C18 4.6 mm 50 mm column

Lycopene was extracted from cultures and analyzed as described previously (McNerney and Styczynski, 2017a (link)). Briefly, 500 μL of bacterial culture was pelleted and resuspended in 50 μL of ultrapure water. Lycopene was extracted with 1 mL of acetone at 50°C for 20 min. Cellular debris was pelleted, and the supernatant was removed for analysis. Sudan I (TCI America, Portland, OR, United States) was used as an internal standard (Xu et al., 2006 (link)) and added to the acetone used for extractions at a concentration of 1 μg/mL.
All HPLC analysis was conducted on a Shimadzu Prominence UFLC using an Agilent C18 4.6 mm × 50 mm column with a 5 μm particle size and a Shimadzu photodiode array detector. A solvent ratio of 50:30:20 acetonitrile:methanol:isopropanol was used as the mobile phase (Lv et al., 2016 (link)) and run at a flow rate of 1 mL/min with a 25 μL sample injection volume. Absorption was detected at 471 nm. Retention times and peak intensities were compared to an analytical lycopene standard (Millipore Sigma, St. Louis, MO, United States) spiked into control extractions from DH10B cells, and the internal standard Sudan I was used to account for acetone evaporation during the extraction protocol and for instrument drift.