Direct yellow 59

Dried lipid extracts were dissolved in chloroform and automatically spotted onto a normal phase HPTLC glass plate (Merck KGaA, Darmstadt, Germany) by a Linomat device (CAMAG, Muttenz, Switzerland). Chloroform/ethanol/water/triethylamine (30:35:7:35, by vol.) was used as the mobile phase. After drying at room conditions for 15 min, the separated lipid fractions were visualized by dipping the entire plate into primuline (Direct Yellow 59, Sigma-Aldrich, Taufkirchen, Germany) solution (50 mg/l in acetone/water (80:20, by vol.)). The lipids in each spot were automatically eluted by a Plate Express™ TLC plate reader (Advion, Ithaca, NY, USA) with methanol as solvent and analyzed by direct transfer into the ESI-IT mass spectrometer.
ESI-IT MS was performed on an Amazon SL mass spectrometer (Bruker Daltonik GmbH) by direct infusion. Conditions were the following: spray voltage 4.5 kV, end plate offset 500 V, nebulizer gas 7 psi, drying gas (N₂) 3 l/min, capillary temperature 180°C, flow rate 3 μL/min, sheath gas (He) flow rate 25 a.U. Spectra were recorded in the enhanced resolution mode by positive or negative ionization with a maximum ionization time of 50 ms.

Lipid extracts (20 µL) were automatically spotted onto an HPTLC glass back plate (Merck KGaA, Darmstadt, Germany) by using a Linomat (CAMAG, Muttenz, Switzerland) and separated with chloroform/ethanol/water/triethylamine (30:35:7:35, by vol.) as the mobile phase. After air-drying for 15 min, the HPTLC plate was dipped into primuline solution (Direct Yellow 59, Sigma-Aldrich, Taufkirchen, Germany, 50 mg/l in acetone/water (80:20, by vol.)). Lipids were visualized under UV light (366 nm) and spots were marked with a pencil. The lipids in each spot were automatically eluted by a Plate Express™ TLC plate reader (Advion, Ithaca, NY, USA) with methanol as solvent and directly analyzed by electrospray ionization mass spectrometry (ESI MS) on an Amazon SL mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) with the following settings: Spray voltage 4.5 kV, end plate offset 500 V, nebulizer gas 7 psi, drying gas (N₂) 3 L/min, capillary temperature 200 °C. For data acquisition and subsequent analysis of lipid spectra, the software “Trap Control” and “Data Analysis” version 4.1 (Bruker Daltonics GmbH) were used, respectively.