Db 23 column

Free fatty acid contents in dry- and wet-aged beef were assessed by the method of Lee et al. [3 (link)]. Briefly, 1 g of lipid was put into the test tube with 1 mL of chloroform and an internal standard (1 mg of triundecanoate/mL isooctane). After removing triglycerides from the samples, free fatty acids were extracted using 2% acetic acid in diethyl ether. The extract was evaporated with nitrogen gas and heated at 85 °C for 10 min. After that, 2 mL of 14% boron trifluoride–methanol was put into the test tube for methylation and heated at the same condition. Then, 2 mL of isooctane and 1 mL of saturated sodium chloride were added into the test tube and centrifuged at 1573× g for 3 min (Continent 512R, Hanil Co. Ltd., Daejeon, Korea). The upper layer containing fatty acid methyl ester (FAME) was dehydrated with anhydrous sodium sulfate. FAME was analyzed using gas chromatography (HP 7890, Agilent Technologies, Santa Clara, CA, USA) with a DB-23 column (60 m × 250 μm × 0.25 μm; length × diameter × thickness) (Supelco, Bellefonte, PA, USA). Each FAME was identified by comparing the retention time of external standards (Supelco^® 37 Component FAME mix, Sigma-Aldrich, St. Louis, MO, USA).

The content of fatty acids was determined in freeze-dried powder flower samples according to Smooker et al. [22 (link)], with some modifications. Powder (0.4 g) was mixed with 1 mL of hexane and 5 μL of internal standard 0.2 M, nonadecanoic acid (C19:0) resuspended in methanol:toluene:2,2-dimethoxypropane:sulfuric acid (33:14:2:1). The mixture was incubated in a water bath for 1 h at 80 °C, then samples were cooled at room temperature and the organic phase was separated, which was injected into the gas chromatograph (GC) (Agilent 6890 coupled 5975 Inert Mass Selective detector (GC-MS, Agilent Technologies, Palo Alto, CA, USA) equipped with a DB-23 column (60 m × 0.25 mm × 0, 25 μm; Supelco) and using nitrogen as carrier gas and a flame ionization detector (FID) at 250 °C. Fatty acid compounds were identified and quantified by comparing with the commercial mix of FAME standards (37 components mix, Supelco Co., Bellefonte, PA, USA). The results were expressed in mg/100 g d.w.