Ferrocene tagging of amines, amino acids and peptides for liquid chromatography with electrochemical detection

Abstract

Ferrocenecarboxylic acid chloride (FAC) and ferrocenesulfonyl chloride (FSC) were investigated as ferrocene tagging reagents for amine and amino acid determination by liquid chromatography with electrochemical detection (LC-EC). Whereas FSC gives low sulfonamide yields, FAC reacts readily with both primary and secondary amines and amino acids to yield stable amides with are reversibly oxidized in mixed acetonitrile—aqueous solutions at potentials 220 mV positive of ferrocene oxidation. A derivatization procedure is presented which yields quantitative derivatization of micromolar concentrations of amines and amino acids and the resulting derivatives are amenable to reversed-phase chromatographic separation coupled with dual-electrode detection schemes. A mixture of fourteen amino acids was derivatized and separated by gradient elution LC-EC within 45 min. FAC derivatization was applied for monitoring the enzymatic hydrolysis of 200 mmol of the tripeptide Gly—Phe—Leu by leucine aminopeptidase M. FAC derivatization of peptides is also demonstrated for the substance P fragment 4–11 ( N-terminus-proline) and subsequent LC-EC determination of the FAC derivative yielded a detection limit of 500 fmol.