Zorbax xdb c18 column

Before lipid extraction, all samples were spiked with 5 μg of the internal standard cholesterol‐d₇ (Avanti Polar Lipids). Lipids were extracted twice in a 1:10 ratio with methyl tert‐butyl ether (VWR). Extracted lipids were dried down and resuspended in high‐performance liquid chromatography grade methanol (Caledon Laboratory Chemicals). Samples were analyzed by liquid chromatography/tandem mass spectrometry with a 6410 Triple Quadropole liquid chromatography/tandem mass spectrometry instrument using an electrospray ionization source and Zorbax XDB‐C18 column (4.6×50 mm, 3.5 μm) (Agilent Technologies). Sample acquisition was performed in multiple reaction monitoring mode using transitions for cholesterol (m/z 404→369) and the internal standard cholesterol‐d₇ (m/z 411→376).

An Agilent 1200 series HPLC instrument equipped with different columns and a reflective index detector was used to determine the concentrations of octyl glucoside, glucose, fructose, sucrose and fermentation products in the E. coli sample every 24 h. One milliliter of liquid cultures was sampled at 24 h and 48 h and centrifuged at 17,000 × g for 15 min to separate the aqueous and hexadecane layers. The supernatant was transferred into a 2-mL HPLC vial. For octyl glucoside detection, samples (20 μL) were analyzed with a Zorbax XDB-C18 column (Agilent). The flow rate was set at 1 mL/min with a column temperature of 30 °C (20 ). For the analysis of sugars (glucose, sucrose, and fructose) from samples supplemented with sucrose, an Aminex HPX-87P column (Bio-Rad) was used to analyze the samples (20 μL) with the flow rate of 0.6 mL/min, and the column temperature was set at 85 °C. For glucose and other fermentation products when sucrose was not supplemented, samples (100 μL) were analyzed with an Aminex HPX-87H column (Bio-Rad), and the flow rate and column temperature were set at 0.6 mL/min and 60 °C, respectively. Serial dilutions of glucose (Sigma-Aldrich), sucrose (Sigma-Aldrich), fructose (Sigma-Aldrich), sodium acetate (Sigma-Aldrich), sodium lactate (Sigma-Aldrich), and absolute ethanol (VWR) were used to determine the amounts of these compounds in the sample.

The Agilent 1200 series HPLC system was also used for quantification analysis. The chromatographic separation was also carried out on the ZORBAX XDB-C₁₈ column (250 mm × 4.6 mm, 5 μm; Agilent, Santa Clara, CA, USA). The mobile phase was composed of acetonitrile (A) and 0.1% formic acid in water (B). The mobile phase was set as follows: 0–30 min, 13%–17% A; 30–32 min, 17%–22% A; 32–42 min, 22%–23% A; 42–45 min, 23%–26% A; and 45–60 min, 26%–30% A. The flow rate was 1.0 mL/min. The column temperature was maintained at 30 °C. The detection wavelength was 265 nm. The injection volume was 10 μL.