Detection of total microcystin in fish tissues based on lemieux oxidation and recovery of 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB) by solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC/MS)

Abstract

Microcystins (MCs) are widespread cyanobacterial toxins in freshwater systems, and have been linked to both acute and chronic health effects. A growing number of studies suggest that MC can bioaccumulate in food webs. Although, several methods (i.e. ELISA, LC-MS) have been developed for analysis of MC in water, extraction (for subsequent analysis) of the toxin from biological matrices (i.e. animal tissues) is impeded owing to covalent binding of toxins and active sites of their cellular targets, i.e. protein phosphatases. As an alternative approach, chromatographic methods for analysis of a unique marker, 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB), the product of the Lemieux oxidation of MCs, have been previously developed, and shown to measure total (bound and unbound) MC. Application, however, has been limited by poor recovery of the analyte. An improved recovery method is proposed – specifically the use of solid-phase microextraction (SPME). The MMPB analogue, 4-phenylbutanoic acid (4PB), and oxidized MC, were used to develop methods, and we specifically investigated several SPME fibres, and post-oxidation steps. Specifically, a method employing post-oxidation methyl esterification, followed by headspace SPME recovery of MMPB, was developed, and subsequently applied to analysis of environmental samples (i.e. fish tissues) previously shown to contain MCs. The method shows high linearity for both water and tissues spiked with MC, and an improved limit of quantitation of approximately 140 ng g⁻¹. Evaluation of field samples by SPME-GC/MS detected considerably higher levels of MC, than detected by conventional methods (i.e. ELISA), and it is proposed that this technique reveals MC (particularly in the bound form) that is not detected by these methods. These results indicate that the developed method provides improved detection capability for MC in biological matrices, and will enhance our ability to understand bioaccumulation in freshwater food webs, as well as monitor exposure.

Keywords:

• solid-phase microextraction (SPME)
• microcystin
• 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB)
• Lemieux oxidation
• cyanobacteria
• bioaccumulation

Acknowledgements

This work was partially supported by a grant from the Great Lakes Fishery Commission. The authors would particularly like to thank Dr Kenneth Furton and his laboratory, and particularly Dr Maiko Kusano, Howard Holness and Katylynn Beltz, at Florida International University (Department of Chemistry and Biochemistry) for use of their GC-MS and general guidance in developing the SPME method. We would also like to the Washington State Department of Ecology, including Art Johnson, and WA State Department of Fish and Wildlife, including Adam Couto and Richard Eltrich, for providing fish tissue from Western Washington Lakes, and Dr Fernando Bernal-Brooks of the Universidad Michoacana de San Nicolás de Hidalgo for assistance in collection of fish from Lago Patzcuaro.