Waters 2795

Cells were washed once in PBS and ice-cold perchloric acid 1M was added. The plates were immediately put on dry ice and stored at −80°. The cells were scraped on ice and the suspension centrifuged at 14,000g at 4° 10 min. The supernatant was neutralized using KOH 20M and phosphate buffer (1.7M KH₂PO₄ and 0.24M K₂HPO₄, pH 7) and stored at −80° for 30 min. The solution was thawed on ice and centrifuged as before. The supernatant was collected and 50 μl were injected in the HPLC. The separation module used was a Waters 2795 (Waters) equipped with Supelcosil LC-18 15 cm × 4.6 mm/3 μm column, guard column, and Waters 2996 photodiode array detector. The filtered (45 μm) mobile phase used for separation consisted of Buffer A (23.3 mM KH₂PO₄ + 1.7 mM K₂HPO₄ + 10 mM tetrabuthylammonium sulfate pH 5.7) and B (Methanol HPLC grade Promochem). The linear gradient used for separation at 0.7 ml/min was from A 91.7% B 8.3% until B 27.7% at 24 min and then immediately B 8.3% until 32 min. The day of the experiment the column was equilibrated with 50 column volumes of buffer A and at the end of the day the column was flushed with 10 volumes of water and 10 volumes of water:methanol 70:30. The peaks were analyzed using Mass Lynx spectrometry software (Waters). The amount of adenosine nucleotides were calculated based on standard curve performed using 100 mM adenosine nucleotides standards.

LC/MS analysis was performed on a waters LCT Premier mass spectrometer (ESI-TOF) and HPLC Waters 2795. Samples were chromatographed on a Reprosil-PUR 2000 C18-AQ column (3 μm, 100 × 2 mm) heated to 50°C using the conditions shown in Table 1:

The rhamnolipid concentration in the various bacterial cultures was determined by LC/MS (Abdel-Mawgoud et al., 2014 (link)). During a period of 6 days, 400 μl culture samples were retrieved at regular time intervals and the OD₆₀₀ was measured (Nanodrop ND-1000, Thermo Fisher Scientific). Then the samples were centrifuged at 16,000 × g for 10 min to remove the bacteria. To 300 μl of supernatant were added 300 μl of acetonitrile and 10 mg/L 5,6,7,8-tetradeutero-4-hydroxy-2-heptylquinoline (HHQ-d4) as an internal standard (Dubeau et al., 2009 (link)). Samples were analyzed by high-performance liquid chromatography (HPLC; Waters 2795, Mississauga, ON, Canada) equipped with a C8 reverse-phase column (Kinetex, Phenomenex) using a water/acetonitrile gradient with a constant 2 mmol/L concentration of ammonium acetate (Dubeau et al., 2009 (link)). The detector was a mass spectrometer (Quattro Premier XE, Waters). Analyses were carried out in the negative electrospray ionization (ESI-) mode.