Determination of dissolved combined amino acids using microwave-assisted hydrolysis and HPLC precolumn derivatization for labeling of primary and secondary amines

Abstract

Abstract Microwave-assisted, vapor phase acid hydrolysis of natural water samples for measurements of individual dissolved combined amino acids (DCAA) was examined and related to standard liquid phase hydrolysis. The microwave technique allowed a labor-saving, simultaneous hydrolysis of 20 samples in 20 min. Relative to liquid phase hydrolysis, a higher reproducibility and lower blanks were obtained with the present procedure. In some samples, the microwave vapor phase hydrolysis produced up to 39% higher DCAA concentrations than did the liquid phase hydrolysis. For HPLC analysis of the DCCA, derivatization with OPA ( o -phthaldialdehyde) and AQC (6-aminoquinolyl- N -hydroxysuccinimidyl carbamat), a new reagent for fluorescent labeling of both primary and secondary amines, was tested and compared. Total DCAA concentrations (proline not included) determined with the two derivatization reagents were similar. Proline made up from 6 to 24% of the DCAA pools. Our results indicate that if DCAA are quantified with a derivatization procedure for only primary amines, concentrations of DCAA in natural waters will be underestimated, due to the occurrence of secondary amines such as proline. A reduced fluorescence intensity of AQC derivatives, relative to OPA, implies that AQC should not be used for quantification of DCAA at concentrations below 200 nM.