Total amino acid analysis using pre-column fluorescence derivatization

Abstract

An approach to total amino acid analysis utilizing off-line pre-column fluorescence derivatization is described. The full array of natural primary amino acids was treated with o-phthaldialdehyde (OPA) in the presence of mercaptans, and the highly fluorescent reaction products were separated on an ODS 5-μm reversed-phase column with gradient elution development. Due to their instability in solution and the relatively poor response of the OPA derivatives, cysteine and cystine were oxidized with performic acid and separated as the OPA cysteic acid reaction adduct, which is highly fluorescent. Secondary amino acids, such as proline and hydroxyproline, were reacted with 4-chloro-7-nitrobenzofurazan, and the separation was carried out on the same column. The stability of the reaction adducts was investigated and the derivatization reactions were optimized with respect to reaction time and temperature. It is shown that the detection limits for most amino acids are in the 0.1–1.0-pmole range. The reproducibility of the method was limited by the derivatization procedure and the gradient elution employed, but, by using an on-line computer data handling system, the retention time could be measured within ± 0.1% relative standard deviation and the relative peak areas, based on the internal standard calculation methodology, were within ± 3% or less. The sensitivity of lysine and hydroxylysine was improved by forming a sodium dodecylsulfate micellar solution around these amino acids to protect the fluorescent adducts from rapid decomposition. The sensitivity and separation appeared to be very much dependent on the pH of the mobile phase. Experimental details for the determination of total amino acid residues in pharmaceutically important proteins, such as thymosin α 1 and insulin, are given. Advantages and disadvantages of the system described are discussed.