Trimethylsilyldiazomethane

Hormone levels were quantified from leaf no. 8. Five leaves were pooled at random to produce four replicates per developmental stage. After harvest, leaves were flash-frozen in liquid nitrogen and stored at −80 °C. The samples were ground twice for 0.5 min at 30 Hz with a Retsch Mixermill and were cooled with liquid nitrogen before and between milling steps. They were then extracted in 2 × 750 µl (total 1500 µl) ethyl acetate with 0.1% formic acid, containing the internal standard (3-hydroxybutyrate 60 ng, 9,10-dihydrojasmonic acid 80 ng, and 3-indole pyruvic acid 50 ng per ml), for 10 min in an ultrasonic bath. After centrifugation, the supernatant was used to quantify soluble hormones, whereas the pellet was hydrolysed to release any bound compounds. After removal of the solvent from the supernatant by using an Eppendorf vacuum concentrator (mode HV) at 30 mbar, 70 µl of a fresh 1:1 mix of methanol and trimethylsilyldiazomethane, Sigma-Aldrich) was added to the dry samples. GC-MS (Shimadzu TQ8040) in the splitless MRM mode was used for sample analyses. Hydrolysis of the dry pellet was performed using 200 µl of 3 M HCl and 200 µl of 3 M NH₃.

We used the same method as previously described [25 (link)]. In brief, total lipids were extracted from the RBC cell membranes using Folch’s method [30 (link)]. Samples were split into 3 parts. Fatty acid levels were measured with gas chromatography [31 (link)]. Phospholipids [32 (link),33 (link)] and sterols (cholesterol and cholestanol) [34 (link)] were measured with liquid chromatography tandem mass spectrometry (LC-MS/MS).
Lipids analysis was conducted at the Mass Spectroscopy Department at the Saint-Antoine Hospital, Paris, France. Internal standards for the determination of phospholipids, sterols, and sphingolipids are from Avanti Polar Lipids (Alabaster, AL, USA). Ammonium acetate and trimethylsilyl diazomethane came from Sigma-Aldrich (Saint Quentin Fallavier, France). Methanol, chloroform, n-hexane, heptane, and isopropanol of mass spectrometry quality were obtained from VWR (Fontenay Sous-Bois, France). Total lipids were extracted from the RBC cell membranes based on the methods of Folch et al. Samples were divided into 3 parts.

Plasma free fatty acids were measured as methyl esters by GC‐MS using internal standard methodology as previously described (15). Briefly, 20 µl of plasma was enriched with extraction surrogates including 20:3n3, and extracted with isopropanol/cyclohexane/ammonium acetate, solvent removed, and residues reconstituted in 1:1 methanol/toluene. Samples were enriched with 15:1n5 and fatty acids methylated with trimethylsilyl‐diazomethane in hexane (Sigma‐Aldrich, St. Louis, MO), dried under vacuum and reconstituted in hexane containing 23:0 for analysis. FAMEs were separated on an HP6890 GC equipped with a 30 m × 0.25 id × 0.25 µm DB‐225ms column (Agilent Technologies) and detected with a 5973N MSD with electron impact ionization. Analytes were quantified with ChemStation vE.02.14 software (Agilent) using internal standard methodologies against a 5‐ to 7‐point calibration curve bracketing all reported concentrations.