Characterization of Avocado (Persea americana Mill) Seed Extract from the Variety Semil 34 Cultivated in the Dominican Republic

Abstract

Avocado (Persea americana Mill.) is a widely cultivated fruit known for its nutritional benefits, with the seed representing a significant portion of the fruit that is often discarded as waste. In the Dominican Republic, the cultivar Semil 34 represents 58% of the national production. This study aimed to explore the potential of Semil 34 avocado seed (AS) as a source of bioactive compounds with applications in the food industry. We conducted the chemical characterization of the seed extract, focusing on its total phenolic content, total flavonoids, and antioxidant capacity. High-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) was employed to identify key phytochemicals, including phenolic acids and flavonoids, that were responsible for the antioxidant properties of the extract. The hydroalcoholic extract of the Semil 34 seeds exhibited an antioxidant capacity of 1743.3 ± 52.3 µM Trolox/g extract, total phenolic content of 25.86 ± 2.17 mg gallic acid equivalents/g extract, and total flavonoid content of 2.09 ± 0.10 mg quercetin equivalents/g extract. However, the extract’s antioxidant capacity was found to be sensitive to pH changes, suggesting the need for stabilization when used in acidic or basic food matrices. The present work identified 53 compounds in the Semil 34 seed extracts; among these, 23 are being reported for the first time in avocado seeds. This study demonstrates the potential of the avocado seed as a source of bioactive compounds and hence a functional ingredient, supporting its value in sustainable production and its possible contribution to environmental goals by reducing waste in the avocado industry.

1. Introduction

Avocado (Persea americana Mill.) is a ubiquitous fruit with origins in Central America. The avocado fruit is known for its high nutritional value and health benefits [1]. Several varieties of avocado have been reported, including Bacon, Fuerte, Gwen, and Hass, with the latter being the most commercialized [2]. In 2022, the global avocado production was approximately 8.98 million metric tons, with the Dominican Republic being the fourth-largest producer of avocado, with 737,200 metric tons [3,4]. In 2023, 21,875 hectares were dedicated to avocado production in the Dominican Republic. Different cultivars, such as Hass, Popenoe, Simmonds, Pollock, Doctor Dupoi, and Carla, are commercially available in different seasons. Regarding exportation, the most popular variety is Semil 34, which accounts for 58% of the total national avocado production [3,5,6]. Semil 34 is a hybrid between the Guatemalan and the West Indian varieties [7]. Previous studies have reported that, on average, the seed, pulp, and peel constitute 13.93, 79.07, and 7.00%, respectively, of the avocado, as well as an oil and dry matter percentage of approximately 9% and 20% [8]. However, there is limited information available about Semil 34.

Throughout the industrialization process of avocados, the pulp is the only part of the fruit utilized, and its byproducts, such as the avocado seed (AS) and peel, are discarded into the environment, contributing to environmental concerns surrounding the produced waste. The AS represents approximately 13% of the fruit [9], which, according to the numbers previously mentioned, translates into 1.16–1.62 million metric tons of waste globally per year and 317,000–439,000 metric tons of AS annually in the Dominican Republic. In the Dominican Republic, initiatives have been undertaken to utilize the avocado residue to produce oil, and this has demonstrated remarkable effectiveness, utilizing approximately 1.5 million kilograms of discarded avocados between 2020 and 2021 [10]. Nevertheless, other management options are to be considered to drive more environmentally friendly production and meet the Sustainable Development Goals proposed by the United Nations, particularly the objectives of sustainable industrialization and production [11].

Numerous studies have explored the potential applications of AS residues in various industries, such as pharmaceuticals, food and cosmetics, highlighting its potential as a source of many bioactive compounds that can provide antioxidant, anticancer, anti-inflammatory, and antibacterial properties [12,13]. AS also contains significant concentrations of carbohydrates, fiber, protein, lipids, and vitamins, such as A, B1, B2, B3, C, and E [14,15,16]. There are several methods available to recover bioactive compounds, including novel green technologies like ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and Ohmic heating-assisted extraction (OHAE), as well as conventional solid–liquid extractions (SLEs) like maceration, cooking, and infusion. Among these, UAE has been widely applied for the recovery of phytochemicals from plant material [17]. This method uses ultrasound waves to facilitate the rupture of cell walls and the release of compounds at moderate temperatures with little solvent, which enables the high recovery of compounds.

The identification of these health-promoting phytonutrients in the AS extract often requires the utilization of sophisticated techniques such as nuclear magnetic resonance and, more recently, high-performance liquid chromatography–electrospray ionization–mass spectrometry in tandem (HPLC-ESI-MS/MS), which allows the separation and identification of molecules. With these techniques, various phenolic acids, including 3-O-caffeoylquinic acid, 3-p-coumaroylquinic acid, caffeoylquinic acid, and derivates of chlorogenic acid, have been characterized from different AS extracts, as well as terpenes and flavonoids like catechin, epicatechin, procyanidins, luteolin, apigenin, kaempferol, and quercetin [18,19,20,21]. Additionally, sodium, calcium, magnesium, zinc, potassium, and other minerals have been found in AS [22,23].

Driven by the promising composition of avocado waste, many authors have studied the potential industrial applications, particularly for avocado seeds. Previous research has highlighted the broad possibilities to repurpose these byproducts. David et al. observed antimicrobial activity in an ethanolic extract of dry avocado seeds [24]. Similarly, avocado seed extracts were reported to reduce the viability of human cancer cell lines through in vitro trials [15]. Previously, in 2011, Dabas et al. studied the potential use of an avocado seed extract as a natural colorant, finding that the development of the orange color is dependent on a polyphenol oxidase reaction [25]. Later, in 2019, Hatzakis et al. isolated the major colored compound in AS extract using high-performance liquid chromatography and identified it as perseorangin, a yellow-orange solid containing glycosylated benzotropone, with a distinctive orange color when in solution [26]. The ability of avocado to produce an orange dye, associated with the many bioactive compounds present in AS, suggests its substantial potential for applications in food, cosmetics, and pharmaceuticals as an added-value natural colorant, as well as a source of antioxidants. This study aims to characterize the phytochemicals present in Semil 34 seed colored extract for potential applications in the food industry. The study also explores the chemical characterization of avocado cultivar Semil 34, focusing on the total phenolic content and total flavonoids and the antioxidant capacity of its seed. We assess the impact of pH variations on the antioxidant activity and identify key phytochemicals present in the extract. Our findings suggest that incorporating this extract into food matrices could enhance their nutritional value, thanks to its diverse phytochemicals. However, stabilizing the extract for use in acidic or basic matrices is crucial, as its antioxidant capacity is influenced by pH variations.

2. Materials and Methods

2.1. Chemical and Reagents

A DPPH antioxidant assay kit, including DDPH reagent, Trolox standard, and assay buffer, was purchased from Dojindo Laboratories (Rockville, MD, USA). Folin–Ciocalteu reagent, gallic acid, quercetin, calcium carbonate anhydrous, and aluminum chloride were purchased from Sigma Aldrich (St. Louis, MO, USA). All solvents (ethanol and methanol) were obtained from Fisher Scientific (Waltham, MA, USA).

2.2. Avocado Samples and Processing

Persea americana Mill. variety Semil 34 samples were collected in the municipality of Tenares (Hermanas Mirabal, Dominican Republic). The avocados were collected through random sampling and transported to the Universidad Nacional Pedro Henríquez Ureña in Santo Domingo for processing. The seeds were manually separated and allowed to dry for 48 h at 55 °C, and the membrane that covered the seed was removed. The seeds were cut into small pieces, crushed into a fine powder, and stored at −20 °C in an amber bottle until further analysis.

2.3. Extraction of Phytochemicals Through Cooking

Five grams of the previously pulverized avocado seed was mixed with 50 mL of solvent (water or water/ethanol mixture) and placed on a hotplate and gently boiled for 5 min. Then, the mixture was removed from the heat source and left to cool down to room temperature. The solution was centrifuged (1500 rpm, 10 min) and vacuum-filtered (0.2 microns, Millipore; Burlington, MA, USA) to remove suspended particles. Samples were run in triplicate.

2.4. Extraction of Phytochemicals Through Infusion

Five grams of the pulverized avocado seed was mixed with 50 mL of solvent (water or water/ethanol mixture) and placed on a hotplate until a temperature of 65 °C was reached. The mixture was then removed from the heat source and left to cool down to room temperature. The solution was centrifuged (1500 rpm, 10 min) and vacuum-filtered to remove suspended particles.

2.5. Extraction of Phytochemicals Through Ultrasound-Assisted Extraction

Five grams of the pulverized avocado seed was mixed with 50 mL of 50% ethanol aqueous solution and placed in the ultrasonic homogenizer cabin (U.S. Solid; Cleveland, OH, USA). The mixture was sonicated for 10 min with the following probe settings: 2 s on time, 3 s off time, maximum temperature 65 °C, power 240 W, and frequency 20 KHz. The solution was then centrifuged (1500 rpm, 10 min) and vacuum-filtered (0.2 microns, Millipore; Burlington, MA, USA) to remove suspended particles.

2.6. Apparent Color Determination

The avocado seed extract was transferred to an empty comparison tube and placed in the space between the Gardner Liquid Standards L230 (Columbia, MD, USA), as recommended by the manufacturer. When the color of the extract is darker than the standard, a (+) is added to the number.

2.7. Antioxidant Activity Assays

The instructions from Dojindo’s DPPH Antioxidant Assay Kit were followed accordingly [27]. A preliminary assay to determine the IC₅₀ was performed. The sample, Trolox standards, and blanks were prepared by mixing the sample solution, solvent, ethanol, assay buffer, and DPPH working solution, following the manufacturer’s protocol. The mixtures were incubated for 30 min, and the absorbance was measured on a microplate reader (ELISA READER ADX-110, Poway, CA, USA) at 492 nm. The antioxidant activity of the seed extract was determined from the calibration plot (Y= −3.0841 × 10⁻⁴x + 0.2573, R² = 0.9608) and expressed as the Trolox-equivalent antioxidant capacity (TEAC)/g seed. All determinations were carried out in triplicate.

2.8. Total Phenolic Compounds

The total phenolic compounds (TPC) were measured following the method proposed by Singleton and Lamuela-Reventós, with a few modifications [28]. One milliliter of avocado seed extract was mixed inside a 100 mL volumetric flask. The solution was treated with 5 mL of Folin–Ciocalteu reagent, followed by 15 mL of sodium carbonate 1.89 M. The mixture was then swirled, filled to volume with distilled water, and left to react for 2 h before recording the absorbance at 760 nm (E-2100UV UV–Vis Spectrometer, Peak Instruments, Inchinnan, UK). Gallic acid (GA) was used as a standard and the TPC was determined using the calibration curve (Y = 0.9225x − 0.0227, R² = 0.9994). The results were expressed as mg of gallic acid equivalents (GAE)/g seed. All determinations were carried out in triplicate.

2.9. Total Flavonoids

The flavonoid content was determined following the method described by Chandra et al., with minor modifications [29]. Methanolic standard solutions of quercetin were created (9–220 μg /mL). Separately, 1 mL of the avocado extract and each of the standards were mixed with 1 mL of aluminum chloride 2% on cuvettes incubated for 60 min. The absorbance of the mixtures was measured against a blank at 420 nm. The concentration of total flavonoid content in the AS extract was calculated using the calibration plot (Y = 0.0089x + 0.0268, R² = 0.9885) and expressed as mg quercetin equivalents (QE)/g seed. All determinations were carried out in triplicate.

2.10. Identification and Characterization of Bioactive Compounds by HPLC-QTOF-MS/MS

The bioactive molecules were analyzed using a quadrupole time-of-flight liquid chromatograph mass spectrometer (LCMS-9050, Shimadzu, Canby, OR, USA). Analyte separation was performed with a Restek Raptor C18 column (2.1 mm ID × 100mm, 1.8 µm particle size). The mobile phases for both positive and negative modes consisted of 0.1% formic acid aqueous solution (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The gradient began at 5% B, was held for 0.6 min, and then increased to 95% B over 27 min. Following the gradient was a 4 min wash at 95% and a 12 min equilibration period. The flow rate was set to 0.3 mL/min, and the column was maintained at 60 °C. Ionization was achieved in positive and negative modes using an electrospray ion source, with nitrogen serving as the drying and nebulizing gas and argon as the collision gas. Data analysis was performed using MZmine [30] and Sirius [31]. The raw files were converted into mzMz files using the POSTRUN Shimadzu software for processing in MZmine (4.3.0). The noise level for mass detection was 3 × 10³ for MS¹ and 2 × 10² for MS². The ion chromatogram was extracted using the ADAP module builder with a minimum absolute height of 6 × 10³ and 5 ppm m/z tolerance (scan-to-scan). The generated features were resolved using the local minimum feature resolver with a chromatogram threshold of 80%, minimum absolute height of 1 × 10³, and peak duration range of 0.1 to 1.12 min/mobility. Finally, the detected features were annotated against multiple spectral libraries, such as NIST and MSbank, with precursor tolerance of 0.01 m/z or 10.0 ppm, a minimum of 5 matched signals, and a cosine similarity score of 0.6 using NIST11 weights. The features extracted from MZmine were further processed in Sirius 5.8.3 for molecular formula assignments and structure predictions for compounds.

2.11. Statistical Analysis

Statistical analysis and plotting were performed using Origin (Origin (Pro), version 2022). Significance was assessed using a two-way two-sample t-test (p < 0.05). The standard deviation was calculated from the triplicates and plotted accordingly.

3. Results and Discussion

3.1. Total Phenolic Compounds, Flavonoids, and Antioxidant Capacity

For the present study, a hydroalcoholic solvent was chosen based on the possibility of industrial applications in food and cosmetic products. Various extraction methods were previously tested (see Supplementary Information) and corroborated with the literature. Different extraction times were tested; however, past 10 min of sonication, the temperature of the mixture would rise beyond our 65 °C cutoff. Utami et al. (2021) previously concluded that ultrasound-assisted extraction (UAE) produced the best avocado dye recoveries at shorter times [32]. On the other hand, Xu et al. (2015) previously observed that greater ethanol concentrations yielded better antioxidant activity in flower extracts [33]. Lastly, Alara et al. (2021) observed that phenolic compounds were easier to extract using semi-polar organic solvents, which agrees with our findings [34].

The Gardner index of the AS extract was 15+, resulting in an orange-colored extract with potential applications in the food industry (see Supplementary Information). Other parameters tested included the recovery yield, pH, and absorbance, with 8.11%, 5.97, and 2.89 (λ_max = 450), respectively. The protection of a biological system against oxidative stress and its harmful effects relies on the antioxidant capacity [19]. Therefore, to assess the potential benefits of the Semil 34 AS extract, we studied the antioxidant capacity (TEAC) as well as the total phenolic content (TPC) and total flavonoid content (TFC).

Table 1. Total phenolic compounds, total flavonoids, and antioxidant capacity of Semil 34 seed extract. Different letters represent different expressions of the concentration: a = (µM/g extract); b = (mg GAE/g extract); c = (mg QE/g extract); d = (mmol Trolox/g dried extract); e = (mg GAE/g dried extract); f = (mMol Trolox eq./100 g of extract); g = mMol GA/100 g of extract; h = mMol Cat. eq./100 g of extract; i = μg TE/g dried extract; j = mg TE/g extract.

Parameter Tested	Semil 34 Seed Extract	[20]	[21]	[19]	[18]
Extraction Method	Ultrasound-assisted extraction (UAE)	Maceration	Maceration	Solid–liquid extraction (SLE)	Microwave-assisted extraction (MAE)
TEAC (µM/g seed)	1743.3 ± 52.3 a	18.4 ± 0.9 d	32.51 ± 9.07 f	500 ± 10 i	266.56 ± 2.76 j
TPC (mg gallic acid equivalents/g seed)	25.86 ± 2.17 b	1303.0 ± 67.7 e	232.36 ± 12.25 g	-	307.09 ± 14.16 b
TFC (mg quercetin equivalents/g seed)	2.09 ± 0.10 c	-	2.13 ± 0.22 h	60 ± 10 e	-

compares the results obtained in this study with those of previous studies found in the literature for other varieties of avocado. The TEAC, TPC, and TFC obtained for the Semil 34 AS extract were 1743.33 µM/g seed, 25.86 mg GAE/g extract, and 2.0911 mg QE/g extract, respectively. In a previous study, Araujo et al. reported TEAC of 1065.00 µM/g seed (units were converted) for a Hass cultivar AS extract [18]. Interestingly, another study by Rosero et al. reported a TEAC value of 18,400 µM/g seed and a TPC value of 1303.0 mg GAE/g extract, which differ from our results (see Table 1). The chemical composition of avocado can vary from cultivar to cultivar, depending on several factors, such as the variety, processing method, region, climate, and precipitation [18,35]; this explains the variability found within the values for the TEAC, TPC, and TFC from different authors, even when the same variety of avocado is evaluated. This also highlights the importance of preliminary studies of the avocado residues prior to processing for the targeted industry.

3.2. Effect of pH on Antioxidant Activity

The effect of the pH on the antioxidant capacity of the AS extracts was tested at pH values of 3 and 9. As shown in Figure 1, a significant drop in the TEAC was observed in both acidic and basic conditions when compared to the unmodified extract. This agrees with previous studies that observed a pH-dependent effect on the antioxidant activity of flavonoids [36]. These results are key when exploring future applications of AS extracts. For example, to benefit from its additional antioxidant properties in the food industry, the colored extract must be incorporated in a matrix with a relatively neutral pH, which excludes processed dairy products such as yogurt. The stabilization of the phytochemical compounds found in the extract through encapsulation and emulsion technologies should be considered to ensure the bioactivity when incorporated into cosmetics or food products [16].

3.3. Proposed Identification of Chemicals in Avocado (P. americana Mill.) Semil 34 Seed

The chemical characterization of Semil 34 was conducted using negative and positive electrospray ionization (ESI) modes via UPLC-MS (Figure S5). A total of 53 putative compounds were identified, based on their predicted chemical formulas and high mass accuracy (Table 2). The chemical classification tools Classyfire and NPClassifier were also utilized to group the previously identified compounds [37,38,39], allowing the classification of the metabolites into carboxylic acids and derivatives, cinnamic acid and derivatives, fatty acyls, flavonoids, furanoid ligans, glycerolipids, glycerophospholipids, organic sulfuric acids and derivatives/arylsulfates/phenylsulfates, organooxygen compounds, and prenol lipids.

Three molecules were tentatively identified among the carboxylic acids and derivates. For example, compound 2 showed a precursor ion at m/z [M-H]⁻ 191.0188, assigned to citric acid. Previous studies have identified citric acid in avocado seeds with precursor ions at m/z 191.021 and 191.0539, which aligns with our findings [19,40]. Within cinnamic acid and derivatives, a coumaroylquinic acid isomer with m/z [M-H]⁻ 337.0922 was found. This compound was previously reported as 5-O-p-coumaroylquinic acid and cis-3-p-coumarylquinic acid at m/z 337.04 and 337, respectively [41,42]. Caffeic acid [M+H-H₂O]⁺ with m/z 163.0387 was also identified, a compound that is widely studied in avocado [20,41,43,44]. An example within the fatty acyl compounds is 2-linoleoylglycerol [M+H-H₂O]⁺ with m/z 337.2732, which has also been previously reported with a fragment ion at 337 [45]. Examples of organooxygen compounds include perseitol, observed at m/z 211.0815 [M-H]⁻, previously identified by Rojas-García et al. at m/z 211.0805, as well as other authors [19,45,46]. Quinic acid with m/z 191.0553 [M-H]⁻ and neochlorogenic acid at m/z 353.0878 [M-H]⁻ were also identified and corroborated by the literature [19,45,47]. Regarding flavonoids, compounds 29 and 30 were found, with proposed compounds Procyanidin B1 and B2, with m/z 579.1490 and 579.1493 [M+H]⁺, respectively, agreeing with previous studies focused on the Hass and Fuerte varieties [48]. Molecules specifically found in avocado, such as compounds 15, 16, 17, and 20, were presented as avocadenol A with m/z 231.2107 [M+H]⁺, 4-AcO-avocadyne with m/z 309.2424 [M+H-H₂O]⁺, avocadene acetate with m/z 293.2472 [M+H-2H₂O]⁺, and avocadyne with m/z 249.2210 [M+H-2H₂O]⁺, which have also been previously reported [45,49,50].

Table 2. Compounds identified from Semil 34 seed extract using HPLC-QTOF-MS/MS.

Peak No.	RT	Proposed Compound Identification	Molecular Formula	Ionization	m/z Observed	m/z Theoretical	Error (ppm)	References
Carboxylic acids and derivatives
1	0.98	gamma-Aminobutyric acid	C4H9NO2	[M+H]+	104.0703	104.0706	−2.8	[51]
2	1.14	Citric acid	C6H8O7	[M-H]−	191.0188	191.0197	−4.7	[40,47]
3	1.36	Pantothenic acid	C9H17NO5	[M+H]+	220.1178	220.1179	−0.4	[51]
Cinnamic acid and derivatives
4	1.64	Neochlorogenic acid	C16H18O9	[M-H]−	353.0873	353.0878	−1.4	[47]
5	2.85	Caffeic acid	C9H8O4	[M+H-H2O]+	163.0387	163.0389	−1.2	[41]
6	2.85	Caffeoylquinic acid	C16H18O9	[M+H]+	355.1022	355.1023	−0.3	[20,41,48]
7	3.53	3-O-p-Coumaroylquinic acid	C16H18O8	[M-H]−	337.0922	337.0929	−2	[18,42]
Fatty acyls
8	15.92	Methyl tetradecanoate	C15H30O2	[M+H]+	243.2316	243.2318	−0.6	[51]
9	17.60	9,10-Methylenehexadecanoic acid	C17H32O2	[M+H-H2O]+	251.2367	251.2369	−0.7
10	27.45	2-Linoleoylglycerol	C21H38O4	[M+H-H2O]+	337.2732	337.2737	−1.4	[52]
11	28.32	1-Palmitoyl-2-linoleoyl-rac-glycerol	C37H68O5	[M+H]+	593.5131	593.5138	−1.17	[52]
12	20.39	Monopalmitolein	C19H36O4	[M+H-H2O]+	311.2569	311.2580	−3.5
13	6.51	3-Hydroxy-3-methylglutaric acid	C6H10O5	[M+H]+	163.0599	163.0601	−1.2	[53]
14	21.99	Linoelaidic acid	C18H32O2	[M+H-H2O]+	263.2367	263.2369	−0.7
15	18.36	Avocadenol A	C17H32O3	[M+H-3H2O]+	231.2105	231.2107	−0.8	[54,55]
16	18.52	4-AcO-avocadyne	C19H34O4	[M+H-H2O]+	309.2423	309.2424	−0.3	[54,55]
17	19.24	Avocadene acetate	C19H36O4	[M+H-2H2O]+	293.2472	293.2475	−1.02	[54,55,56]
18	13.58	16-Heptadecyn-4-one, 1,2-dihydroxy	C17H30O3	[M+H-H2O]+	265.2160	265.2162	−0.7
19	18.03	9E,11E-Octadecadienoic acid	C18H32O2	[M+H-H2O]+	263.2367	263.2369	−0.7	[57]
20	18.62	Avocadyne	C17H32O3	[M+H-2H2O]+	249.2210	249.2213	−1.2	[58]
21	18.63	9,10-Methylenehexadecanoic acid	C17H32O2	[M+H-2H2O]+	233.2262	233.2272	−4.2
22	9.96	Nonadecanedioic acid	C19H36O4	[M+H-H2O]+	311.2575	311.2581	−1.9
23	23.66	Oleoyl ethylamide	C20H39NO	[M+H]+	310.3101	310.3103	−0.6
24	25.47	cis-13-Docosenoic acid	C22H42O2	[M+H-H2O]+	321.3149	321.3152	−0.9	[59]
25	10.62	(9Z)-5,8,11-Trihydroxyoctadec-9-enoic acid	C18H34O5	[M-H]−	329.2329	329.2333	−1.2
26	14.84	(12Z)-9,10,11-Trihydroxyoctadec-12-enoic acid	C18H34O5	[M-H-H2O]−	311.2221	311.2228	−2.2
27	25.47	Erucamide	C22H43NO	[M+H]+	338.3435	338.3417	5.3
28	23.0805	1,2,4-nonadecanetriol	C19H40O3	[M+H]+	299.2945	299.2945	0.0
Flavonoids
29	2.41	Procyanidin B2	C30H26O12	[M+H]+	579.1493	579.1497	−0.6	[48]
30	3.82	Procyanidin B1	C30H26O12	[M+H]+	579.1490	579.1497	−1.2	[48]
31	4.04	Procyanidin A1	C30H24O12	[M+H]+	577.1338	577.1341	−0.5	[58]
32	3.54	Pavetannin B6	C45H36O18	[M+H]+	865.1968	865.1974	1.3
Furanoid ligans
33	8.16	Syringaresinol	C22H26O8	[M+H-H2O]+	401.1592	401.1595	0.3	[20]
Glycerolipids
34	18.57	Lichesterinic acid	C19H32O4	[M-H]−	325.2377	325.238	−0.9
Glycerophospholipids
35	18.11	1-Oleoyl-sn-glycero-3-phosphocholine	C26H52NO7P	[M+CHO2]−	566.3457	566.3452	0.8	[45]
36	15.84	D-myo-Inositol, 1-[2-hydroxy-3-[(1-oxo-9,12-octadecadienyl)oxy]propyl hydrogen phosphate], [S-(Z,Z)]-	C25H49O12P	[M-H]−	595.2885	595.2889	0.4
37	15.35	1-Myristoyl-sn-glycero-3-phosphocholine (LPC 14:0)	C22H46NO7P	[M+H]+	468.3079	468.3085	−1.2
38	16.01	1-palmitoleoyl-glycerophosphocholine (LPC 16:1)	C24H48NO7P	[M+H]+	494.3237	494.3241	−0.8
39	17.08	1-(10Z-heptadecenoyl)-2-hydroxy-sn-glycero-3-phosphocholine (17:1 Lyso PC)	C25H50NO7P	[M+H]+	508.3393	508.3397	−0.7
40	17.11	1-Palmitoyl-sn-glycero-3-phosphocholine (LPC 16:0)	C24H50NO7P	[M+H]+	496.3394	496.3398	−0.8	[60]
41	17.40	1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (LPE 16:0)	C21H44NO7P	[M+H]+	454.2923	454.2928	−1.1
42	19.73	1-Stearoyl-sn-glycero-3-phosphocholine (LPC 18:0)	C26H54NO7P	[M+H]+	524.3715	524.3710	0.9	[60]
Organic sulfuric acids and derivatives/arylsulfates/phenylsulfates
43	3.04	3-[3-(Sulfooxy)phenyl]propanoic acid	C9H10O6S	[M-H]−	245.0117	245.0125	−3.2	[61]
Organooxygen compounds
44	0.75	Perseitol	C7H16O7	[M-H]−	211.0815	211.0823	−3.7	[62]
45	26.67	Soyacerebroside I	C40H75NO9	[M-H]−	712.5361	712.5369	−1.1
46	5.12	Phenetyl rutenoside	C20H30O10	[M+NH4]+	448.2170	448.2188	−4.0
47	0.94	Quinic acid	C7H12O6	[M-H]−	191.0553	191.0561	0.8	[41]
48	0.99	Volemitol	C7H16O7	[M-H]−	211.0814	211.082	0.6	[63]
49	1.22	D-Picose	C6H12O6	[M-H2O-H]−	161.0446	161.0455	−5.5
50	6.16	Isomaltulose	C12H22O11	[M+H-3H2O]+	289.0917	289.0928	−3.8
51	6.16	Sucrose	C12H22O11	[M+H-2H2O]+	307.1022	307.1034	3.9	[64]
52	2.95	(1r,3R,4s,5S)-4-(((2E)-3-(3,4-Dihydroxyphenyl)prop-2-enoyl)oxy)-1,3,5-trihydroxycyclohexanecarboxylic acid	C16H18O9	[M-H]−	353.0873	353.0878	−1.4
Prenol lipids
53	7.52	Abscisic acid	C15H20O4	[M+H-H2O]+	247.1326	247.1329	0.3	[65]

Table 2. Compounds identified from Semil 34 seed extract using HPLC-QTOF-MS/MS.

Peak No.	RT	Proposed Compound Identification	Molecular Formula	Ionization	m/z Observed	m/z Theoretical	Error (ppm)	References
Carboxylic acids and derivatives
1	0.98	gamma-Aminobutyric acid	C4H9NO2	[M+H]+	104.0703	104.0706	−2.8	[51]
2	1.14	Citric acid	C6H8O7	[M-H]−	191.0188	191.0197	−4.7	[40,47]
3	1.36	Pantothenic acid	C9H17NO5	[M+H]+	220.1178	220.1179	−0.4	[51]
Cinnamic acid and derivatives
4	1.64	Neochlorogenic acid	C16H18O9	[M-H]−	353.0873	353.0878	−1.4	[47]
5	2.85	Caffeic acid	C9H8O4	[M+H-H2O]+	163.0387	163.0389	−1.2	[41]
6	2.85	Caffeoylquinic acid	C16H18O9	[M+H]+	355.1022	355.1023	−0.3	[20,41,48]
7	3.53	3-O-p-Coumaroylquinic acid	C16H18O8	[M-H]−	337.0922	337.0929	−2	[18,42]
Fatty acyls
8	15.92	Methyl tetradecanoate	C15H30O2	[M+H]+	243.2316	243.2318	−0.6	[51]
9	17.60	9,10-Methylenehexadecanoic acid	C17H32O2	[M+H-H2O]+	251.2367	251.2369	−0.7
10	27.45	2-Linoleoylglycerol	C21H38O4	[M+H-H2O]+	337.2732	337.2737	−1.4	[52]
11	28.32	1-Palmitoyl-2-linoleoyl-rac-glycerol	C37H68O5	[M+H]+	593.5131	593.5138	−1.17	[52]
12	20.39	Monopalmitolein	C19H36O4	[M+H-H2O]+	311.2569	311.2580	−3.5
13	6.51	3-Hydroxy-3-methylglutaric acid	C6H10O5	[M+H]+	163.0599	163.0601	−1.2	[53]
14	21.99	Linoelaidic acid	C18H32O2	[M+H-H2O]+	263.2367	263.2369	−0.7
15	18.36	Avocadenol A	C17H32O3	[M+H-3H2O]+	231.2105	231.2107	−0.8	[54,55]
16	18.52	4-AcO-avocadyne	C19H34O4	[M+H-H2O]+	309.2423	309.2424	−0.3	[54,55]
17	19.24	Avocadene acetate	C19H36O4	[M+H-2H2O]+	293.2472	293.2475	−1.02	[54,55,56]
18	13.58	16-Heptadecyn-4-one, 1,2-dihydroxy	C17H30O3	[M+H-H2O]+	265.2160	265.2162	−0.7
19	18.03	9E,11E-Octadecadienoic acid	C18H32O2	[M+H-H2O]+	263.2367	263.2369	−0.7	[57]
20	18.62	Avocadyne	C17H32O3	[M+H-2H2O]+	249.2210	249.2213	−1.2	[58]
21	18.63	9,10-Methylenehexadecanoic acid	C17H32O2	[M+H-2H2O]+	233.2262	233.2272	−4.2
22	9.96	Nonadecanedioic acid	C19H36O4	[M+H-H2O]+	311.2575	311.2581	−1.9
23	23.66	Oleoyl ethylamide	C20H39NO	[M+H]+	310.3101	310.3103	−0.6
24	25.47	cis-13-Docosenoic acid	C22H42O2	[M+H-H2O]+	321.3149	321.3152	−0.9	[59]
25	10.62	(9Z)-5,8,11-Trihydroxyoctadec-9-enoic acid	C18H34O5	[M-H]−	329.2329	329.2333	−1.2
26	14.84	(12Z)-9,10,11-Trihydroxyoctadec-12-enoic acid	C18H34O5	[M-H-H2O]−	311.2221	311.2228	−2.2
27	25.47	Erucamide	C22H43NO	[M+H]+	338.3435	338.3417	5.3
28	23.0805	1,2,4-nonadecanetriol	C19H40O3	[M+H]+	299.2945	299.2945	0.0
Flavonoids
29	2.41	Procyanidin B2	C30H26O12	[M+H]+	579.1493	579.1497	−0.6	[48]
30	3.82	Procyanidin B1	C30H26O12	[M+H]+	579.1490	579.1497	−1.2	[48]
31	4.04	Procyanidin A1	C30H24O12	[M+H]+	577.1338	577.1341	−0.5	[58]
32	3.54	Pavetannin B6	C45H36O18	[M+H]+	865.1968	865.1974	1.3
Furanoid ligans
33	8.16	Syringaresinol	C22H26O8	[M+H-H2O]+	401.1592	401.1595	0.3	[20]
Glycerolipids
34	18.57	Lichesterinic acid	C19H32O4	[M-H]−	325.2377	325.238	−0.9
Glycerophospholipids
35	18.11	1-Oleoyl-sn-glycero-3-phosphocholine	C26H52NO7P	[M+CHO2]−	566.3457	566.3452	0.8	[45]
36	15.84	D-myo-Inositol, 1-[2-hydroxy-3-[(1-oxo-9,12-octadecadienyl)oxy]propyl hydrogen phosphate], [S-(Z,Z)]-	C25H49O12P	[M-H]−	595.2885	595.2889	0.4
37	15.35	1-Myristoyl-sn-glycero-3-phosphocholine (LPC 14:0)	C22H46NO7P	[M+H]+	468.3079	468.3085	−1.2
38	16.01	1-palmitoleoyl-glycerophosphocholine (LPC 16:1)	C24H48NO7P	[M+H]+	494.3237	494.3241	−0.8
39	17.08	1-(10Z-heptadecenoyl)-2-hydroxy-sn-glycero-3-phosphocholine (17:1 Lyso PC)	C25H50NO7P	[M+H]+	508.3393	508.3397	−0.7
40	17.11	1-Palmitoyl-sn-glycero-3-phosphocholine (LPC 16:0)	C24H50NO7P	[M+H]+	496.3394	496.3398	−0.8	[60]
41	17.40	1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (LPE 16:0)	C21H44NO7P	[M+H]+	454.2923	454.2928	−1.1
42	19.73	1-Stearoyl-sn-glycero-3-phosphocholine (LPC 18:0)	C26H54NO7P	[M+H]+	524.3715	524.3710	0.9	[60]
Organic sulfuric acids and derivatives/arylsulfates/phenylsulfates
43	3.04	3-[3-(Sulfooxy)phenyl]propanoic acid	C9H10O6S	[M-H]−	245.0117	245.0125	−3.2	[61]
Organooxygen compounds
44	0.75	Perseitol	C7H16O7	[M-H]−	211.0815	211.0823	−3.7	[62]
45	26.67	Soyacerebroside I	C40H75NO9	[M-H]−	712.5361	712.5369	−1.1
46	5.12	Phenetyl rutenoside	C20H30O10	[M+NH4]+	448.2170	448.2188	−4.0
47	0.94	Quinic acid	C7H12O6	[M-H]−	191.0553	191.0561	0.8	[41]
48	0.99	Volemitol	C7H16O7	[M-H]−	211.0814	211.082	0.6	[63]
49	1.22	D-Picose	C6H12O6	[M-H2O-H]−	161.0446	161.0455	−5.5
50	6.16	Isomaltulose	C12H22O11	[M+H-3H2O]+	289.0917	289.0928	−3.8
51	6.16	Sucrose	C12H22O11	[M+H-2H2O]+	307.1022	307.1034	3.9	[64]
52	2.95	(1r,3R,4s,5S)-4-(((2E)-3-(3,4-Dihydroxyphenyl)prop-2-enoyl)oxy)-1,3,5-trihydroxycyclohexanecarboxylic acid	C16H18O9	[M-H]−	353.0873	353.0878	−1.4
Prenol lipids
53	7.52	Abscisic acid	C15H20O4	[M+H-H2O]+	247.1326	247.1329	0.3	[65]

3.4. Proposed Identification of Novel Chemicals in Avocado (P. americana Mill.) Semil 34 Seed

We also present 23 tentative compounds that, to our knowledge, have not been previously reported in the literature. These compounds belong to various classes, including 10 fatty acyl compounds, five glycerophospholipids, six organooxygen compounds, one glycolipid, and one flavonoid. Among the fatty acyl compounds, pavetannin B1 was provisionally identified with a precursor ion at m/z 865.1968 [M+H]⁺, displaying major fragment ions at m/z 695, 575, 533, and 287. Another example is linoelaidic acid, detected at m/z 263.2367 [M+H-H2O]⁺ with a mass error of −0.7 ppm. Its fragmentation spectrum exhibited major product ions at m/z 245, 109, and 95. An example of an organooxygen compound is (1r,3R,4s,5S)-4-(((2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl)oxy)-1,3,5 trihydroxycyclohexanecarboxylic acid, with m/z of 353.0878 [M-H]⁻ and major ions such as 191, 179, and 134. Other proposed compounds include isomaltulose, with a precursor ion at m/z 289.0917 [M+H-3H2O]⁺, D-picose at m/z 161.0446 [M-H2O-H]⁻, and LPE 16:0 with m/z 454.2923 [M-H]⁺, among others.

4. Conclusions

The current research targeted the sustainability of the agroindustry through the valorization of the residues produced during its operations. Understanding the phytochemical composition of avocado seeds is key when considering their further applications in food, cosmetics, and/or pharmaceuticals. Our results indicate that seed extracts from the cultivar Semil 34 offer promising health benefits due to their antioxidant capacity and the various chemicals present. These compounds have been associated with antidiabetic, anti-inflammatory, antioxidant, and antimicrobial properties that could benefit several industries, particularly the food sector. Further research is needed to completely characterize the cultivar Semil 34 and fully comprehend all of the possible benefits within this variety. Moreover, prior to its incorporation in food matrices, more research is required to determine the cytotoxicity and negative health impacts and to assess the appropriate technology needed to stabilize the extract and prevent the loss of antioxidants.