4.4.1. Sample Extraction

The cryopreserved samples were freeze-dried and then ground for 1 min at 30 Hz using a MixerMill MM400 (Retsch Technology, Haan, Germany). Subsequently, 100 mg powder was weighed and extracted for 24 h at 4 ◦C in 1.0 mL of 70% methanol. During this period, the samples were vortexed (10 s, 40 Hz) once per 10 min for a total of three times. After extraction, the pellets were centrifuged at 10,000× *g* for 10 min at 4 ◦C and the extracts were then filtered through a 0.22 μm microporous membrane and stored in a sample vial. The QC was prepared by mixing all the samples. In order to examine the repeatability of the analysis process, a QC sample was injected after every five test samples during the instrumental analysis.
