Selective determination of total normal microcystin by colorimetry, LC/UV detection and/or LC/MS

Microcystins, hepatotoxic cyclic heptapeptides, are produced by freshwater cyanobacteria, and are classified four groups according to the amino acid structure at unit 7. Normal microcystins contain N-methyldehydroalanine (Mdha) or dehydroalanine (Dha) at unit 7, and command the great part of all microcystins. As unusual microcystin classes, Dhb⁷]microcystins, - and -Ala⁷, or N-MeAla⁷]microcystins and -Ser⁷]microcystins have been found.

On tumor initiation and/or promotion activities of microcystins, the tumor promotion activity of normal microcystins has been found, but cancer-related activities of microcystins belonging in the other classes have not been clear.

To determine normal microcystins as hepatotoxic tumor promoters, a selective determination method was developed. Only Mdha or Dha in normal microcystins was reacted with glutathione (GSH). The GSH-normal microcystins conjugates were reacted with trinitrobenzene sulfonate (TNBS). The TNB–GSH-normal microcystin conjugate can be determined as the total normal microcystin by colorimetry. After methanolysis of the conjugate, dimethyl TNB–glutamate from the conjugate was determined by liquid chromatography/ultraviolet detection (LC/UV) and/or liquid chromatography/mass spectrometry (LC/MS). The detection limits of the total normal microcystin by colorimetry, LC/UV and/or LC/MS were 1 μg, 10 and 0.1 ng, respectively.