Synergi 4 fusion rp 80a column

Light cells and dark cells isolated from wild-type colonies grown in different media were rapidly harvested and metabolites were extracted as described earlier (Walvekar et al., 2018 (link)). Metabolites were measured using LC-MS/MS method as described earlier (Walvekar et al., 2018 (link)). Standards were used for developing multiple reaction monitoring (MRM) methods on Sciex QTRAP 6500. Metabolites were separated using a Synergi 4µ Fusion-RP 80A column (100 × 4.6 mm, Phenomenex) on Agilent’s 1290 infinity series UHPLC system coupled to the mass spectrometer. For positive polarity mode, buffers used for separation were- buffer A: 99.9% H₂O/0.1% formic acid and buffer B: 99.9% methanol/0.1% formic acid (Column temperature, 40°C; Flow rate, 0.4 ml/min; T = 0 min, 0% B; T = 3 min, 5% B; T = 10 min, 60% B; T = 11 min, 95% B; T = 14 min, 95% B; T = 15 min, 5% B; T = 16 min, 0% B; T = 21 min, stop). For negative polarity mode, buffers used for separation were- buffer A: 5 mM ammonium acetate in H₂O and buffer B: 100% acetonitrile (Column temperature, 25°C; Flow rate: 0.4 ml/min; T = 0 min, 0% B; T = 3 min, 5% B; T = 10 min, 60% B; T = 11 min, 95% B; T = 14 min, 95% B; T = 15 min, 5% B; T = 16 min, 0% B; T = 21 min, stop). The area under each peak was calculated using AB SCIEX MultiQuant software 3.0.1.

Light cells and dark cells isolated from wild-type colonies grown in different media were rapidly harvested and metabolites were extracted as described earlier (Walvekar et al., 2018 (link)). Metabolites were measured using LC-MS/MS method as described earlier (Walvekar et al., 2018 (link)). Standards were used for developing multiple reaction monitoring (MRM) methods on Sciex QTRAP 6500. Metabolites were separated using a Synergi 4µ Fusion-RP 80A column (100 × 4.6 mm, Phenomenex) on Agilent’s 1290 infinity series UHPLC system coupled to the mass spectrometer. For positive polarity mode, buffers used for separation were- buffer A: 99.9% H₂O/0.1% formic acid and buffer B: 99.9% methanol/0.1% formic acid (Column temperature, 40°C; Flow rate, 0.4 ml/min; T = 0 min, 0% B; T = 3 min, 5% B; T = 10 min, 60% B; T = 11 min, 95% B; T = 14 min, 95% B; T = 15 min, 5% B; T = 16 min, 0% B; T = 21 min, stop). For negative polarity mode, buffers used for separation were- buffer A: 5 mM ammonium acetate in H₂O and buffer B: 100% acetonitrile (Column temperature, 25°C; Flow rate: 0.4 ml/min; T = 0 min, 0% B; T = 3 min, 5% B; T = 10 min, 60% B; T = 11 min, 95% B; T = 14 min, 95% B; T = 15 min, 5% B; T = 16 min, 0% B; T = 21 min, stop). The area under each peak was calculated using AB SCIEX MultiQuant software 3.0.1.

Microcystis aeruginosa PCC 7806 can produce two kinds of MAAs: shinorine and porphya-334, with the maximal absorption at 334 nm [25 (link)]. Therefore, the dried cell powder of M. aeruginosa PCC7806 was used for the preparation of MAA standards. The semi-preparation column Synergi 4µ Fusion-RP 80A column (250 × 10 mm, Phenomenex) was used to separate the two kinds of MAAs. A total of 500 µL of the aqueous sample was injected and the flow rate was set at 5.0 mL/min with the mobile phase of 25% methanol containing 0.1% acetic acids under isocratic conditions. The detection wavelength was set as 330 nm. The MAA eluates were collected and lyophilized according to their retention time. The pellets were dissolved by 25% methanol. All the aqueous solutions were transferred to a Strata-X 33 µ high-performance polymeric solid phase extraction column (200 mg/6 mL, Phenomenex). Solid phase extraction was conducted according to the protocols set by the manufacturer. The last eluate containing concentrated MAAs was collected and used as a standard.