Determination of microcystins and nodularins in ambient freshwater and seawater by liquid chromatography- mass spectrometry including toxin screening and identification

Abstract

Background

Microcystins (MCs) and nodularins (NODs) produced by cyanobacteria occur in ambient freshwaters and across the freshwater-marine continuum, and pose health threats through drinking and recreational waters, as well as food resources. Approximately 300 MC and NOD toxins have been published, but less than 15 of them are commercially available as toxin standards. Our aim herein was to rapidly identify and quantify all toxin congeners, including those without standards, in water samples even at low abundance by reversed-phase solid phase extraction (SPE)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) to provide insights into toxin levels and potential toxicity.

Results

Trizma instead of acid was used as an ion pairing reagent to increase retention of MCs without arginine residues on Strata X SPE. Toxin elution from Oasis HLB SPE was complicated by their hydrophilic and lipophilic interactions with HLB sorbent. Three stable isotope-labeled (SIL) toxin standards representing MCs carrying 2, 1, and 0 arginine residues served as internal standards (IS) for toxin determination and evaluation of cyanobacterial cell lysis methods. Average recoveries of toxin standards in lake water and seawater using the HLB sorbent for validation ranged from 90 to 109% except MC-WR & LW (71 to 87%) with detection limits from 1.3 to 23.7 ng L^-1. Doubly charged protonated toxin molecular ions were employed as precursors for tandem MS to screen and identify toxin congeners using a hybrid triple quadrupole linear ion trap MS. More than 30 (4 new) MCs were detected in Microcystis aeruginosa strain LE-3 culture.

Significance

This is the first report to explain the issues with and possible mechanisms for SPE extraction of MCs from water, to use SIL-IS to evaluate methods for lysing cyanobacterial cells for MC release, and to show that doubly rather than singly charged toxin molecular ions as MS/MS precursors enabled efficient screening, identification, and quantification of all toxins at low levels. A MC with homoalanine residue at position 1 was reported for the first time.