*4.2. GC-MS Analysis*

Frozen sirloin samples were powdered with liquid nitrogen and weighed (100 mg) in the frozen state. Frozen samples were plunged into 80% methanol and homogenized using zirconia beads and an ultrasonic homogenizer for 5 min. The samples were centrifuged at 15,000 rpm for 5 min. The supernatant was filtered using a Mono-Spin C18 column (GL Science, Tokyo, Japan), and then the filtration (50 μL) was dried by a nitrogen gas flow. Methoxyamine hydrochloride solubilized with pyridine (20 mg/mL, 50 μL) was added to each sample, and oxime formation was achieved by reacting at 30 ◦C for 90 min. Trimethylsilyl-trifluoroacetamide (50 μL) was then added to each sample, and trimethylsilylation was carried out by reacting at 37 ◦C for 30 min. Analyses were performed on a gas chromatography–mass spectrometer (GC-MS, QP2010Ultra, Shimadzu, Kyoto, Japan) using a DB-5 column (Agilent Technologies, Santa Clara, CA, USA) at the Kazusa DNA Research Institute. The carrier gas was helium in a flow of 1.1 mL/min. The injection temperature was 280 ◦C, and the injection volume was 0.5 μL. The temperature program was isothermal for 4 min at 100 ◦C, then raised at a rate of 4 ◦C / min to 320 ◦C and held for 8 min. The ion source temperature and scan speed were set to 200 ◦C and 2500 u/sec, respectively. Sample peaks were recorded over the mass range of 45–600 *<sup>m</sup>*/*<sup>z</sup>*. The retention time correction of peaks was carried out based on the retention time of a standard alkane series mixture (C-7 to C-33). Annotation and relative quantification of metabolite was measured by each peak using the GC-MS solution (Shimadzu) and GC/MS Metabolite Database Ver. 2 (Shimadzu). The relative area was calculated using the peak area of each metabolite relative to the analyzed sample weight at Kazusa DNA Research Institute.
