Noriyuki Nimura

o-Phthalaldehyde—N-acetyl-L-cysteine as a chiral derivatization reagent for liquid chromatographic optical resolution of amino acid ernantiomers and its application to conventional amino acid analysis

Abstract A useful fluorescence derivatization reagent, o -phthalaldehyde-N-acetyl- l -cysteine, has been developed for the optical resolution of enantiomeric amino acids and for conventional amino acid analysis. Amino acids rapidly reacted with o -phthalaldehyde in the presence of N-acetyl- L -cysteine to give intensely fluorescent products, which were diastereoisomers. When this reaction was used in the precolumn derivatization of amino acid enantiomers, their diastereomeric derivatives were efficiently resolved on a reversed-phase column. Simultaneous analysis of common protein amino acid enantiomers was achieved by gradient elution within 70 min. In addition, the reagent was applied to the post-column derivatization of protein amino acids, combined with hypochlorite oxidation for the detection of amino acids, such as proline. Seventeen protein amino acids were sufficiently separated in 17 min by ion-pair chromatography with sodium dodecyl sulphate as a counter-ion, and they were determined at the same level of sensitivity by the above post-column fluorimetric detection system. Applications of both the precolumn and the post-column derivatization methods with the new reagent to hydrolysed protein samples are also described.

Fluorescent Labeling of Fatty Acids with 9-Anthryldiazomethane (ADAM) for High Performance Liquid Chromatography

Abstract A novel method for rapid and simple fluorescent labeling of carboxylic acids with 9-anthryldiazomethane (ADAM) for HPLC is described. This reagent is stable in solution and highly reactive with carboxylic acids. Esterification proceeds at room temperature without catalyst. The labeled compounds are separated using a reversed-phase system and determined with a flow-through flurometer. Picomole level of fatty acids are determined by this method.

Reversed-phase liquid chromatographic resolution of amino acid enantiomers by derivatization with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate

Abstract A novel method for reversed-phase high-performance liquid chromatographic resolution of amino acid enantiomers by the formation of diastereomers using a new chiral reagent, 2,3,46-tetra-O-acetyl-β- d -glucopyranosyl isothiocyanate (GITC), is described. GITC reacts readily with enantiomeric amino acids at room temperature and the reaction mixture can directly be injected into the chromatograph. The derivatives were detected spectrophotometrically at 250 nm. Complete resolutions were observed for all enantiomers examined o a reversed-phase column eluted with aqueous methanol.

Reversed-phase high-performance liquid chromatographic resolution of non-esterified enantiomeric amino acids by derivatization with 2,3,4,6-tetra-o-acetyl-β-d-glucopyranosyl isothiocyanate and 2,3,4-tri-o-acetyl-α-d-arabinopyranosyl isothiocyanate

Abstract Novel methods for reversed-phase high-performance liquid chromatographic resolution of non-esterified amino acid enantiomers by the formation of diastereomers using two chiral reagents, 2,3,4,6-tetra-O-acetyl-β d -glucopyranosyl siothiocyanate and 2,3,4,-tri-O-acetyl-α- d -arabinopyranosyl isothiocyanate, are described. These compounds react readily with enantiomeric free amino acids at room temperature and the reaction mixture can be injected directly into the chromatograph. The separation of the enantiomers was monitored spectrophotometrically at 250 nm. Complete resolutions were observed for all enantiomers examined on a reversed-phase column eluted with methanol-10 m M potassium phosphate (pH 2.8).

Postcolumn fluorometric detection system for liquid chromatographic analysis of amino and imino acids using o-phthalaldehyde/N-acetyl-L-cysteine reagent

A high-performance liquid chromatographic system for the determination of amino acid and imino acid was developed using a two-step reaction with sodium hypochlorite and o-phthalaldehyde/N-acetyl-L-cysteine (OPTA/AcCys) reagent. This reagent improved the sensitivity in the analysis of proline presumably because the fluorophore is more stable to hypochlorite which has been used for the oxidative cleavage of the imino linkage. The use of OPTA/AcCys facilitated the detection of imino acids at the same concentration level as that of amino acids. The detection limit for all the amino and imino acids was a few picomoles. This detection system, together with cation-exchange chromatographic separation, was applied to the determination of amino and imino acids in biological samples.

Amino acid analysis by reversed-phase high-performance liquid chromatography : Automatic pre-column derivatization with activated carbamate reagent

Succinimido alpha-naphthylcarbamate, an activated carbamate reagent, facilitated the simple and rapid pre-column derivatization of amino acids for fluorimetric detection. The method is based on the formation of naphthylcarbamyl derivatives of amino acids. The carbamylation of amino acids is completed within 1 min at room temperature, and the reaction mixture can be injected directly into a liquid chromatograph equipped with an octadecylsilyl reversed-phase column. The effluent stream is monitored fluorimetrically at 370 nm excited at 290 nm at the sub-picomole level. Naphthylcarbamyl derivatives of common protein amino acids were separated within 30 min by gradient elution with aqueous sodium acetate and acetonitrile. Excess of the reagent does not interfere with the analysis of amino acids, because it is hydrolysed in 2-3 min to give naphthylamine, which is more strongly retained than any amino acid derivatives.

Optical resolution of amino acid enantiomers by high-performance liquid chromatography

Abstract Simultaneous analysis of common protein amino acid enentiomers was achieved by a chiral derivatization and a chiral mobile phase method. In the chiral derivatization method, 2,3,4,6-tetra-O-acetyl-β- d -glucopyranosyl isothiocyanate was chosen as the reagent for the derivatization of enantiomeric amino acids to give diastereomeric thiourea derivatives. These derivatives were efficiently separated on a octadecylsilyl silica gel column by gradient elution and were detected by their absorbance at 250 nm. Derivatives of all common protein amino acid racemates, except cysteine, were resolved within about 2 h, although a few peaks were slightly overlapped. In the chiral mobile phase method, the optically active binary copper complex with N( p toluenesulphonyl)- d -phenylglycine was used as a chiral additive in the mobile phase for the ligand-exchange chromatographic resolution of underivatized d , l -amino acids. Simultaneous resolution of common protein amino acid enantiomers on a resversed phase was achieved by a column-switching technique, utilizing two ODS columns of different lengths, and by gradient elution with acetonitrile. The column eluate was monitored fluorometrically after reaction with o -phthalaldehyde.

New preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for fatty acids and derivatives

A preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for carboxylic acids is described. 9-Anthraldehyde hydrazone is oxidized with an organic oxidant, N-chlorosuccinimide, in an organic solvent such as ethyl acetate to give ADAM, and then the reaction mixture is directly used as the reagent solution for the derivatization of carboxylic acids. Both the oxidation and the derivatization reaction are carried out at room temperature, and an aliquot of the derivatization mixture is directly injected into a chromatograph. 9-Anthrylmethyl ester derivatives formed from ADAM and various carboxylic acids are sufficiently separated on a reversed-phase column and are sensitively detected fluorometrically. The present method was applied to the high-performance liquid chromatographic determination of long and short chain fatty acids, keto acids, and hydroxy acids.

Phorbol myristate acetate-stimulated release of cyclooxygenase products in rat pleural cells: derivatization of prostaglandins with 9-anthryldiazomethane for fluorometric determination by high performance liquid chromatography

White cells were collected from the wash of rat pleural cavity after exsanguination. The incubation mixture of the pleural cells with 1 microM phorbol myristate acetate (PMA) was extracted with acidified ethanol and purified with a Sep-pak C18. The resultant fraction containing prostaglandins (PG) and thromboxane (TX) was allowed to react with 9-anthryldiazomethane (ADAM). After removing contaminants and degraded reagent by silica gel Sep-pak, samples were applied to reversed phase high performance liquid chromatography of octadecylsilyl silica gel and monitored by a fluorescent detector. ADAM derivatives of the authentic PGD2, PGE2, PGF2 alpha, 6-keto-PGF1 alpha, 6-keto-PGE1, TXB2, 15-keto-PGE2, 13,14-dihydro-15-keto-PGF2 alpha and 13,14-dihydro-15-keto-PGE2 showed linear regression lines of peak heights within a range of 0.5-25 ng. By using this method PGD2, 6-keto-PGF1 alpha and TXB2 were detected in the incubation mixture of the rat pleural cells with PMA. The result clarified the origin of these PGs and TX found in the exudate of rat pleurisy induced by PMA. ADAM method for HPLC with a help of clean-up by Sep-pak could be a useful tool for detection of a series of arachidonate metabolites in biological materials.

Investigation of enantioselective separation of quinolonecarboxylic acids by capillary zone electrophoresis using vancomycin as a chiral selector

When a chiral selector that is a pharmaceutical compound is added to the separation buffer in capillary electrophoresis, the enantioselectivity and the mobility of analytes which interact with that chiral selector may be altered. The changes in enantioselectivity and mobility of the analyte are a function of the strength of the affinity interaction, which depends on the structure of each. The macrocyclic antibiotic vancomycin contains a variety of functionalities that are known to be useful for enantioselective interactions (e.g., hydrogen bonding groups, hydrophobic pockets, aromatic groups, amide linkages). Capillary electrophoresis with vancomycin as a buffer additive was used to separate the enantiomers of different compounds. In this study, the chiral separation of quinolonecarboxylic acids that exhibit marked antibacterial activity and of related compounds was achieved by capillary electrophoresis using vancomycin. The correlations between the separation parameters and analyte structures were investigated. The molecular interaction, which is based on the differences of structure, and the effect of experimental parameters on the enantioselective separation between the quinolonecarboxylic acids and vancomycin are discussed.