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.
Fürst
P
P
Institute for Biological Chemistry and Nutrition, University of Hohenheim, Stuttgart, F.R.G.
Pollack
L
L
Graser
T A
TA
Godel
H
H
Stehle
P
P
D000596:Amino Acids / Q000032:analysis*; D001826:Body Fluids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003619:Dansyl Compounds; D005449:Fluorenes; D006801:Humans; D017879:Isothiocyanates; D013861:Thiocyanates; D009764:o-Phthalaldehyde
D000596:Amino Acids; D003619:Dansyl Compounds; D005449:Fluorenes; D017879:Isothiocyanates; D013861:Thiocyanates; C005441:phenylisothiocyanate; D009764:o-Phthalaldehyde; C054007:1-(9-fluorenyl)methyl chloroformate