LC-MS Analysis with Thiol Derivatization to Differentiate [Dhb7]- from [Mdha7]-Microcystins: Analysis of Cyanobacterial Blooms, Planktothrix Cultures and European Crayfish from Lake Steinsfjorden, Norway

Kinetic studies showed that [Asp(3), Dhb(7)]MC-RR reacted with mercaptoethanol hundreds of times more slowly than MC-RR and a range of other [Mdha(7)]-containing microcystin congeners. The difference in reaction rate was sufficiently large that derivatization of microcystin-containing samples with mercaptoethanol, followed by LC-MS analysis, clearly discriminated between microcystins containing the isobaric [Dhb(7)]- and [Mdha(7)]-groups. Application of this approach, using LC-MS with both ion trap and triple-quadrupole mass spectrometers, to water samples and Planktothrix cultures from Lake Steinfjorden, Norway, demonstrated the presence of [Asp(3), Dhb(7)]MC-RR (5), [Asp(3)]MC-RY (14), and [Asp(3)]MC-LY (16), as well as analogues tentatively identified as [Asp(3)]MC-RR (4), [Asp(3), DMAdda(5), Dhb(7)]MC-LR (6), [Asp(3), Dhb(7)]MC-HtyR (8), [Asp(3)]MC-HtyR (9), [Asp(3), Dhb(7)]MC-LR (10), [Asp(3)]MC-LR (11), [Asp(3), Dhb(7)]MC-RY (15), and [Asp(3), Dhb(7)]MC-LY (17), together with low levels of several other analogues. This is the first use of this thiol-based LC-MS approach to identify Dhb-containing microcystins, and allowed identification of LC-MS peaks in a mixture of [Mdha(7)]- and [Dhb(7)]-congeners of [Asp(3)]MC-RR (4, 5), -RY (14, 15), and -LY (16, 17) in the samples from L. Steinsfjorden. This is also the first report of MC-RY-congeners outside of Africa, or in Planktothrix spp. Analysis of European crayfish (Astacus astacus) taken from L. Steinsfjorden revealed the presence of only trace levels of microcystins in the edible parts.