Lc mini pal

Lipid extracts were dissolved in chloroform, and appropriate amounts of internal standards were introduced. The lipids were detected by triple quadrupole mass spectrometer (Applied Biosystems API 4000) with an autosampler (LC Mini PAL; CTC Analytics)^{59 (link)}. Data processing was performed in a Lipidome DB Data Calculation Environment (http://lipidome.bcf.ku.edu:9000/Lipidomics) with normalization to internal standards.

Samples were prepared by adding internal standards (Additional file 3: Table S3) and 20 µl (Ryu and Wang and single-extraction) or 40 µl (single-extraction with repeat extraction) of sample corresponding to 0.2 mg extracted leaf dry mass. Samples were brought to 1.4 ml by adding chloroform: methanol: 300 mM ammonium acetate in water (30/66.5/3.5, v/v/v) for mass spectrometric analysis. Analysis was performed on a triple quadrupole mass spectrometer with an electrospray ionization source (API 4000 QTRAP, Applied Biosystems, Foster City, CA) in direct infusion mode. Samples were introduced using an autosampler (LC Mini PAL, CTC Analytics AG, Zwingen, Switzerland) fitted with the required injection loop for the acquisition time and presented to the electrospray ionization needle at 30 µl/min. Samples were analyzed with neutral loss and precursor scans. Most instrument settings were as indicated by Xiao et al. [19 (link)], but scan-specific analytical parameters are listed in Additional file 4: Table S4.