3 n methanolic hcl

Total lipids were extracted from 100 μL serum into chloroform:methanol (2:1 v/v) according to Folch et al. method [32 (link)]. Previous to the extraction, 0.05 mg pentadecanoic acid was added to the samples as internal standard. FA methyl esters were produced according to Stoffel et al. [33 (link)] by adding 1 mL of 3 n methanolic HCl (Supelco, Sigma-Aldrich, St. Louis, MO, USA) and heating at 90 °C for 1 h. The derivatives were extracted into hexane and stored at −20 °C until gas chromatographic analysis.
FA methyl esters were analyzed by gas chromatography using a SP-2560 capillary column (100 m × 0.25 mm i.d. × 20 µm) (Supelco, Sigma-Aldrich, St. Louis, MO, USA) in a Hewlett-Packard 6890 gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector. The temperature of the detector and the injector was 240 °C. The oven temperature was programmed at 175 °C 30 min and increased at 2 °C/min to 230 °C and held at this temperature for 17 min. Helium was used as the carrier gas at a pressure of 45 psi. ChemStation software (Agilent Technologies, Santa Clara, CA, USA) was used to analyze FA data. Peaks were identified by comparison of their retention times with appropriate FA methyl esters standards (Sigma-Aldrich, St. Louis, MO, USA) and FA concentrations determined in relation to peak area of internal standard.

Metabolite standards, 3 N methanolic HCl, and toluene were purchased from Sigma. [U-¹³C]Glucose, [U-¹³C]glycine, and [U-¹³C]fumarate were obtained from Isotec. Potassium hydroxide (KOH), methylene chloride, ethoxyamine hydrochloride, and MSTFA+1% TMCS (N-methyl-N-trimethylsilytrifluoroacetamide plus 1% trimethylchlorosilane), solvents for GC-MS and LC-MS/MS, were purchased from Fisher Scientific. Gibberellins (GA4/GA7) and Murashige and Skoog basal salt were ordered from PhytoTechnology Laboratories.