Stable-isotope dilution LC-MS/MS method for quantitative determination of microcystin conjugates with cysteine and glutathione in biotic matrices
Microcystins are cyclic peptide toxins with hepatotoxic and tumor-promoting properties, which are produced in significant quantities (up to tens of μg/L) in freshwater cyanobacterial water blooms. Several studies reported microcystin accumulation in fish with possible food transfer to humans. These compounds are further metabolized to cysteine and glutathione conjugates which can be present in tissues in significant concentrations. In this study, we focused on the development and evaluation of robust and highly sensitive SPE-LC-MS/MS method for the analysis of microcystin conjugates in fish tissue samples. For the first time, we demonstrate the use of isotopically labeled internal standards which are essential for accurate and precise determination of analytes in complex biotic matrices. LLOQs of respective microcystin conjugates (signal-to-noise ratio; S/N > 10, peak-to-peak method) ranged from 3.3 to 5.0 ng/g of tissue fresh weight (FW). The calibration was linear within a range of concentrations from 1 to 70 ng/mL for all analyzed conjugates. The precision and repeatability of the method were very good with recoveries in the range of 88.5–107.6% and relative standard deviations between 8.8 and 13.2% for all analytes. In the follow-up study, fully validated method was used for the determination of microcystin conjugate levels in common carp exposed to microcystin-containing cyanobacterial biomass under controlled conditions. Significant amounts of microcystin conjugates (up to 55 ng/g) were found in the tissues of fish after 7 weeks of exposure. Our method was shown to be robust, sensitive, selective, and suitable for the determination of trace levels of microcystin conjugates in fish tissues.
KeywordsMicrocystin l-Cysteine l-Glutathione LC-MS/MS
The research activities were carried out in the RECETOX Research Infrastructure supported by the Czech Ministry of Education, Youth and Sports (LM2015051) and the European Structural and Investment Funds, Operational Programme Research, Development, Education (CZ.02.1.01/0.0/0.0/16_013/0001761).
This study was also supported by the project PROFISH CZ.02.1.01/0.0/0.0/16_019/0000869. The project is financed by European Regional Development Fund in the Operational Programme Research, Development and Education and The Czech Ministry of Education, Youth and Sports.
Compliance with ethical standards
All in vivo experiments were performed in compliance with Czech laws for the protection of animals against cruelty (Act No. 246/1992 and amendments), as approved by the Ethics Committee of the University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic.
Conflict of interest
The authors declare that there are no conflicts of interest.
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