Hss t3 1.8 μm column

As described in former studies [39 (link)], we also used an ACQUITY UPLC coupled to a SYNAPT G2 HDMS system (Waters, Milford, MA, USA), which was equipped with an HSS T3 1.8 μm column (2.1 × 150 mm; Waters, Manchester, United Kingdom). The same applies to flow rates and the used gradient and mobile phases, respectively (Phase A: ACN:MetOH:MTBE = 70:20:10 (v:v:v); Phase B: water with 10mM Ammoniumacetate).
Ten concentration steps (1.7–400mg/L) of standards of β-carotene (CAS-no.: 7235-40-7), fucoxanthine (CAS-no.: 3351-86-8) and chlorophyll a (CAS-no.: 479-61-8) were created by diluting pure substances in pure isopropanol in order to calibrate those three substances as well as to validate the accuracy of our detection according to the formerly described methods [39 (link)]. In the beginning of daily LC-MS-batches, 10 measurements of pooled samples were performed to equilibrate the column. Through this, we were given proof that retention time shifts and decreased sensitivity have not occurred within daily batches. Between measurements’ runs of pure isopropanol were performed to prevent carry-over-effects. Identifications of carotenoids other than the calibrated ones were realized using m/z-ratios and retention times formerly described in studies using the same or similar LC-MS-methods [3 (link),39 (link)].

The detailed procedure for LC–MS detection was described previously [13 (link), 14 (link)]. Briefly, 0.5 mL aliquot of cell culture was centrifuged 10 min at 2000×g. The cell pellet was collected and resuspended with ethanol and hexane mixture (2:1 v/v with 3.0 mL in total). De-ionized water at 2.0 mL and 4.0 mL of hexane was then added into the mixture. The samples were then vigorously vortexed and re-centrifuged for 5 min at 2000×g. Hexane layer was transferred to miVac Quattro (Genevac, England) and evaporated at room temperature, then re-dissolved with methyl tertiary butyl ether (MTBE): acetonitrile (ACN) (1:1 v/v). The liquid was analyzed by ultra-high performance liquid chromatography, coupled with UV and mass spectrometer (UPLC-UV–MS). UPLC separation was performed on ACQUITY UPLC (Waters, Milford, USA) and an HSS T3 1.8 μm column (2.1 × 150 mm; Waters, UK) was applied for reversed phase chromatography.