Simultaneous Detection of 14 Microcystin Congeners from Tissue Samples Using UPLC- ESI-MS/MS and Two Different Deuterated Synthetic Microcystins as Internal Standards

Abstract

Cyanobacterial microcystins (MCs), potent serine/threonine-phosphatase inhibitors, pose an increasing threat to humans. Current detection methods are optimised for water matrices with only a few MC congeners simultaneously detected. However, as MC congeners are known to differ in their toxicity, methods are needed that simultaneously quantify the congeners present, thus allowing for summary hazard and risk assessment. Moreover, detection of MCs should be expanded to complex matrices, e.g., blood and tissue samples, to verify in situ MC concentrations, thus providing for improved exposure assessment and hazard interpretation. To achieve this, we applied two synthetic deuterated MC standards and optimised the tissue extraction protocol for the simultaneous detection of 14 MC congeners in a single ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) run. This procedure was validated using plasma and liver homogenates of mice (male and female) spiked with deuterated MC standards. For proof of concept, tissue and plasma samples from mice i.p. injected with MC-LR and MC-LF were analysed. While MC-LF was detected in all tissue samples of both sexes, detection of MC-LR was restricted to liver samples of male mice, suggesting different toxicokinetics in males, e.g., transport, conjugation or protein binding. Thus, deconjugation/-proteinisation steps should be employed to improve detection of bound MC.

Development and Application of Extraction Methods for LC-MS Quantification of Microcystins in Liver Tissue

“…MCs in tissue can be quantified using enzyme-linked immunosorbent assays (ELISA) [32], protein phosphatase inhibition assays (PPIA) [20,33], Lemieux oxidation (also known as MMPB) method [34,35], and LC-MS [22,[36][37][38]. ELISA, PPIA, and Lemieux oxidation methods cannot distinguish between different MC congeners and adducts.…”

“…Furthermore, a combination of poor recoveries, extensive sample preparation, and inadequate limits of detection (LODs) hinder the convenience and efficacy of those methods. LC-MS is robust and precise, but published methods for extraction of MCs from tissue have been validated for few congeners [36,37] or require complex sample preparation [38]. The lowest limit of quantification (LOQ) achieved previously, 17 ng of MC-LR per g of dried liver tissue [22], may not be adequate to study the effects of chronic exposure to MCs in low doses.…”

Is Toxin-Producing Planktothrix sp. an Emerging Species in Lake Constance?

“…Corresponding to the presence of mcyE in 2019, low amounts of the microcystins MC-LR and MC-YR were detected using UPLC–MS/MS. In 2020, although mcyE amplicons were detectable in some of our samples, concentrations of microcystins were below the detection levels of the UPLC–MS/MS that was used (LOD of UPLC–MS/MS method: 0.5 ng/mL [ 65 ]; therefore, the resulting LOD in our water samples: 62 pg/L lake water). Despite the low abundance of Planktothrix spp.…”

Toxic benthic freshwater cyanobacterial proliferations: Challenges and solutions for enhancing knowledge and improving monitoring and mitigation

“…This is a compound nearly identical to the compound being analysed but distinguishable by several mass units. This strategy was recently applied to the analysis of MCYs in another complex matrix—plasma and liver samples (Altaner et al., 2019)—and might be of benefit for the analysis of benthic cyanobacteria samples. The main drawback with this approach is the limited availability of internal standards.…”