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

Two new reagents, NDTE (2,5-dihydroxyphenylacetic acid, 2,5-bis-tetrahydrop yranyl ether p-nitrophenyl ester) and HLTE (homogentisic gamma-lactone tetr ahydropyranyl ether), are described for the chemical derivatization of prim ary and/or secondary amines to form an electro! chemically active product. These reagents undergo reaction with the aforementioned analytes to form a product possessing the hydroquinone moiety, thus allowing for reversible el ectrochemical detection at mild oxidation potentials. The reactivity of eac h reagent was demonstrated by using N-ethylbenzylamine (EBzA) and the dipep tide isoleucine leucine methyl ester as model analytes. The investigation i ncluded the isolation and identification of the intermediates and final pro ducts from derivatization of EBzA. These isolated standards were subsequent ly characterized with respect to electrochemical properties by means of cyc lic voltammetry. In LC-EC experiments, the concentration limit of detection (CLOD) of the purified EBzA product tvas determined to be 5 nM (100 fmol) at a detection potential of +200 mV vs Ag/AgCl ([Cl-] = 3 M). The CLOD valu es 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), respect ively.