UPDATE AND EVALUATION OF THE EFFECTIVENESS OF DIFFERENT THIOLS AND MICELLAR MEDIA IN ROTHS FLUOROMETRIC METHOD FOR THE DETERMINATION OF PRIMARY AMINO-COMPOUNDS

The reaction time profiles, stabilities, and relative fluorescence res ponses of isoindoles formed from reaction of 12 different primary amin o compounds with Roth's reagent, i.e., a-phthalaldehyde (OPA) in conju nction with a thiol such as 2-mercaptoethanol (2-ME), 3-mercaptopropio nic acid (S-RIPA), N-acetyl-L-cysteine (NAG), and 1-dhio-D-glucose (TG TA), were determined and discussed in the context of available literat ure. The effects of surfactant micelles in the derivatization solution were also ascertained. Results indicate that the substitution of eith er NAC or 3-MPA for 2-ME in the OPA reagent formulation leads to super ior performance in terms of the isoindole stability and fluorescence r esponse. The addition of surfactant micelles resulted in much greater stability and enhanced fluorescence response for isoindoles formed fro m reaction of OPA and 2-ME with such primary amines as methylamine, ty ramine, aminomethylphosphonic acid, and gamma-aminobutyric acid as wel l as diamino analytes such as putrescine, spermine, ornithine, and lys ine. With 3-MPA or NAC as the thiol in Rath's reagent, the addition of surfactant micelles improved isoindole stability and fluorescence res ponse 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 isoind ole stability and fluorescence response in Roth's method. For derivati zations conducted with TGTA as the thiol, the addition of micelles is not recommended as the resulting isoindole fluorescence response is di minished. It is thought that the favorable micellar effects upon isoin dole stability and fluorescence response. arises as a consequence of i soindole binding to the micellar aggregate where the molecule experien ces a less polar microenvironment (which leads to enhanced fluorescenc e) and is compartmentalized and isolated within a micelle such that th ere is a low probability that the same micelle would contain any exces s OPA reagent molecules (which leads to improved stability), A brief d egradation 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. (C) 1997 Academic Press .