Determination of toxic cyclic heptapeptides by liquid chromatography with detection using ultra-violet, protein phosphatase assay and tandem mass spectrometry

Microcystins, toxic cyclic heptapeptides and nodularin-R, a toxic cyclic pentapeptide, were determined using liquid chromatography (LC) with detection using photo-diode array ultra-violet (PDA-UV) and protein phosphatase (PP) assay. Positive fractions were analysed for toxins using collision-induced dissociation (CID) and tandem MS/MS experiments which were carried out simultaneously using electrospray ion-trap instrumentation. Reversed-phase liquid chromatography (LC) using an acetonitrile/water gradient was used for the LC-MS(2) determination of six microcystins standards and nodularin. The molecular related ion species, [M+H](+)([M+2H](2+) in the case of MC-RR), were used as the precursor ions for MS(2) experiments. Optimum calibration and reproducibility data were obtained for MC-LR using LC-MS(2); 0.1-5.0 microg/ml, r2 = 0.992 (n = 3); % RSD < or =7.3 at 0.25 microg MC-LR/ml (n = 3). The detection limit (S/N = 3) was better than 0.1 ng. Water samples for microcystin analysis were first screened using protein phosphatase (PP) assays and positives were concentrated using C-18 solid-phase extraction. The developed method was applied to examine a lake in Ireland contaminated by Microcystis sp. and MC-LR and MC-LA were identified.