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.

H Brückner, T Westhauser, H Godel

Index: J. Chromatogr. A. 711 , 201, (1995)

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Abstract

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.