Update and Evaluation of the Effectiveness of Different Thiols and Micellar Media in Roth's Fluorimetric Method for the Determination of Primary Amino Compounds

Abstract

The reaction time profiles, stabilities, and relative fluorescence responses of isoindoles formed from reaction of 12 different primary amino compounds with Roth's reagent, i.e.,o-phthalaldehyde (OPA) in conjunction with a thiol such as 2-mercaptoethanol (2-ME), 3-mercaptopropionic acid (3-MPA),N-acetyl-l-cysteine (NAC), and 1-thio-d-glucose (TGTA), were determined and discussed in the context of available literature. The effects of surfactant micelles in the derivatization solution were also ascertained. Results indicate that the substitution of either NAC or 3-MPA for 2-ME in the OPA reagent formulation leads to superior performance in terms of the isoindole stability and fluorescence response. The addition of surfactant micelles resulted in much greater stability and enhanced fluorescence response for isoindoles formed from reaction of OPA and 2-ME with such primary amines as methylamine, tyramine, aminomethylphosphonic acid, and γ-aminobutyric acid as well as diamino analytes such as putrescine, spermine, ornithine, and lysine. With 3-MPA or NAC as the thiol in Roth's reagent, the addition of surfactant micelles improved isoindole stability and fluorescence response only for diamino-containing analytes. It is important to realize that the surfactant purity and final surfactant micelle concentration govern the extent by which any micelle system will enhance the isoindole stability and fluorescence response in Roth's method. For derivatizations conducted with TGTA as the thiol, the addition of micelles is not recommended as the resulting isoindole fluorescence response is diminished. It is thought that the favorable micellar effects upon isoindole stability and fluorescence response arises as a consequence of isoindole binding to the micellar aggregate where the molecule experiences a less polar microenvironment (which leads to enhanced fluorescence) and is compartmentalized and isolated within a micelle such that there is a low probability that the same micelle would contain any excess OPA reagent molecules (which leads to improved stability). A brief degradation study revealed that the presence of Brij-35 micellar media enhanced the stability of the isoindole and diminished the rate of its postulated autoxidation degradation reaction.