**1. Introduction**

Avocado (*Persea americana* Mill.) is a member of the Lauraceae family. Although avocado trees are native to Central America, they are also widely distributed in tropical and subtropical countries. Anatomically, the avocado fruit can be distinguished into three regions - the innermost seed that constitutes 20% of the fruit, the pulp covering the major portion (65%) and the outermost peel (15%) [1,2]. Popularly known as "vegetable butter" or "butter pear", the fruit contains a substantial amount of triglycerides (TGs) along with a high content of unsaturated fatty acids. It is also rich in many other bioactive phytochemicals such as carotenoids, tocopherols, phytosterols, aliphatic alcohols and hydrocarbons [3,4].

Unlike oil extracted from other fruits, the oil from avocado fruit is often extracted from the mature fruit flesh [4], and its lipid content has been reported as the highest among all known fruit and vegetable varieties [5–7]. Avocado oil has a multitude of applications such as a culinary oil and as an ingredient in healthcare products, cosmetics, pharmaceuticals and nutraceuticals. The consumption of avocado oil has become popular owing to its high nutritional value and potential benefit to human health, including the management of hypercholesterolemia [8,9], hypertension [10], diabetes and fatty liver disease [11]. The oil can also reduce cardio-metabolic risk [12] and possesses anti-cancer and antimicrobial properties [13,14]. Over the last decade, the production of avocado oil worldwide has grown steadily and currently accounts for about 4.4 million tons of fresh fruit [15,16].

TGs are the most important nutritive group of compounds in avocado oil and represent a significant amount (~90%) of the entire oil composition. Chemically, TGs are complex hydrophobic molecular species formed by the esterification of three fatty acids (FAs) with a glycerol backbone under enzymatic catalysis. The complexity of TGs is due to a large number of possible FA combinations attached to the glycerol skeleton, which can differ in the number of acyl carbon atoms (CNs), the degree of unsaturation, and the position and configuration (cis/trans) of the double bonds (DBs) in each FA. Furthermore, the TG molecule demonstrates optical activity (enantiomers) when the two primary hydroxyl groups are esterified with different FAs, and the stereo-specific distribution (regioisomers) can vary when stereo-chemical positions (sn-1, 2 or 3) on the glycerol skeleton are attached by various combinations of FAs. Several analytical techniques have been employed for the qualitative and quantitative determination of TGs in edible oils, ranging from spectroscopy methods such as infrared spectroscopy [17,18] and nuclear magnetic resonance [19] to chromatographic techniques including gas chromatography (GC), liquid chromatography (LC) [20] and supercritical fluid chromatography (SFC) coupled with mass spectrometry/tandem mass spectrometry [21]. Non-aqueous reversed-phase liquid chromatography coupled with positive-ion atmospheric pressure chemical ionization (APCI) mass spectrometry has become increasingly popular and currently is the most widely used separation technique for TGs analysis. By using this technique, the separation of TGs is governed by the equivalent carbon number (ECN) defined as ECN = CN – 2DB. Separations of TGs within the same ECN group [22,23], cis/trans isomers and isomers with different positional DB have been reported [24]. In contrast, GC is the most commonly used method for the analysis of FAs, but it requires transesterification to convert TGs to its corresponding fatty acid methyl esters (FAMEs). Although high-temperature GC for the direct determination of intact TGs has been reported [25], samples subjected to this technique must be thermally stable and resistant to isomerization.

In recent years, the popularity of avocado oil in the US market has been promoted with oils extracted from alternative sources such as avocado seed. Some manufacturers and consumers have considered avocado seed oil as a source of fatty acids, carbohydrates, dietary fiber and a broad range of phytochemicals. Unfortunately, there is no sufficient evidence to support the claimed health benefits and safe use of such oils. In addition, vegetable oils are among the top 25 ingredients that are most susceptible to adulteration and represent 24% of reported fraud cases [18]. Thus, avocado oil could be a target for fraudulent practices such as adulteration with low-cost oils. Therefore, the development of accurate and reproducible methods for TG and FA analysis in avocado oil is needed for characterization and quality control of this valuable commodity.

As part of an ongoing research program on the authentication, safety and biological evaluation of phytochemicals and dietary supplements, an in-depth chemical investigation of avocado oil was performed. The current study aimed to establish the comprehensive profile of TGs in oils extracted from avocado peel, pulp and seed. A UHPLC/ESI-MS method was developed for the identification and quantification of 13 TGs present in authenticated and commercial avocado oils. Furthermore, the hydrolysis and transesterification products of avocado oils were analyzed for FAMEs using a GC/MS method. To verify the precision and accuracy of the developed methods, the results from GC/MS and UHPLC/ESI-MS were compared. Finally, the TG and FA compositional data, along with chemometric analysis, was used for quality evaluation and identification of possible adulteration in commercial oils.
