairing process. This method represents an alternative to ion-exchange HPLC. If offers a practical advantage when detection of radiolabeled samples by in-line radioactive flow detectors is required, because low-quenching solvents with good miscibility with scintillant fluids are employed. The analysis of various chromatographic conditions, the system reproducibility and its application to the analysis of biological samples are described.     

High-performance liquid chromatographic separation of enantiomeric amines

Summary Separations of twelve different racemic amines were studied by high-performance liquid chromatography (HPLC) in several column-solvent combinations. Relative retentions, which show some dependence upon the substitution at the asymmetric centers, are reported for the amines which were examined as the (+)-10-camphorsulfonamides.     

High-performance liquid chromatographic enantioseparation of beta-amino acids

Direct and indirect high-performance liquid chromatographic methods were developed for the enantioseparation of beta-amino acids (beta-substituted-beta-alanines). Direct separation involved the application of chiral columns: Crownpak CR(+), Chirobiotic T and Chirobiotic R. Indirect separation was based on precolumn derivatization with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate or N-alpha-(2,4-dinitro-5-fluorophenyl)-L-alanineamide (Marfey's reagent), with subsequent separation on an achiral column. The chromatographic conditions were varied to achieve optimum separation.     

High-performance liquid chromatographic separation of bile acids and bile alcohols diastereoisomeric at C-25

The high-performance liquid chromatographic separation of the 25R and 25S diastereoisomers of the bile alcohols 5 beta-cholestane-3 alpha,7 alpha,26-triol and 5 beta-cholestane-3 alpha,7 alpha, 12 alpha, 26-tetrol and the bile acids, 3 alpha,7 alpha-dihydroxy-5 beta-cholestane-26-oic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestane-26-oic acid is described. A Radial-Pak microBondapak C18 reversed-phase cartridge was used for the separations and elutions were carried out with acetonitrile-water-methanol-acetic acid mixtures. All eight diastereoisomeric compounds showed baseline separation when up to 200 micrograms of the isomeric mixtures were injected into the column and the method can be used for isolation of pure diastereoisomers of these bile acids and bile alcohols.     

High-performance liquid chromatographic separation of novel atropic alpha,alpha-disubstituted-beta-amino acids, either on different beta-cyclodextrin-bonded phases or as their 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide derivatives.

The high-performance liquid chromatographic enantioresolution of free and N- and/or C-protected derivatives of (R,S)-2',1':1,2;1",2":3,4-dinaphthcyclohepta-1,3-diene-6-aminometh yl-6- carboxylic acid (beta 2-Bin) by direct and indirect methods is reported. The direct separation was carried out on native and different derivatized beta-cyclodextrin-bonded phases. The indirect resolution was achieved by applying pre-column derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide. The effects of different parameters such as the mobile phase composition and the structures of the compounds on the enantiomeric resolution are discussed.     

High-performance liquid chromatographic separation of polyolefin antioxidants and light-stabilisers

Summary A scheme was devised for the identification of 22 common antioxidants and light-stabilisers in polyolefins. The separation of these stabilisers was performed by isocratic reversed phase high-performance liquid chromatography on a RP-18 column. Three different separation conditions have been used: the mobile phase composition was 100% acetonitrile (MeCN), 90/10 meCN/H₂O and 80/20 MeCN/H₂O. The UV₂₅₄/UV₂₈₀ ratio and the elution time of each stabiliser were determined for these three mobile phase compositions. The values of UV₂₅₄/UV₂₈₀ ratios may be used together with the retention time values for the identification of unknown stabilisers in polyolefin samples.     

High-performance liquid chromatographic separation of subcomponents of antimycin A

Using a reversed-phase high-performance liquid chromatographic (HPLC) technique, a mixture of antimycins A was separated into eight hitherto unreported subcomponents, A1a, A1b, A2a, A2b, A3a, A3b, A4a, and A4b. Although a base-line resolution of the known four major antimycins A1, A2, A3, and A4 was readily achieved with mobile phases containing acetate buffers, the separation of the new antibiotic subcomponents was highly sensitive to variation in mobile phase conditions. The type and composition of organic modifers, the nature of buffer salts, and the concentration of added electrolytes had profound effects on capacity factors, separation factors, and peak resolution values. Of the numerous chromatographic systems examined, a mobile phase consisting of methanol-water (70:30) and 0.005 M tetrabutylammonium phosphate at pH 3.0 yielded the most satisfactory results for the separation of the subcomponents. Reversed-phase gradient HPLC separation of the dansylated or methylated antibiotic compounds produced superior chromatographic characteristics and the presence of added electrolytes was not a critical factor for achieving separation. Differences in the chromatographic outcome between homologous and structural isomers were interpreted based on a differential solvophobic interaction rationale. Preparative reversed-phase HPLC under optimal conditions enabled isolation of pure samples of the methylated antimycin subcomponents for use in structural studies.     

High-performance liquid chromatographic separation of chiral metallocenic ketones and alcohols

The enantiomers of chiral (arene)tricarbonylchromium ketones and alcohols were separated by high-performance liquid chromatography with a Chiralcel OD column. The absolute configuration of the ketones was assigned on the basis of the sign of optical rotation determined with an on-line detector.     

High-performance liquid chromatographic separation and indirect fluorescence detection of thiols

A fluorescent post-column reaction detection scheme has been devised for selective determination of thiols. The post-column reagent is 40 microM Cd2+ and 100 microM 8-hydroxyquinoline-5-sulfonic acid (HQS) in non-complexing buffer at pH 10. HQS complexes Cd2+ to form a fluorescent product. Thiols in the HPLC effluent compete for complexation of the Cd2+, resulting in a decrease in the fluorescence response. Detection limits of 0.2 microM (0.04 ppm) are achieved for cysteine, homocysteine and glutathione in a 5 min separation. Recoveries from spiked synthetic urine samples are 87-120%.