258 Introduction Toxic cyanobacteria waterblooms in lakes, rivers and reser- voirs have become a common occurence in many countries (Skulberg et al., 1984). These toxic waterblooms have caused the death of wild and domestic animals, plus illness and death in humans (Chorus and Bartram, 1999), (Pouria et al., 1999). The hepatotoxins produced by cyanobacteria in- clude the cyclic peptide toxins microcystins and nodularins. At least 65 microcystin analogs are known (Carmichael, 1997). Microcystins (Nishiwaki-Matsushima et al., 1992) are liver tumour promoters (Ohta et al., 1994). Epidemio- logical studies of certain areas in China show positive correlation between the presence of microcystins in water supplies and the incidence of human primary liver cancer (Yu, 1989). Thus, both acute and chronic exposure to cyanobacterial hepatotoxins may be significant human health risks. Electrospray ionization mass spectrometry (ESIMS) coupled with tandem mass spectrometry (MS/MS) is capable of detecting microcystins (Zweigenbaum et al., 2000). Low-energy collision-induced dissociation (CID) mass spectra of microcystin –LR and –RR (Edwards et al., 1993) and electrospray MS/CID of microcystins (Yuan et al., 1999), (Robillot et al., 2000) have been described. In this paper, an electrospray ion-trap mass spectrometry method (MS/MS) was developed for the detection and char- acterization of microcystins in water samples. Full characterization of microcystins was achieved using an or- thogonal hybrid quadrupole time-of-flight (QqTOF) mass spectrometer. Materials and Methods Purchased chemicals included Microcystin-RR, Micro- cystin-YR, Microcystin-LR, Microcystin-LA (Calbiochem, Nottingham, UK); Microcystin-LW, Microcystin-LF (Alexis Corporation, Nottingham, UK). LC-spectrograde trifluo- roacetic acid (TFA) was purchased (Sigma-Aldrich, Dorset, UK). LC-grade solvents were purchased from Labscan (Dublin, Ireland). Lakewater/algae samples (50 mL) were repeatedly freeze-thawed. The samples were then filtered and applied to a solid phase extraction (SPE) column. The SPE method utilised Bakerbond C18 Polarplus cartridges and was similar to a previously developed procedure (Keshavanath et al., 1994). The cartridge was conditioned with methanol and water. A filtered lakewater sample (50 mL) was applied and the cartridge was washed with 10 mL portions of 5%, 15% and 25% methanol/water. Toxins were eluted using methanol (6 mL) containing 0.1% trifluoroacetic acid (TFA) and the eluent was evaporated to dryness and reconsti- tuted in water (1 mL) for analysis. The LC system was a Waters 2690 Alliance (Waters Corporation, Milford, MA, USA) that was linked to a Finnigan MAT LCQ ion-trap mass spectrometer (Thermo-Finnigan, San Jose, CA, USA). Gradient chromatography was performed using acetonitrile- water (30:70 to 100:0) containing 0.05% TFA, over 42 minutes at a flow rate of 200 μL/min. The analytical col- umn (Luna (2) C 18 , 150 × 2.0 mm, 3 μm, Phenomenex, Macclesfield, UK) was operated at 35°C. Mass spectrometric analysis was carried out at atmospheric pressure using an electrospray ionisation (ESI) source and data were acquired in positive mode. MS/MS experiments were obtained by trapping the [M+H] + ion for each toxin, for subsequent frag- mentation experiments to produce characteristic spectra for each toxin. Nano electrospray (ESI) quadrupole hybrid time-of-flight (QqTOF) MS was obtained using a QSTAR spectrometer (Applied Biosystems). Results and Discussion For method development, a mixture of six commercially available standard MCs and the pentapeptide, nodularin- R were used. The MS/MS spectrum for each standard was obtained. The optimized relative collision energies (RCE) were 35% for MC-RR, 35% for nodularin, 35% for MC-YR, 37% for MC-LR, 27% for MC-LA, 36% for MC-LW and 25% for MC-LF. The microcystins were determined using Multiple Tandem Mass Spectrometry Methods for the Determination of Toxic Cyclic Heptapeptides, Microcystins Ambrose Furey, Paloma Muniz-Ortea, Janet Crowley, Orla Allis, Brett Hamilton, Monica Diaz Sierra, Mary Lehane, and Kevin J. James PROTEOBIO, Mass Spectrometry Center for Proteomics and Biotoxin Research, Cork Institute of Technology, Bishopstown, Cork, Ireland Abstract Microcystins are chemically a very diverse group of heptapeptide toxins, with over 65 characterised to-date. Microcystins are specific inhibitors of protein phosphatases, PP1 and PP2A, and they can also act as potent tumour promoters. The challenges facing scientists in the detection of these toxins include the lack of commercially available toxin standards, poor recoveries from clean-up steps, interfering matrix compounds and inconclusive identification of unknowns. Here, we report the application of liquid chromatography-mass spectrometry (LC-MS) techniques for the trace detection and structural characterization of these microcystins. The MS techniques applied included electrospray ion-trap mass spec- trometry and quadrupole time-of-flight (QqTOF) mass spectrometry.These techniques complement each other and allow the rapid identification and quantitation of trace cyanobacterial toxins in algae and drinking water samples. Steidinger, K. A., J. H. Landsberg, C. R. Tomas, and G. A. Vargo (Eds.). 2004. Harmful Algae 2002. Florida Fish and Wildlife Conservation Commission, Florida Institute of Oceanography, and Intergovernmental Oceanographic Commission of UNESCO.