2.3. Sample Preparation and GC-MS Derivatization

Serum (100 µL) was thawed at 4°C and mixed with methanol (300 µL) to precipitate proteins. The mixture was vigorously vortexed for 15 s. After centrifuging samples at 13,000 rpm for 5 min, 300 µL of the supernatant was lyophilized. One hundred µL of O-methoxamine hydrochloride in pyridine (15 mg/mL) was then added into the freeze-dried sample. After vortex-mixing each sample for 3 min at room temperature, 100 µL of N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) was added and incubated at 70°C in a water bath for 1 h. Finally, 600 µL of methyl stearate (10 mg/L in heptane) was added as an internal standard. After centrifuging at 13,000 rpm for 5 min, supernatant was transferred to a 1.5 mL glass vial for GC/MS analysis.