Liquid Chromatography-Tandem Mass Spectrometry Application, for the Determination of Extracellular Hepatotoxins in Irish Lake and Drinking Waters Orla Allis, † Justine Dauphard, † Brett Hamilton, † Aine Ni Shuilleabhain, ‡ Mary Lehane, †,§ Kevin J. James, † and Ambrose Furey* ,† PROTEOBIO, Mass Spectrometry Centre for Proteomics and Biotoxin Research, Cork Institute of Technology, Bishopstown, Cork, Ireland, Northern Regional Fisheries Board, Corlesmore, Ballinagh, Co. Cavan, Ireland, and Department of Applied Sciences, Limerick Institute of Technology, Moylish Park, Limerick, Ireland A novel method for the determination of hepatotoxins; microcystins (MCs), and nodularin (Nod) in lake water and domestic chlorinated tap water has been developed using liquid chromatography hyphenated with electro- spray ionization triple quadrupole mass spectrometry (LC- ESI-MS/MS). Optimization of the mass spectrometer parameters and mobile-phase composition was performed to maximize the sensitivity and reproducibility of the method. Detection of the hepatotoxins was carried out using multiple reaction monitoring experiments, thus improving the selectivity of the method. A total ion chromatogram and a precursor ion scan on ion m/z 135 was also applied to all samples to detect unknown micro- cystins or microcystins for which there are no standards available. A comprehensive validation of the LC-ESI-MS/ MS method was completed that took into account matrix effects, specificity, linearity, accuracy, and precision. Good linear calibrations were obtained for MC-LR (1- 200 μg/L; R 2 ) 0.9994) in spiked lake and tap water samples (1-50 μg/L; R 2 ) 0.9974). Acceptable interday repeatability was achieved for MC-LR in lake water with RSD values (n ) 9) ranging from 9.9 (10 μg/L) to 5.1% (100 μg/L). Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with spiked MCs and Nod samples; LOD ) 0.27 μg/L and LOQ ) 0.90 μg/L for MC-LR in the “normal linear range” and LOD ) 0.08 μg/L and LOQ ) 0.25 μg/L in the “low linear range” in both lake and chlorinated tap water. Similar results were obtained for a suite of microcystins and nodularin. This sensitive and rapid method does not require any sample preconcentration, including the elimination of solid-phase extraction (SPE) for the effective screening of hepatotoxins in water below the 1 μg/L WHO provisional guideline limit for MC-LR. Furthermore, SPE techniques are time- consuming, nonreproducible at trace levels, and offer poor recoveries with chlorinated water. The application of this LC-ESI-MS/MS method for routine screening of hepatotoxins in lake and chlorinated tap water (average Cl 2 ) 0.23 mg/L) is achieved and this study represents the first direct method for the screening of hepatotoxins in chlorinated tap water. Cyanobacteria (blue-green algae) are very important freshwater phytoplanktonic organisms due to their ability to potentially produce hepatotoxins and neurotoxins. 1 These potent toxins produced by various cyanobacteria genera represent a serious health and environmental threat to water bodies, in particular those that supply drinking water treatment plants. The most common class of hepatotoxins are the microcystins, of which to date there are over 70 isolated and structurally elucidated. 1,2 Perhaps the most common microcystin producers are members of the Microcystis spp. genera; in particular Microcystis aeruginosa. However, Planktothrix is also a common producer of microcystins, especially in Central Europe. 3 Microcystins are cyclic heptapep- tides with the following general structure: cyclo(D-alanine-X-D- MeAsp-Z-Adda-D-glutamate-Mdha). These large compounds con- tain two variable L-amino acids at the X and Z positions, which give rise to the naming of the compounds; for example, micro- cystin-LR (MC-LR) contains the amino acids; leucine (L) and arginine (R) (Figure 1a, Table 1). Nodularin (Nod) is a cyclic pentapeptide and, like the microcystins, adversely affects hepa- tocytes by binding to the protein phosphatases 1 and 2A, thus inhibiting normal cell regulation 4,5 (Figure 1b, Table 1). At high toxin concentrations, the disruption of the hepatocyte structure leads to a complete collapse of liver function followed by death; at subacute levels, the microcystins are potent tumor promoters. 6,7 * To whom correspondence should be addressed. Phone: (+353)-21-4326701. E-mail: ambrose.furey@cit.ie. Fax: (+353)-21-4345191. † Cork Institute of Technology. ‡ Northern Regional Fisheries Board. § Limerick Institute of Technology. (1) Sivonen, K.; Jones, G. Cyanobacterial toxins; E and FN Spon: London, 1999. (2) Spoof, L.; Vesterkvist, P.; Lindholm, T.; Meriluoto, J. J. Chromatogr., A 2003, 1020, 105-119. (3) Ernst, B.; Hoeger, S. J.; O’Brien, E.; Dietrich, D. R. Aquat. Toxicol. 2006, 79, 31-40. (4) Yoshizawa, S. R.; Matsushima, M. F.; Watanabe, M. F.; Harada, K.-I.; Ichihara, A.; Carmichael, W. W.; Fujiki, H. Cancer Res. Clin. Oncol. 1990, 116, 609-614. (5) Dawson, R. M. Toxicon 1998, 36, 953-962. (6) Falconer, I. R.; Buckley, T. H. Med. J. Aust. 1989, 150, 351. Anal. Chem. 2007, 79, 3436-3447 3436 Analytical Chemistry, Vol. 79, No. 9, May 1, 2007 10.1021/ac062088q CCC: $37.00 © 2007 American Chemical Society Published on Web 04/03/2007