Derivatisation of microcystin with a redox-active label for high-performance liquid chromatography/electrochemical detection

Abstract

Microcystins are a group of low molecular weight, cyclic peptide hepatotoxins. The most common detection and quantitation methods for these toxins are liquid chromatography with UV or mass spectrometric detections, phosphatase inhibition assays and enzyme-linked immunosorbent assays. In addition, derivatisation of these toxins with organic fluorophores followed by CE/laser induced fluorescence detection and HPLC/chemiluminescence detection; and with luminescent lanthanide chelates for competition assays have also been reported. However, the use of an electrochemical-active unit as a tag for microcystins has never been explored. Since the sulfhydryl group of 6-ferrocenylhexanethiol (Fc-C6-SH) can undergo a facile addition reaction with the α,β-unsaturated carbonyl group, this compound has been used as a redox-active labelling agent for a derivative of microcystins, microcystin-LR (MC-LR). The conjugate, Fc-MC-LR, has been isolated by high-performance liquid chromatography with electrochemical detection. The peak height-concentration curve was linear in the test range 20-400 ng of MC-LR (r value for linear regression > 0.9987). The detection limit was determined to be ca. 18 ng MC-LR (S/N ≈ 3). Meanwhile, the conjugate Fc-MC-LR has also been characterised by positive-ion electrospray-ionisation mass spectrometry. Electrochemical studies show that the adduct displays a reversible ferrocenium/ferrocene couple at ca. -0.040 V vs. SCE (scan rate = 50 mV s-1) in 0.1 M aqueous ammonium acetate-acetonitrile (55:45 v/v).