Serum amino acid analysis with pre-column derivatization: comparison of the o-phthaldialdehyde and N,N-diethyl-2,4-dinitro-5-fluoroaniline methods

Abstract

We compared two pre-column derivatization methods, o-phthaldialdehyde (OPA) and N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA), for analysis of serum amino acids by reversed-phase high-performance liquid chromatography. Separations took 102 and 106 min for FDNDEA and OPA (reconditioning included), respectively, allowing a very good resolution of 30 amino acids by the former process and 38 by the latter. Linearity, within- and between-day variability and advantages in terms of accuracy and speed were studied for both methods. Twenty serum samples from healthy volunteers were assayed with OPA, FDNDEA and with the reference method of ion-exchange and post-column ninhydrin reaction (amino acid analyser), which took 170 min. The correlation between OPA and ninhydrin was good for all the amino acids ( r = 0.959) except for the last-eluting lysine. Good agreement was found for FDNDEA ( r = 0.987), which appeared in general to be a highly reproducible technique. Both pre-column methods were more sensitive than the post-column ninhydrin method.