Liquid chromatographic determination of D- and L-amino acids by derivatization with o-phthaldialdehyde and N-isobutyryl-L-cysteine. Applications with reference to the analysis of peptidic antibiotics, toxins, drugs and pharmaceutically used amino acids.

In order to evaluate and extend the applicability of an analytical method that enables the quantitative and simultaneous high-performance liquid chromatographic determination of D- and L-amino acids (DL-AAs) by automated precolumn derivatization with o-phthaldialdehyde together with the chiral thiol N-isobutyryl-L-cysteine [J. Chromatogr., 666 (1994) 259] selected natural and synthetic bioactive peptides, as well as pharmaceutically used formulations of AA, were investigated and the amounts of D- and L-AA determined by fluorescence detection. Peptides containing cys(e)ine were oxidized with performic acid prior to hydrolysis with 6 M HCl, and those containing Trp were hydrolyzed with 4 M methanesulfonic acid (24 h at 110 degrees C in both cases). Peptides analyzed were the peptide antibiotics bacitracin, gramicidins A and S, polymyxin B, metanicin C, the peptide toxin malformin A and the peptide drugs D-Arg-[Hyp3,Thi5,8,D-Phe7]-bradykinin, beta-casomorphin and alpha s1-exorphin. Further, the enantiomeric ratios of pharmaceutically used AA formulations containing racemic DL-Ser, DL-Asp and DL-Met were determined, and the AA drugs L-Asp and L-Trp were tested negatively for the presence of the respective D-enantiomers. In two aqueous formulations of L-AA used for parenteral nutrition, low amounts of D-AA (0.1-0.9% with respect to certain L-AA enantiomers and of totally 128 mg and 149 mg D-AAs per liter infusion solution) were determined.

Liquid chromatographic determination of D-amino acids in cheese and cow milk. Implication of starter cultures, amino acid racemases, and rumen microorganisms on formation, and nutritional considerations.

Free L- and D-amino acids (L-AA, D-AA) were isolated from an Appenzeller cheese, from raw milk, and from an ethanolic extract as well as a total hydrolysate of cow's rumen microorganisms, and their relative amounts were determined by reversed-phase high-performance liquid chromatography after derivatization witho-phthaldialdehyde together withN-isobutyryl-L-(or D)-cysteine. D-Ala, D-Asp and D-Glu were found, among other D-AA in all cases and a microbial origin of free D-AA found in cheese and milk was rationalized. From the results, and taking other findings of the occurrence of D-AA in food and beverages into account, the highest intake of D-AA is to be expected from the consumption of ripened cheeses. From the presence of D-amino acid oxidases in human kidney, liver, and brain and from reports on the intravenous administration of racemic AA to humans and their metabolisation it is concluded that intake of free D-AA found in food is no threat for human beings.

Appraisal of four pre-column derivatization methods for the high-performance liquid chromatographic determination of free amino acids in biological materials.

Reversed-phase high-performance liquid chromatography (RP-HPLC) is a powerful method for assaying physiological amino acid concentrations in biological fluids. Four pre-column derivatization methods, with o-phthaldialdehyde (OPA), 9-fluorenylmethyl chloroformate (FMOC-Cl), phenyl isothiocyanate (PITC) and 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl-Cl), were assessed with respect to their applicability in biological research. The methods permit the measurement of 21-26 major amino acids in 13-40 min. The superior sensitivity favours the use of OPA, FMOC-Cl and dansyl-Cl techniques. Because of instability of the OPA adducts, automated on-line derivatization is required when using this method in general practice. Application of the PITC method, although less sensitive, is useful in clinical chemistry, where sample availability is rarely a problem. Cystine determination is not feasible when using OPA or FMOC-Cl and with PITC the reproducibility and linearity are poor, whereas the dansyl-Cl method allows reliable quantitation. The four methods are currently used to perform ca. 8000 OPA and 5000-6000 FMOC-Cl, PITC and dansyl-Cl analyses of biological samples per year. The results obtained with the RP-HPLC methods compare favourably with those derived from conventional ion-exchange amino acid analyses. When the guard column is regularly changed after 120 analyses, the separation remains satisfactory for at least 700 OPA, 800 FMOC-Cl, 150 PITC and 500 dansyl-Cl analyses. Careful control of factors and limitations inherent in the various methodologies is a prerequesite for proper identification and appropriate quantitation.

Automated enantioseparation of amino acids by derivatization with o-phthaldialdehyde and n-acylated cysteines.

The enantioseparation of standard mixtures composed of protein DL-amino acids was performed by reversed-phase high-performance liquid chromatography of the corresponding diastereomeric isoindolyl derivatives, formed by automated precolumn derivatization with o-phthaldialdehyde (OPA) and a series of N-acyl-L-cysteines(Acyl-Cys). A photodiode-array detector, operating at 338 nm, was used for detection. In order to evaluate systematically the influence of the structures of the acyl group in the chiral thiol reagents, a series of novel N-acyl-L-cysteines was synthesized [acyl = n-butyryl, isobutyryl (i-But), pivaloyl, benzoyl) and the chromatographic behaviour of the diastereomers formed was compared with those of already known reagents, N-acetyl-L-cysteine and N-ter.-butyloxycarbonyl-L-cysteine. All Acyl-Cys derivatives of DL-amino acids were resolved. In particular, i-But-Cys gave the highest resolutions for most of the amino acid enantiomers in comparison with the other Acyl-Cys. Investigation of yoghurt using OPA-acetyl-Cys demonstrated the applicability of the method to a complex food matrix and the occurrence of D-Asp, D-Glu and D-Ala in this dairy product.

An ultra rapid and sensitive high-performance liquid chromatographic method for determination of tissue and plasma free amino acids.

An ultra rapid and sensitive HPLC method for measuring individual free amino acids in biological fluids has been developed by using o-phthaldialdehyde/3-mercaptopropionic acid as derivatization agent and employing 3-micron-particle-size reversed-phase columns. Resolution of the amino acid derivatives is accomplished with an acetonitrile gradient in 12.5 mM sodium phosphate buffer, pH 7.2. These conditions facilitate separation of the 23 major tissue free physiological amino acids in the lower picomole range in less than 13 min. Muscle, liver, and kidney free amino acid concentrations, as determined by HPLC, are in the expected physiological range and compare favorably with those obtained by conventional amino acid analyzer.

Measurement of free amino acids in human biological fluids by high-performance liquid chromatography.

Favourable analytical conditions allowing amino acid analysis in biological fluids, acquired from small human biopsy specimens, were achieved by considering various derivatization methods, the mode of detection and the column used. By using o-phthalaldehyde-3-mercaptopropionic acid as derivatization agent (high sensitivity and stability) and fluorescence detection (excitation at 330 nm, emission at 450 nm), excellent separation of 26 amino acids was obtained in the lower pmol range (1-10 pmol). The reproducibility of the retention times was better than 1.0% for the majority of amino acids and the results from high-performance liquid chromatography (HPLC), compared favourably with those of conventional amino acid analysis (r = 0.97). HPLC technology facilitates amino acid analysis in biopsy specimens of less than 1 mg of tissue.