Of particular note is the presence selleck products of MC-RY (9) as the dominant microcystin, together with less common or unreported analogues such as MC-RA (10), MC-RL

(28) and MC-RF (13) and some of their [Dha7]- and [Asp3]-variants. As reported earlier (Miles et al., 2012), there was a marked effect from Arg-substitution on retention times for microcystins during LC–MS2 analysis (Table 1). Analogues containing two Arg groups (Arg2 and Arg4) eluted around 2 min (e.g. 3). For microcystins containing a single Arg, those containing an Arg4 group (e.g. 1) eluted at 3.3–4.1 min, whereas those containing an Arg2 group (e.g. 9) eluted at 4.8–6.4 min. Microcystins without Arg groups (e.g. 4) eluted at 7.6–10.5 min. Apart from [Mser7]-microcystins, all of the analogues in the present study reacted with mercaptoethanol and (MEMHEG), indicating the presence of Mdha or Dha (rather than Mdhb or Dhb) at position 7. The presence in African samples of MC-RY (9) and [Asp3]MC-RY (16) has recently been reported, based on mass spectral analyses (Miles et al., 2012; Okello et al., 2010a), although their identities were not confirmed by methods capable of discriminating structural and stereochemical isomers, such as NMR spectroscopy or amino

acid analysis. The MC-RY (9) used in the present study was therefore purified and its structure verified by one- and two-dimensional NMR spectroscopy, thus greatly strengthening interpretation of buy PF-02341066 its MS2 fragmentation patterns (which were largely consistent with those reported by Okello et al. (2010a)). This in turn strengthens the interpretation of the MS2 spectra (Fig. 4 and Supplementary data) leading to the tentative identification of the less common Arg2-containing analogues for which standards are not available, such as MC-RA (10), MC-RL (28) and MC-RF (13) and their [Dha7]-, [Asp3]-, and [Mser7]-congeners. Comparison of the MS2 spectrum of MC-RY (9) with that of MC-YR (2) (Fig. 4) cAMP reveals that while a number of prominent fragment ions (e.g. m/z

916, 911, 638, 620, 603, 602 and 375) are common to both compounds, several prominent fragment ions from 2 (e.g. m/z 728, 710, 682, 571, and, most notably, 599) are absent in the MS2 spectrum of 9. The MS2 spectrum of 9 also contained several fragment ions (m/z 865, 595, 368 and, most notably, 440) that were not present in the MS2 spectrum of 2. The fragment ion at m/z 440 is characteristic for Arg2-containing microcystins (weak fragment ions at both m/z 440 and 599 were present in the MS2 spectrum of MC-RR (3)), and appears to arise from amino acids 7 and 1–3 ( Supplementary data). Thus, [Asp3]MC-RY (16) and [Dha7]MC-RY (23) both showed prominent fragment ions at m/z 426, whereas [Asp3, Dha7]MC-RY ( Miles et al., 2012) and [Mser7]MC-RY (22) showed this fragment at m/z 412 and 458, respectively ( Supplementary data).