Hydroquinone-based derivatization reagents for the quantitation of amines using electrochemical detection

Two new reagents, NDTE (2,5-dihydroxyphenylacetic acid, 2,5-bis-tetrahydropyranyl ether p-nitrophenyl ester) and HLTE (homogentisic gamma-lactone tetrahydropyranyl ether), are described for the chemical derivatization of primary and/or secondary amines to form an electrochemically active product. These reagents undergo reaction with the aforementioned analytes to form a product possessing the hydroquinone moiety, thus allowing for reversible electrochemical detection at mild oxidation potentials. The reactivity of each reagent was demonstrated by using N-ethylbenzylamine (EBzA) and the dipeptide isoleucine leucine methyl ester as model analytes. The investigation included the isolation and identification of the intermediates and final products from derivatization of EBzA. These isolated standards were subsequently characterized with respect to electrochemical properties by means of cyclic voltammetry. In LC-EC experiments, the concentration limit of detection (CLOD) of the purified EBzA product was determined to be 5 nM (100 fmol) at a detection potential of +200 mV vs Ag/AgCl ([Cl-] = 3 M). The CLOD values obtained by LC-EC after derivatization of aqueous solutions of EBzA and Ile-Leu-OMe with NDTE were 25 nM (250 fmol) and 250 nM (2.5 pmol), respectively.