Supplementary Information

The mix of calibration standards used in the LC-MS/MS analysis includes ten MC variants and nodularin. Table 1 shows the LC-MS/MS parameters established for the SRM acquisition mode:

Analyte / precursor ion
m/z / TL (V) / quantitation / confirmation
product ion
m/z / CE (V) / product ion
m/z / CE (V)
MC-dmRR / 512.8 / 130 / 135.0 / 26 / 102.8 / 51
MC-RR / 520.0 / 28 / 127.0 / 41
Nodularin (I.S.) / 825.5 / 165 / 50 / 227.1 / 50
MC-LA / 910.6 / 180 / 40 / 375.3 / 31
MC-dmLR / 981.5 / 190 / 58 / 213.0 / 53
MC-LF / 986.6 / 37 / 248.9 / 26
MC-LR / 995.7 / 200 / 55 / 213.1 / 45
MC-LY / 1002.6 / 45 / 265.1 / 31
MC-LW / 1025.7 / 205 / 51 / 213.0 / 29
MC-YR / 1045.5 / 57 / 213.0 / 40
MC-WR / 1068.7 / 210 / 63 / 53

TL: Tube Lens; CE: Collision Energy; I.S.: internal standard. Scan time: 100 msec.

Quantification of analytes was performed by internal calibration using nodularin as internal standard in SRM mode. For identification purposes the following criteria were accomplished: i) the ratio of the chromatographic retention time of the analyte to that of the internal standard, shall correspond to that of the calibration solution at a tolerance of ±2.5%; ii) two m/z transitions were confirmed for each analyte; iii) the ratio between the two transitions in the sample compared to ratio in the calibration curve should be in agreement to [calibration curve average ± maximum permitted tolerances for relative ion intensities as proposed in European Commission Decision 2002/657/CE]. The optimum MS/MS conditions are also showed in Table 1. The MC from which there is no commercial standard was estimated using the response factor of the variant with an analytical behaviour most similar.

Data were acquired in full scan mode (m/z 400-1200, 1.2 sec/scan) for screening of MC variants and MS/MS spectra were performed for confirmation of MC identity when commercial standards were not available. For identification purposes important diagnostic ions of each MC variant and generic characteristic fragments of MCs were observed such as m/z 135, 213 and 375.

Figures 1S and 2S display a chromatogram of a mixed standard and a chromatogram of the field sample, respectively.

Figure 1S: Chromatogram of MC-dmRR, -RR, -YR, -LR, -dmLR, -WR, -LA, -LY, -LW, -LF and nodularin standard mixture.

Figure 2S: Chromatogram of MCs detected in Alcántara bloom sample.