(Miles et al., 2012) The elution gradient and HPLC column were identical to those used for method A. LC–MS scans were acquired over m/z 900–1100, and data-dependent (m/z 900–1150) LC–MS2 scans were obtained for selected samples with CID settings as for method A ( Miles et al., 2012). Proposed identities of microcystin contaminants detected in standards (Miles
et al., 2012), and of microcystins detected in algal sample BSA6, were based CX-5461 mouse on LC–MS2 analysis and thiol-derivatization, aided by comparison with published data, and are presented in Table 1. Observed MS2 spectra for 1–9, 11, 12, 14–16, 17, 19–21, 29, and 31 were consistent with published mass spectral information (Bateman et al., 1995; del Campo and Ouahid, 2010; Diehnelt et al., 2006; Krishnamurthy et al., 1989; Mayumi et al., 2006; Miles et al., 2012; Namikoshi
et al., 1995, 1992; Okello et al., 2010a; Okello et al., 2010b; Robillot et al., 2000; Welker et al., 2004; Zweigenbaum et al., 2000), and all compounds displayed the expected molecular ions during high-resolution MS (Supplementary Data). It should be noted that mass spectrometric methods alone cannot differentiate between isobaric amino acids (e.g. Aba and isoAba) or stereochemistry (e.g. E- vs. Z-Adda, or between l- and d-amino GSK 3 inhibitor acids). Therefore, compounds in Table 1 are listed as tentative unless an authentic standard was used to establish its identity by both retention time and MS/MS comparisons. BSA6 was one of a series of microalgal concentrates collected during a Microcystis bloom event in Lake Victoria in 2010 ( Nonga, 2011). Initial LC–MS analysis ( Fig. 3a) revealed a number of candidate microcystin peaks in the range m/z 900–1100. Examination of the apparent molecular ion clusters (ratio of [M + H]+:[M + NH4]+:[M + Na]+:[M−H+2Na]+:[M−2H+3Na]+) and MS2 spectra of their [M + H]+ ions revealed which of the major peaks were clearly microcystins,
and which probably arose from other compounds. However, derivatization with mercaptoethanol ( Fig. 3b), and comparison of the chromatogram with that of the underivatized sample, allowed identification of peaks with MH+m/z values that increased Carbachol by 78 Da and with slightly altered retention times, and thus potentially contained Mdha or Dha (and therefore were probably microcystins), and of peaks that did not change (and thus probably were not microcystins). Although software could be used to align the two chromatograms and then to identify components that do, and do not, change with derivatization, even visual comparison revealed a large number of minor candidate-microcystins ( Fig. 3a,b and Table 1). Subsequently, LC–MS2 spectra were used to establish which peaks were probably not microcystins, and the fragmentation patterns revealed tentative structures for the putative microcystins.
Other articles you might like;