Musk Sample Preparation and GC-MS Analysis

Sample preparation was performed as previously described with minor modifications (Li et al., 2016 (link)). Twenty milligrams of musk sample from each group were pooled, divided into 2 parts and dissolved in 2.5 mL of diethyl ether and ether alcohol, followed by a 2-h extraction by ultrasonication and centrifugation at 13,000 × g for 5 min. Two milliliters of supernatant was placed into a GC–MS instrument (GCMS-QP2010 Plus, Shanghai, China) for chemical composition detection with the chromatographic conditions as follows: column temperature: 40°C, inlet temperature: 290°C, interface temperature: 220°C, split injection with the pressure of 49.5 kPa. The column temperature program was as follows: 40°C (2 min), 200°C (5 min, 10°C/min), 240°C (5 min, 5°C/min), maintained at 290°C for 15 min. The total and column flow rates were 9.0 and 1.0 mL/min, respectively; the linear velocity was 36.1 cm/s; the purge flow rate was 3.0 mL/min; and the mass scanning range was 33–600 m/z. The acquired data in total ionic chromatograms (TICs) were compared with those in the mass spectral library of the National Institute of Standards and Technology (NIST). A confidence coefficient of above 80% was adopted for the data, and peak area % values were used to determine component variations. Finally, to discriminate the chemical compositions among 6 groups, we performed Partial Least Squares Discriminant Analysis (PLS-DA) after Pareto scaling.