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

✍ Scribed by P. Fürst; L. Pollack; T.A. Graser; H. Godel; P. Stehle

• Publisher: Elsevier Science
• Year: 1990
• Tongue: English
• Weight: 864 KB
• Volume: 499
• Category: Article
• ISSN: 1873-3778
• DOI: 10.1016/s0021-9673(00)97000-6

✦ Synopsis

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), 9fluorenylmethyl chloroformate (FMOC-Cl), phenyl isothiocyanate (PITC) and ldimethylaminonaphthalene-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 cu. 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.