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The concentrations of catecholamine-related compounds in body fluids reflect sympathetic nerve functions. Measuring the enzyme activity of these metabolic pathways will improve diagnosis since a variety of symptoms are reported. An isocratic elution system with two column switching valves was developed using three types of semi-micro columns for fast chromatographic analysis of catecholamine related compounds. Columns are a pentyl-bonded phase, 50 x 2.1 mm i.d., a phenylhexyl-bonded phase, 100 x 2.1 mm i.d. and an octadecyl-bonded phase, 100 x 2.1 mm i.d. The separation of 20 standard compounds was achieved within 25 min using reversed-phase ion-pair liquid chromatography with an electrochemical detector. This new system was applied for analysis of catecholamine-related compounds in pig brain, since pigs are a widely used animal model for transgenic manipulation of neural genes, and MHPG (or VMA), DOPAC, DOPA, NE, EP, DA, 5HTP and 5HIAA were quantified.

Enantiomeric separation and detection of D,L-aspartic acid (Asp) derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) by capillary electrophoresis (CE) using modified cyclodextrins as chiral selectors was studied. Heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin(TM-β-CD) was most effective for enantiomeric separation of NBD-D,L-Asp with optimum conditions of 30 mM TM-β-CD in 50 mM phosphate buffer (pH 4.0) and the limit of detection (LOD) attained was 100 nM for each enantiomer. The method proposed in the present study was convenient for both D- and L-Asp determination since the other amino compounds migrated differently and D-Asp in bio-samples such as rat pineal gland and foods was determined with a simple sample pretreatment and a short analysis run time. Copyright © 1999 John Wiley & Sons, Ltd.

The methodologies for the analysis of D-amino acids in biological materials have been reviewed, including the use of enzymes, gas and liquid chromatography with chiral stationary phases and diastereomer derivatization with chiral reagents followed by GC or HPLC separation. The distribution of D-amino acids in the body, their origin, metabolism and possible roles in human diseases are discussed.

Enantiomeric separation of d-/l-norepinephrine (NE) and d-/l-epinephrine (E) was investigated with various mobile phases by high-performance liquid chromatography using a beta-cyclodextrin type chiral stationary phase as a chiral column. The mobile phase of 2.5 M phosphate buffer (pH 3.0) gave separation factors (alpha s) of 1.04 and 1.06 for NE and E, respectively.

A new fluorescent reagent for carboxylic acids, 4-(N-hydrazinoformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulphonyl-2,1,3-benzoxadiazole (DBD-CO-Hz) was synthesized and its applicability as a precolumn derivatization reagent in high-performance liquid chromatography (HPLC) was examined. DBD-CO-Hz reacted with saturated fatty acids in the presence of a condensing agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and pyridine at room temperature for 30 min to give fluorescent products. The reaction solution was subjected to a reversed-phase HPLC and detected fluorometrically at a wavelength of 550 nm with an excitation of 440 nm. The detection limits of the derivatives were at low fmol range on column.

A novel method for the amino acid sequence and configuration determination of peptides containing D- or L-amino acids is presented. An enkephalin analogue, [D-Ala2, D-Leu5]enkephalin (Tyr-Ala-Gly-Phe-Leu) was derivatized with a fluorogenic Edman reagent, 7-N,N-dimethylaminosulphonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS), cleaved and cyclized with trifluoroacetic acid at 50 degrees C for 1 min and the resultant thiazolinone derivative (DBD-thiazolinyl-L-Tyr) was separated from the racemized DBD-thiazolinyl-D-Tyr (about 20% as compared to the L-isomer) on a phenylcarbamylated cyclodextrin column: The separation factor (alpha) for D- and L-isomers was 1.09. The column eluate was monitored fluorometrically at 524 nm with excitation at 387 nm. The detection limit was about pmol range. The same treatment adopted for the residual peptide, Ala-Gly-Phe-Leu, gave DBD-thiazolinyl-D-Ala (alpha = 1.09 for D,L-Phe) with the lesser amount of L-analogue (about 20%). In the same manner, Gly and L-Phe (alpha = 1.09) were detected. The method might be useful for the study of aging of proteins such as eye lens and amyloid proteins derived from Alzheimer's disease.