Dataset Information

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.

SUBMITTER: Altaner S

PROVIDER: S-EPMC6669509 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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

Altaner Stefan S Puddick Jonathan J Fessard Valerie V Feurstein Daniel D Zemskov Ivan I Wittmann Valentin V Dietrich Daniel R DR

Toxins 20190702 7

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 ...[more]

PMID: 31269739

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Project description:Human-driven environmental change has increased the occurrence of harmful cyanobacteria blooms in aquatic ecosystems. Concomitantly, exposure to microcystin (MC), a cyanobacterial toxin that can accumulate in animals, edible plants, and agricultural soils, has become a growing public health concern. For accurate estimation of health risks and timely monitoring, availability of reliable detection methods is imperative. Nonetheless, quantitative analysis of MCs in many types of biological and environmental samples has proven challenging because matrix interferences can hinder sample preparation and extraction procedures, leading to poor MC recovery. Herein, controlled experiments were conducted to enhance the use of ultra-performance liquid-chromatography tandem-mass spectrometry (UPLC-MS/MS) to recover MC-LR and MC-RR at a range of concentrations in seafood (fish), vegetables (lettuce), and environmental (soil) matrices. Although these experiments offer insight into detailed technical aspects of the MC homogenization and extraction process (i.e., sonication duration and centrifugation speed during homogenization; elution solvent to use during the final extraction), they centered on identifying the best (1) solvent system to use during homogenization (2-3 tested per matrix) and (2) single-phase extraction (SPE) column type (3 tested) to use for the final extraction. The best procedure consisted of the following, regardless of sample type: centrifugation speed = 4200 × g; elution volume = 8 mL; elution solvent = 80% methanol; and SPE column type = hydrophilic-lipophilic balance (HLB), with carbon also being satisfactory for fish. For sonication, 2 min, 5 min, and 10 min were optimal for fish, lettuce, and soil matrices, respectively. Using the recommended HLB column, the solvent systems that led to the highest recovery of MCs were methanol:water:butanol for fish, methanol:water for lettuce, and EDTA-Na4P2O7 for soils. Given that the recommended procedures resulted in average MC-LR and MC-RR recoveries that ranged 93 to 98%, their adoption for the preparation of samples with complex matrices before UPLC-MS/MS analysis is encouraged.

Project description:Diacyl glycerophospholipids (GPs) belong to the most abundant lipid species in living organisms and consist of a glycerol backbone with fatty acyl groups in sn-1 and sn-2 and a polar head group in the sn-3 position. Regioisomeric mixed diacyl GPs have the same fatty acyl composition but differ in their allocation to sn-1 or sn-2 of the glycerol unit. In-depth analysis of regioisomeric mixed diacyl GP species composed of fatty acyl moieties that are similar in length and degree of saturation typically requires either chemical derivatization or sophisticated analytical instrumentation, since these types of regioisomers are not well resolved under standard ultra-performance liquid chromatography (UPLC) conditions. Here, we introduce a simple and fast method for diacyl GP regioisomer analysis employing UPLC tandem mass spectrometry (MS/MS). This GP regioisomer analysis is based both on minor chromatographic retention time shifts and on major differences in relative abundances of the two fatty acyl anion fragments observed in MS/MS. To monitor these differences with optimal precision, MS/MS spectra are recorded continuously over the UPLC elution profile of the lipid species of interest. Quantification of relative abundances of the regioisomers was performed by algorithms that we have developed for this purpose. The method was applied to commercially available mixed diacyl GP standards and to total lipid extracts of Escherichia coli (E. coli) and bovine liver. To validate our results, we determined regioisomeric ratios of phosphatidylcholine (PC) standards using phospholipase A2-specific release of fatty acids from the sn-2 position of the glycerol backbone. Our results show that most analyzed mixed diacyl GPs of biological origin exhibit significantly higher regioisomeric purity than synthetic lipid standards. In summary, this method can be implemented in routine LC-MS/MS-based lipidomics workflows without the necessity for additional chemical additives, derivatizations, or instrumentation.

Dataset Information

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

Publications

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

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