Some of the most hazardous chemicals are natural toxins such the cyanotoxin, microcystin (MC). Microcystin is a cyclic heptapeptide hepatotoxin produced by cyanobacteria. Anthropogenic actions have increased the number and intensity of freshwater toxic cyanobacteria blooms increasing the exposure risk through drinking water and recreational waters. US EPA has released a drinking water health advisory at 0.3 ppb MC for pre-school aged children and a draft recreational health advisory at 4.0 ppb MC. With over 150 MCs and only 13 congeners commercially available as standards; quantitation by ELISA is recommended by US EPA. Therefore, the goal of our research was to develop target and nontarget MC MS workflows to vet the ELISA method. Thirty four of 168 samples analyzed by ELISA and LC/MS/MS met a &gt; 30% disagreement criteria. Nontargeted MCs were detected by PDA and a minor shift in chromatography. Based on the PDA, the nontarget MCs concentration were at concentrations around 250 ppt. Nominal mass data suggests that the nontarget MCs are isobaric to D-Asp3 MC LR and MC-YR. LC/IT/HRMS and chemical derivatization enabled identification of nontarget MCs. Several freshwater bacteria strains degrade MC. Bench experiments were designed to mimic natural waters biodegradation. Initial the experiments were analyzed by ELISA and no microbial degradation was reported. However, analyses by LC/MS/MS showed decreased concentrations of MC-LR. Two degradation products were identified by LC/IT/HRMS, 615 m/z and 1013 m/z as the tetramer and linearized MC, respectively. Samples were fractionated by HPLC/PDA. The fractions were analyzed by ELISA and LC/MS/MS and fractions with 615 m/z tetramer and 1013 m/z linear MC tested MC positive by ELISA. The presence of these biodegradation by-products will cause the MC concentration to be artificially inflated. Oxidative pretreatment of water with permanganate is a useful tool in the drinking water industry. The kinetics of MC destruction determined by ELISA vs LC/MS/MS is different; the ELISA concentrations predict slower reaction rates. Two products formed by oxidative cleavage of MC have been detected by LC/MS. Based on the kinetics by LC/MS, these byproducts are interfering with the ELISA. Suspect ELISA byproducts have been identified by LC/IT/HRMS. Drinking water and recreational water managers are using MC ELISA results to determine human exposure, it is important to understand the strengths and weaknesses in different applications.

Using mass spectrometry to vet microcystin concentrations by enzyme-linked immunosorbent assay

2019 Fall National Meeting

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