Detection of free and covalently bound microcystins in animal tissues by liquid chromatography–tandem mass spectrometry

Microcystins are cyanobacterial hepatotoxins capable of accumulation into animal tissues. The toxins act by inhibiting specific protein phosphatases and both non-covalent and covalent interactions occur. The 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) method determines the total, i.e. the sum of free and protein-bound microcystin in tissues. The aim of the method development in this paper was to tackle the problems with the MMPB methodology: the rather laborious workflow and the loss of material during different steps of the method. In the optimised workflow the oxidation recovery was of acceptable level (29–40%), the extraction efficiency good (62–97%), but the signal suppression effect from the matrix remained severe in our system (16–37% signal left). The extraction efficiency for the determination of the free, extractable microcystins, was found to be good, 52–100%, depending on the sample and the toxin variant and concentration. The study concerns method development for the LC–MS–MS analysis of both free and protein-bound microcystin in tissue materials.