Poroshell 120 phenyl hexyl column

Ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) was realized on Agilent 1290 Infinity II UHPLC (Agilent Technologies) with diode array detector (DAD) coupled to an Agilent 6545 QTOF with an electrospray ionization source. Analyses were performed in negative and positive ion mode. Compound 2 was prepared at 250 uM in ethanol and 1 μL was used for injection. The analyses were performed on a reversed-phase column Agilent Poroshell 120 Phenyl Hexyl column (150 × 2.1 mm, 1.9 µm), using water/acetonitrile mobile phase, both containing formic acid at 20 mM (phase A/B respectively). Phase B increased from 10% to 100% in 10 min, then held at 100% B for 2 min, returned to 10% in 0.1 min, and equilibrated for 2 min at a flow rate of 350 µL/min, and column temperature of 40 °C.
The raw data were processed by MassHunter workstation software (Agilent Technologies), Qualitative Analysis (version B.07.00).

The intracellular extracts were analyzed by ultra high performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) according to Klitgaard et al. [24 (link)]. Liquid chromatography was performed on an Agilent 1290 Infinity LC system with a DAD detector, coupled to an Agilent 6550 iFunnel Q-TOF with an electrospray ionization source (Agilent Technologies, Santa Clara, CA, USA). The separation was performed on a 2.1 mm × 250 mm, 2.7 μm Poroshell 120 Phenyl-Hexyl column (Agilent) at 60 °C with a water-acetonitrile gradient (both buffered with 20 mM formic acid) going from 10 % (v/v) to 100 % acetonitrile in 15 min, followed by 3 min with 100 % acetonitrile. The flow rate was kept constant at 0.35 mL/min throughout the run. The injection volume, depending on the sample concentration, typically varied between 0.1 and 1 μL. Mass spectra were recorded as centroid data for m/z 85–1700 in MS mode and m/z 30–1700 in MS/MS mode, with an acquisition rate of 10 spectra/s. The lock mass solution in 95% acetonitrile was infused in the second sprayer using an extra LC pump at a flow of 10–50 μL/min, and the solution contained 1 μM tributyle amine (Sigma-Aldrich), 10 μM Hexakis(2,2,3,3-tetrafluoropropoxy) phosphazene (Apollo Scientific Ltd., Cheshire, UK), and 1 μM trifluoroacetic acid (Sigma-Aldrich) as lock masses.

HPLC-MS-MS-DAD analysis was run on an Agilent Q-TOF 6545 using an Agilent Poroshell 120 Phenylhexyl column (2.1mm diameter × 150 mm length, with 1.9 μm particle size) in eluent A consisting of 100% water with 20 mM formic acid and eluent B 100% acetonitrile with 20 mM formic acid, in a gradient running from 10% eluent B to 100% B in 10 min, held at 100% B at 2 min, back to 10% B over 0.1. min, and held at 10% B for 1.9 min. The column temperature was 40°C, and the eluent flow rate was 350 μl/min. The MS range measured was from 100 to 1700 Dalton, with a scan rate of 10 spectra/s, using positive and negative ionization separately. Fragmentation (for MS-MS) was done at 10, 20 and 40 eV [44 (link)]. The UV spectrum was compared to the published spectrum [14 (link)] and to a standard of purpurogallin (purpurogallin standard purchased from Sigma-Aldrich, as nr. P7380 MSDS), the latter having a slightly different UV spectrum (and retention time) than purpurogallin carboxylic acid. The UV spectra of purpurogallin carboxylic acid and purpurogallin carboxylic acid glycopyranoside were identical (Fig. S3).