Liquid chromatographic determination of d- and l-amino acids by derivatization with o-phthaldialdehyde and chiral thiols
1994; Elsevier BV; Volume: 666; Issue: 1-2 Linguagem: Inglês
10.1016/0021-9673(94)80388-9
ISSN1873-3778
AutoresHans Brückner, S Haasmann, Matthias Langer, T. Westhauser, Robert Wittner, H. Godel,
Tópico(s)Biopolymer Synthesis and Applications
ResumoA high-performance liquid chromatographic procedure was developed for the fully automated precolumn derivatization of amino acids by derivatization with o-phthaldialdehyde (OPA) together with the chiral thiols N-isobutyryl-l-cysteine (IBLC) and N-isobutyryl-d-cysteine (IBDC) [(CH3)2CHCONHCH(CH2SH)COOH]. The derivatization is completed within 2 min at room temperature and the resulting diastereomeric isoindol derivatives exhibit linearity with respect to fluorescence in the range 25–1000 pmol. The derivatives are completely separated within 75 min on an octadecylsilyl stationary phase using a linear gradient generated by sodium acetate buffer (pH 5.95) and methanol-acetonitrile. Fluorescence detection at an excitation wavelength of 230 nm and an emission wavelength of 445 nm makes possible the detection of 1–2 pmol of an amino acid enantiomer. Derivatization with OPA-IBLC permits the separation of a 41-component standard containing seventeen protein l-amino acids and, in addition, glycine, dl-Cysteic acid, the internal standard l-homo-arginine and the non-protein amino acids α-aminoisobutyric acid and dl-isovaline. Replacement of OPA-IBLC with OPA-IBDC (or vice versa) leads to a reversal in the elution of the derivatives of d- and l-amino acids. The applicability of the method to various fields of biosciences is demonstrated with the detection and determination of d-amino acids in bacteria, microfungi, higher plants, invertebrates and vertebrates, including man, and in the amino acid-containing Murchison meteorite.
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