**3. Materials and Methods**

#### *3.1. Chemicals and Materials*

*n*-Hexane (GC grade), 2-propanol (Optima LC/MS grade) and ammonium formate (HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Formic acid obtained from Honeywell (Waltham, MA, USA) was of HPLC grade. Water was obtained from a Milli-Q system (Millipore, Burlington, MA, USA). The reference standards of soybean oil and sesame oil (Analytical grade) were also obtained from Sigma-Aldrich.

TG standards: OOO was purchased from Nu-Chek-Prep, Inc. (Elysian, MN, USA). LLO, LLP, OLO, PLO, OOP, PPO, PPOO, LOL, OPL, OPO and POL (Table 2) were purchased from Larodan (Monroe, MI, USA). The purities of all the TGs standards were >99.0% as per the label and further confirmed by peak area normalization with LC/MS analysis.

FAMEs standards: the methyl esters of undecanoic acid, palmitic acid, palmitoleic acid, steric acid, oleic acid, vaccenic acid, linoleic acid and linolenic acid were obtained from Nu-Chek-Prep, Inc. The purities of all the FAMEs standards were >99.0% by the label and further confirmed by peak area normalization with GC/MS analysis. The FAMEs mixture (C4–C24) consisting of 36 compounds with the positional DB and cis/trans configuration isomers was purchased from Sigma-Aldrich and used for further compound identification.

Mature fresh fruits of avocado (*P. americana*) were purchased from different local grocery stores located in Oxford, MS, USA. All the fruits were selected manually with good morphological integrity. The authenticity of the avocado fruits was confirmed by Dr. John Sabestian, a taxonomist at the National Center of Natural Products Research (NCNPR), University of Mississippi. After washing and drying at room temperature, the peel, pulp and seed were separated and cut into small pieces. The isolated parts were freeze-dried for 24 h until constant weights were obtained. The dehydrated samples were then kept in sealed containers and stored at –20 ◦C to avoid any possible degradation and content loss.

Nineteen avocado oil products claimed to contain avocado pulp or seed oil were purchased from different grocery stores in the US or via various online commercial vendors (Table S1). Each of these 19 commercial samples, along with the authenticated avocado fruits, was assigned a unique identification code, and representative voucher samples were deposited in the Botanical Repository of NCNPR at the University of Mississippi.

#### *3.2. Sample Preparation*

Two extraction methods, viz. solvent extraction and SFE, were performed and evaluated. The solvent extraction was conducted by following the method described in AOAC 920.39 [28]. Five grams of each avocado peel, pulp or seed (dried powder) was extracted using a Soxhlet apparatus with 200 mL n-hexane at 70 ◦C for 4 h, and the procedure was repeated once. For the SFE, the same amount of each sample was loaded into the extraction vessel and mixed with glass beads. The extraction parameters, such as CO2 flow rate, extraction time, pressure and temperature as well as co-solvent were optimized to obtain the highest oil yields possible. Finally, the CO2 flow rate of 10 mL/min, 250 bar, 50 ◦C and 40 min were adopted. For both extraction methods, the oil yields were calculated as the percentage of oil obtained based on the weight of the sample used. The solvent extraction was used as a reference method for the comparison of oil yields obtained from SFE method.
