PNNL

Abstract

Background: Microcystins are an emergent public health problem. These toxins are secondary metabolites of harmful cyanobacterial blooms, with blooms becoming more prevalent with eutrophication of water. Exposure to microcystins can result in sickness, liver damage, and even death. Over 300 microcystins have been identified to date, with differences in toxicity based on the specific amino acid composition. Because of this diversity in microcystins, as well as the likelihood of detecting yet undiscovered microcystins, it is vital to establish a method to confidently identify any microcystin in a complex sample, regardless of the availability of a reference standard. In the case of a public health emergency, this method would facilitate unbiased detection of microcystins involved in an exposure event, even for novel microcystins. Methods: A liquid-chromatography–high-resolution mass spectrometry method is utilized to identify microcystins present in cyanobacterial extracts from a strain of Microcystis aeruginosa and an Aphanizomenon sp. First, microcystin congeners with available standards were identified in the cyanobacterial extract. These were considered to have the highest confidence identifications due to availability of accurate mass, retention times, and library spectra for comparison. Utilizing the spectra of these microcystins, relatively high-abundance diagnostic product-ions were identified and employed to screen the data for additional candidate microcystins. The identities of the microcystins determined herein were additionally supported by product-ion searches, thiol reactivity, esterification reactions, and neutral loss analysis. Results: In total, 23 microcystins were identified in the M. aeruginosa culture, including two not published previously: [D-Asp3]MC-LCit and the incompletely identified MC-L(C7H11NO3). Extracts of the Aphanizomenon sp. culture, intended as a cyanobacterial matrix blank, were positive for four identified microcystins. However, the relative abundance of these microcystins was at least 25-fold lower than in the M. aeruginosa culture.