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Home , Corylus avellana, Obtusifoliol, Oleic acid

Hindawi Journal of Chemistry Volume 2017, Article ID 2609549, 11 pages https://doi.org/10.1155/2017/2609549

Research Article Chemical Characterization of Major and Minor Compounds of Oils: , , and Nut

Gabriel D. Fernandes,1 Raquel B. Gómez-Coca,2 María del Carmen Pérez-Camino,2 Wenceslao Moreda,2 and Daniel Barrera-Arellano1

1 and Oils Laboratory, Department of Food Technology, University of Campinas, Avenida Bertrand Russell, 13081-970 Campinas, SP, Brazil 2Department of Characterization and Quality of , Instituto de la Grasa, CSIC, Campus of Universidad Pablo de Olavide, Building 46, 41013 Seville, Spain

Correspondence should be addressed to Gabriel D. Fernandes; [email protected]

Received 5 February 2017; Accepted 6 April 2017; Published 2 May 2017

Academic Editor: Maria B. P. P. Oliveira

Copyright © 2017 Gabriel D. Fernandes et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The aim of this work was to characterize the major and minor compounds of laboratory-extracted and commercial oils from sweet almond, hazelnut, and pecan nut. Oils from sweet almond, hazelnut, and pecan nut were obtained by means of an expeller system, while the corresponding commercial oils were provided from Vital Atmanˆ (BR). The contents of triacylglycerols, fatty acids, aliphatic and terpenic alcohols, desmethyl-, methyl-, and dimethylsterols, , and tocopherols were determined. Oleic, palmitic, and linoleic acids were the main fatty acids. Desmethylsterols were the principal minor compounds with 𝛽-sitosterol being the most abundant component. Low amounts of aliphatic and terpenic alcohols were also found. The major tocopherol in hazelnut and sweet almond oils was 𝛼-tocopherol, whereas 𝛾-tocopherol prevailed in pecan nut oil. Principal component analysis made it possible for us to differentiate among samples, as well as to distinguish between commercial and laboratory-extracted oils. Heatmap highlighted the main variables featuring each sample. Globally, these results have brought a new approach on nut oil characterization.

1. Introduction The almond group is composed of two species, namely, Prunus dulcis (sweet ) and Prunus amara (bit- Nuts belong to various families, although they have ter almond). Almond oil is extracted mainly from sweet special common features such as high oil content and large almonds, which contain around 50% oil. This extraction is size when compared to other oilseed species. Almonds commercially conducted by the cold press and/or solvent (Prunus dulcis, family Rosaceae), (Corylus avellana, extraction [3]. According to FAO [4], USA is the main family ), and pecan nuts (Carya illinoinensis, almond producer in the world (∼62% of the total production) family Juglandaceae) are part of the main group of tree nuts followed by Spain and Australia (∼5% each). Chemically and nut oil sources. In many parts of the world such as the speaking, sweet almond oil has been described as an unsat- Mediterranean countries and North America, tree nuts are urated oil, with oleic acid (O, C18:1) being the main fatty not only an important oil crop but also an essential dietary acid (∼65%) [5], with 𝛽-sitosterol as the most representative component, acting as energy and functional compound and 𝛼-tocopherol as the major tocopherol [6, 7]. Table 1 sources. Actually, nut oils have been widely enjoyed for food shows the detailed composition of almond oil based on applications, mainly due to their particular flavor and, more bibliographic research [1–3, 5–7]. recently, because of their relationship with health-promoting Hazelnut (Corylus avellana) is a nut included in the effects. Besides, tree nut oils are also widely used in the Mediterraneandiet,whosemainworldwideproduceris cosmetic industry [1, 2]. , with just about 63% of the total production 2 Journal of Chemistry in 2012 [4]. Hazelnut kernels contain around 60% oil, which individual sample of each commercial nut oil from sweet is obtained by cold press and/or solvent extraction. Hazelnut almonds, hazelnut, and pecan nut were provided by Vital oil has been frequently compared to due to their Atmanˆ (Uchoa, SP, Brazil). Both nuts and nut oils were ∘ similar compositions: oleic acid as the main and 𝛽- properly stored at 4 C until extraction and analysis. Each sitosterol as the main minor compound (Table 1) [8, 9]. Actu- chemical characterization was performed in triplicate. ally, hazelnut oil is commonly used in the cosmetic industry, although its current prominence as health-promoting oil has 2.3. Extraction of Nut Oils. For each extraction, 1000 g nuts increased its uses as food and even gourmet oil. were milled in a knife mill and the obtained products were Pecan nut (Carya illinoinensis) originates from the USA taken to an expeller press system, Komet Oil Press (IBG but is well adapted in several countries including Aus- Monforts Oekotec GmbH & Co.KG., Germany), and then tralia, South Africa, and different parts of South America. filtered through filter paper to remove any solid material. Nowadays, more than 80% of the total world production is Each laboratory sample was a pooled sample from the whole produced in the USA. This nut contains 70% oil, which is extraction process, which was performed separately for each ∘ easily extracted by means of an expeller press. Also here, oleic nut. The oil obtained was stored at 4 Cuntilanalysis. acid is the predominant fatty acid (∼60%), and, curiously, 𝛾- tocopherol has been reported as the main tocopherol, as can 2.4. Chemical Characterization be seen in Table 1 [10–12]. All these nuts are perennial crops; thus, there is a partic- 2.4.1. Fatty Acid Composition. The fatty acid composition was ular concern about the sustainability of these crops. Most of determined according to IUPAC Standard Methods [15, 16], these crops are related to some specific regions in the world, as the composition of fatty acid methyl (FAME) by GC. especially USA Midwest and Mediterranean east. In this way, Transesterification of the oils was carried out with KOH in they are very important to support the commercial balance of methanol at a concentration of 2 mol/L. The chromatographic these regions/countries, enabling the economic development analysis was done using an Agilent 5890 GC system (Agilent of some communities. Additionally, from the environmental Technologies, Santa Clara, California) equipped with an point of view, nut have been considered an excellent automated liquid sampler (1 𝜇L injections), split injector option for native reforestation with commercial exploitation (1 : 50 split ratio), polar capillary column (SPTM-2380, 100 m capacity [2, 13]. × 0.25 mm internal diameter (i.d.) × 0.20 𝜇m film thickness), Nut oils are getting an outstanding position as gourmet and flame ionization detector (FID). Hydrogen was used as carrier gas at a flow rate of 1.0 mL/min. The initial and health-promoting oils, for both their sensory and their ∘ oven temperature was 180 C, and the temperature gradient nutritional characteristics. Actually, as far as the nutritional ∘ ∘ ∘ was from 180 C to 220 Cat3C/min. The detector and features are concerned, the high amounts of oleic acid, as well ∘ ∘ as those of , allow frequent comparisons of these injector temperatures were 225 Cand250C, respectively. oils with olive oil. However, nut oils have also been indicated Peak identification was carried out by means of comparison as inducers of allergic reactions in consumers [14]. with a standard chromatogram. Data were described as fatty Theaimofthisworkistocharacterizethemajorand acid profiles by peak area normalization. minor compounds of laboratory-extracted and commercial nut oils from sweet almond, hazelnut, and pecan nut in order 2.4.2. Triacylglycerol Composition. This determination was to increase the knowledge about the chemical composition of done following the procedure established by Moreda et al. nutoilsaswellastoestablish,basedonastatisticalapproach, [17]. For oil purification, a Si-SPE cartridge was washed the main compounds which would allow for distinguishing withoutvacuumwith6mLhexane.Afterthat,asolutionof among oil samples and their origin. the oil (0.12 g) in 0.5 mL hexane was added. The solution was pulled through the cartridge and then eluted with 10 mL 2. Materials and Methods hexane-diethyl ether (87 : 13 v/v) solution. The eluted solvents were evaporated to dryness under reduced pressure at room 2.1. Chemicals. , diethyl ether, hexane, propionitrile, temperature. The residue was dissolved in 2 mL acetone. For and tetrahydrofuran (THF) were supplied by VWR Interna- triacylglycerol (TAG) analysis, 10 𝜇Lofthissolutionwas tional (West Chester, PA, USA). Silica solid phase extrac- injected directly, using the autosampler (508 system), in a tion (Si-SPE) cartridges were from Varian (EA Middelburg, RP-HPLC system. The separation was done on a Merck Li- The Netherlands). Potassium hydroxide (KOH) was from Chrospher 100 RP-18 column (250 mm × 4mmi.d. × 4 𝜇m ∘ Panreac (Montcada i Reixac, Barcelona, Spain). Hexamethyl- particle size) thermostated at 20 C. The liquid chromato- disilazane, pyridine, trimethylchlorosilane, and standards graph (Beckman Coulter, Fullerton, CA, USA) was equipped 𝛼 𝛾 𝛽 𝛿 of -, -, -, and -tocopherol were from Merck (Merck with a pumping unit (118 solvent module) and propionitrile 𝛼 𝛽 Group, Darmstadt, Germany). Standards of 5 -cholestan-3 - was used as mobile phase at a flow rate of 0.6 mL/min. ol, squalane, and n-eicosanol were from Sigma-Aldrich Co. Detection was done with a PerkinElmer 200 RI detector. (St. Louis, MO, USA). All chemical reagents were at least of Identification of TAG peaks was done by comparison with the analytical grade. chromatograms established by the authors’ method, as well as the Supelco 37 Component FAME Mix (47885-U SUPELCO). 2.2. Samples. Nuts from sweet almonds, hazelnuts, and pecan The data were processed by peak area normalization and nuts were obtained in local grocery stores in Brazil. One expressed as TAG percentage. Journal of Chemistry 3

Table 1: Bibliographic information on the chemical composition of almond, hazelnut, and pecan nut oils. References [1–3, 5–8, 10, 21–29].

Nut oil Almond Hazelnut Pecan nut −1 Oil amount (g.100 g ) 25.1–60.7 8.10–67 58-74 - 14:0 0–0.07 0–0.1 0.05–0.09 - 16:0 4.7–15.8 4.5–6.5 6.4–7.6 - 16:1 0.1–2.5 0.1–0.3 0.1–0.2 - 18:0 0.3–2.5 0.4–3.8 2.2–2.8 Fatty acid composition (% area) Oleic acid - 18:1 50.4–81.2 76.3–86.5 49.6–62.1 - 18:2 6.21–37.1 6.5–15.6 27.2–37.7 Linolenic acid - 18:3 0–11.1 0.1–1.9 1.4–1.9 - 20:0 0.04–0.2 0–0.2 0.34 LLLn 0.1 — LLL 8.7 3.7 — OLLn 0.1 0.5 — OLLn 27.6 12.3 — OLnO — 0.7 — LLP 4.8 1.6 — Triacylglycerol composition (% area) OLO 28.0 28.2 — LOP 11.3 5.2 — PLP 0.5 0.2 — OOO 13.3 36.5 — SLO 1.8 1.4 — OOP 2.7 6.1 — SOO 0.6 2.8 — −1 Total mg⋅kg 2178–2777 1096–6031 1899 — 0.8–2.3 — 24-Methylene-cholesterol — 0–0.1 — Campesterol 2.5 4.8–7.4 2.7 Campestanol — 0–0.2 — 2.5 1.3–2.1 17.9 Δ7-Campesterol — 0–0.4 — Sterol composition (% area) Δ5,23-Stigmastadienol + Clerosterol — 0.9–1.3 — 𝛽-Sitosterol 55.9–95.1 78.1–90.4 82.8 Sitostanol — 1.8- 3.6 — Δ5-Avenasterol 8.5–28.2 1.3–5.2 — Δ5,24-Stigmastadienol — 0.3–1.1 — Δ7-Stigamastenol — 0.3–2.3 — Δ7-Avenasterol — 0.5–1.9 — −1 Squalene mg⋅kg 95.0 186.0–371.0 152.0 −1 Totaltocopherolmg⋅kg 451.0 25.8–690.8 180.0 𝛼-Tocopherol 97.3 53.8–90.6 12.0 𝛽-Tocopherol — 2.1–4.2 — Tocopherol and tocotrienol composition (% of total content) 𝛾-Tocopherol 2.8 3.1–41.9 168.0 𝛿-Tocopherol ——— 𝛼-Tocotrienol — 0–7.1 — —: not determined and/or evaluated by the authors. P: palmitic acid; S: stearic acid; O: oleic acid; L: linoleic acid; Ln: linolenic acid.

2.4.3. Sterol Composition and Aliphatic Alcohols. Sterols and bytheInternationalOliveCouncil[18,19].Tosummarize, aliphatic alcohols are components of the unsaponifiable frac- 5 g of oil was saponified with 50 mL ethanolic KOH solution, tion. Therefore, removing the saponifiable compounds previ- at a concentration of 2 mol/L, during 1 h under reflux. The ously to their determination is essential. In this line, the sam- unsaponifiable compounds were then extracted with diethyl ples were analyzed according to the methodology proposed ether (3 × 80 mL) and the organic phase was washed with 4 Journal of Chemistry distilled water until complete neutralization. After drying, i.d. × 4 𝜇mparticlesize).Theelutionsolventwasamixture the unsaponifiable matter was fractionated by thin layer of hexane : 2-propanol (99 : 1, v/v) at a flow rate of 1 mL/min. chromatography (TLC) using silica plates impregnated with Detection was done by means of a RF-10AXL Shimadzu KOH.Eachplatewasdevelopedtwicewithamixtureof fluorescence detector, setting excitation at 𝜆 =290nmand petroleum ether : diethyl ether (87 : 13, v/v). After separation, emission at 𝜆 =330nm.Theanalyticalcurveforquantitative four bands could be observed, corresponding to desmethyls- and qualitative determinations was performed by means terols, methylsterols, aliphatic alcohols, and dimethylsterols. of injections of tocopherol standards at concentrations of Each band was then scratched off and extracted with hot 4–6 𝜇g/mL in hexane. Results were expressed in mg/kg for chloroform and diethyl ether. The solutions were evaporated each tocopherol compound. to dryness, derivatized with 500 𝜇L of a 1 : 3 : 9 (v/v/v) trimethylchlorosilane : hexamethyldisilazane : pyridine solu- 2.4.6. Stigmastadienes. In order to verify the presence of tion,andanalyzedbyGC.Thegaschromatograph(Agilent refined oils, stigmastadienes were determined only in com- 6890N) was equipped with an automated liquid sampler mercial samples. The method described in COI/T.20/Doc. (1 𝜇L injections), split injector (1 : 50 split ratio), a fused silica number 11 was followed for this determination [31]. Oil low-polarity capillary column (DB-5HT, 30 m × 0.25 mm samples (20 g) together with 1 mL internal standard solution i.d. × 0.25 𝜇m film thickness, Agilent Technologies, Santa (3,5-cholestadiene, 20 𝜇g/mL) were saponified with 75 mL Clara, California), and FID. The oven program for the alcoholic KOH (10%) during 30 min under reflux. The determination of desmethylsterols was set isothermally at ∘ unsaponifiable compounds were then extracted with hexane 260 C.Hydrogenwasusedascarriergasataflowrateof (2 × 100 mL), and the organic phase neutralized washing 1 mL/min. For the analysis of the other fractions, a temper- ∘ it with an -water (1 : 1 v/v) solution. The solvent was ature gradient was applied: starting at 220 C(2min)until ∘ ∘ ∘ then evaporated to dryness in a rotary evaporator at 30 C. 295 Cat2C/min. The temperatures of injector and detector ∘ After this preparation, the obtained unsaponifiable matter were 300 C. The quantitative determinations were done using was fractionated by silica gel column chromatography using internal standards: 𝛼-cholestanol for desmethylsterols and n- hexane as mobile phase. The first 30 mL eluate were discarded eicosanol for aliphatic alcohols, methyl- and dimethylsterols. and the following 40 mL were collected, dried, and injected Data were always expressed as the total (mg/kg oil) of each into the chromatograph. The GC system was an Agilent compound class, and the profile of each class was described as 6890N Gas Chromatograph (Agilent Technologies, Santa the percentage of the area of each compound within the class, Clara, California) equipped with an automatic liquid sampler, according to the method recommendation. Peak assignments split injection (15 : 1 ratio), and a FID. The parameters for were carried out by relative retention time calculation and the GC assays were DB5-HT column; 30 m × 0.25 mm i.d. × comparison with reference chromatograms as described in 0.10 𝜇m film (Agilent Technologies, Santa Clara, California); each method. 1.0 𝜇L injection volume and hydrogen as carrier gas at ∘ 1 mL/min. The injector and detector temperatures were 300 C ∘ 2.4.4. Squalene. This procedure derives from that published and 320 C, respectively. The oven temperature program was ∘ ∘ ∘ previously [20]. Oil samples (0.04 g) together with 40 𝜇L 235 C for 6 minutes, rising at 2 C/minupto285C. For peak internal standard (squalane 5 mg/mL) were dissolved in 1 mL identification, the retention time along with a comparison hexane and saponified at room temperature with 200 𝜇L with the standard chromatogram described in the method methanolic KOH at a concentration of 2 mol/L. After sep- was evaluated. For quantitative evaluation, 3,5-cholestadiene aration (by gravity), the upper phase was washed with 3 × was used as internal standard. Data were expressed in mg/kg. 400 𝜇L ethanol : water 1 : 1, v/v, and 1 𝜇Lofthesupernatant was analyzed by GC. GC analyses were carried out with 2.5. Statistical Analysis. In order to verify significant dif- an Agilent 6890N Gas Chromatograph (Agilent Technolo- ferences (𝑝 < 0.05) among samples, for each feature, an gies, Santa Clara, California) equipped with an automatic ANOVA test was accomplished in Metaboanalyst 3.0 web- liquid sampler, split injection (20 : 1 ratio), and a FID. The based tool [32]. After that, a multivariate statistical analysis conditions for the GC assays were DB5-HT column; 30 m was performed with full information of the chemical charac- × 0.25 mm i.d. × 0.10 𝜇m film (Agilent Technologies, Santa terization; data files were saved as.csv format and uploaded Clara, California); hydrogen carrier gas at 0.8 mL/min. The ∘ into the Metaboanalyst 3.0 web-based tool. A principal oven worked isothermally at 250 Cfor10minutes.The ∘ ∘ component analysis (PCA) was performed considering all injector and detector temperatures were 300 Cand345C, features and their relationship. To make the features more respectively. Peak identification was conducted by relative comparable, a range scaling (mean-centered and divided by retention time calculation, based on the internal standard. the value range of each variable) was applied. The quantitative evaluation was carried out using squalane as Additionally, cluster hierarchical analysis was also per- an internal standard, and the data was expressed in mg/kg oil. formed, using a word clustering algorithm and a Euclidean distance measure. The cluster was then plotted with a 2.4.5. Tocopherols. Tocopherols were determined following heatmap composed of the 15 most important characteristics IUPAC Standard Method 2432 [30], according to which 10 mg of the samples. These characteristics were selected by random oilwasdilutedwith1mLhexaneanddirectlyinjectedintoa forest analysis using random features selection from a boot- liquid chromatograph with an Si-column (250 mm × 4mm strap sample until the best grouping was reached. Journal of Chemistry 5

3. Results and Discussion abundant class of compounds in the unsaponifiable matter [20]. In this work, desmethyl-, methyl-, and dimethylsterols 3.1. Major Component, Fatty Acid, and Triacylglycerol Profiles. have been analyzed. Desmethylsterols are the most com- Fatty acids composition is the most common feature for monly analyzed group. The total amount of desmethylsterols and oil characterization. It is related to oxidative stability was higher for both commercial and extracted sweet almond as well as to some nutritional characteristics. The fatty acid oils. As expected, 𝛽-sitosterol was the main desmethylsterol, profile has been widely described for almonds, hazelnuts, and followed by Δ5-avenasterol, which was higher in pecan nut pecan nuts, as Table 1 shows. However, in this work, it was oil (around 15%) than in sweet almond and hazelnut oils, possible to bring some new information about this feature around 9% and 5%, respectively (Table 3). Among all samples, (Table 2), as it is the case of the description of some isomers cholesterol and stigmasterol concentrations did not show present in each sample. According to ANOVA analysis, the significant statistical differences (𝑝 > 0.05). amount of 𝜔-9 palmitoleic acid (C16:1𝜔-9), 𝜔-7 and 𝜔-11 𝜔 𝜔 In the case of methylsterols (Table 3), citrostadienol is oleic acids (C18:1 -7, C18:1 -11), (C22:0), and the main species in all samples. Sweet almond oil shows (C24:0) did not differ statistically among the higher obtusifoliol presence (reaching 28%) than hazelnut samples (𝑝 > 0.05), whereas for all other fatty acids a 𝑝 ≤ 0.05 and pecan nut oils, whose concentrations do not exceed 20% statistical difference was found ( )forthissetof and 10%, respectively. The total amount of methylsterols is samples. 𝜔 𝜔 at least three times higher in pecan nut oil samples than For all the samples, -9 oleic acid (C18:1 -9) was the in the other samples. Regarding dimethylsterols, the total main fatty acid, with its concentration being around 80% amount in pecan nut oil was at least six times higher than that in hazelnut, 70% in pecan nut, and 60% in sweet almond. ⋅ −1 𝜔 𝜔 in the other samples, reaching 200 mg kg , while in sweet Other isomers of oleic acid like C18:1 -7 and C18:1 -11 were ⋅ −1 also found, although their sum never reached 2% of the total almond and hazelnut oils it was around 30 mg kg .Forsweet fatty acids. In the case of palmitoleic acid isomers, their sums almondandhazelnutoils,themaindimethylsterolwas24- did not exceed 1% in each sample, and C16:1𝜔-7 was the methylencycloartanol, followed by butyrospermol in - most abundant one. Linoleic acid (C18:2𝜔-6) was the second nut oil, while in sweet almond oil the profile changed between most abundant fatty acid, with the highest presence in sweet commercial and extracted samples. However, in pecan nut almond, almost 30%, followed by pecan nut with around 20% oil samples, it is possible to see a very particular behavior, and hazelnut with approximately 10%. sinceinpecannutoilsamplesthemaindimethylsterolwas Table 2 provides the complete fatty acid composition , reaching 70% of the total dimethylsterols. for both sweet almond and hazelnut. These results are in Squaleneisaterpenichydrocarbon,aprecursorofsterols, general within the ranges shown in Table 1. Exceptions can be which has been highlighted due to its health benefits [20]. In found regarding pecan nut, where lower amounts of palmitic general, the total amount of squalene in commercial oils was (C16:0), linoleic (C18:2𝜔-6), and linolenic (C18:3𝜔-3) acids always higher than in laboratory-extracted samples (Table 3). were found, as well as higher concentrations of stearic (C18:0) Terpenic alcohols’ presence in sweet almond oil was −1 −1 and oleic (C18:1) acids. Although statistically different, the below 10 mg⋅kg ,whereasitwentbeyond20mg⋅kg in −1 fatty acid compositions of the three nut oils are quite similar, hazelnut oil and 30 mg⋅kg in pecan nut oil. Actually, it was −1 calling to mind the similarities between olive and higher than 60 mg⋅kg in the extracted samples of pecan nut. oils, both of them oils [33, 34]. In the case of aliphatic alcohols in sweet almond samples, −1 Although the fatty acid composition is quite similar, the the total amount did not exceed 7 mg⋅kg , while in hazelnut −1 distributions of the fatty acids in the triacylglycerol molecules and pecan nut oils the total amount was above 12 mg⋅kg , are very different (Table 2). While in hazelnut and pecan nut with the content in the extracted samples being higher than oil samples there is a clear predominance of PLP + OOO + in commercial oils (Table 3). Differences can be noted in the PoPP,in sweet almond samples, this predominance is equally profile of both terpenic and aliphatic alcohols and seem to shared between PLP + OOO + PoPP and OOL + LnPP. be related to sample processing and origin, although there Another particularity of the sweet almond is the amount of is no concrete evidence to prove it. Tocotrienols were not OLL, near 18%, whereas it does not reach 5% and 10% in present in any of the samples; however, the total amount −1 hazelnut and pecan nut, respectively. of tocopherols exceeded 200 mg⋅kg inallcases,withno The presence of SOL (around 12%) is a particularity of statistical difference𝑝 ( > 0.05). The amount of 𝛿-tocopherol hazelnut and it does not exceed 3% in sweet almond and was not statistically different as well. When it comes to pecannut,incontrasttoPOO,whichdoesnotsurpass sweet almond and hazelnut oils, the main compound was 1% in hazelnut and is around 7 and 8% in sweet almond 𝛼-tocopherol (Table 3), the same as in most vegetable oils. and pecan nut, respectively. Fatty acid and TAG profiles are Regarding pecan nut samples, the main tocopherol was 𝛾- complementary since TAG profile shows how fatty acids are tocopherol, which is also present in and corn oils grouped. [20].

3.2. Minor Compounds and Unsaponifiable Matter. Minor components are commonly known as the fingerprint of some 3.3. Statistical Grouping Analysis. Principal component anal- vegetable oils. In this way, they have been widely related to ysis (PCA) was performed in order to establish a statistical oil identity in many regulations [33]. Sterols are the most relationship among samples. Figure 1 shows results plotted by 6 Journal of Chemistry — — 0.19 0.21 0.01 0.01 0.10 0.01 0.01 0.01 0.03 0.03 0.02 0.07 0.26 0.26 0.05 0.02 0.20 0.08 0.69 0.26 0.08 0.00 0.00 0.40 0.00 0.00 0.04 0.00 0.00 0.00 0.00 0.00 0.04 0.00 0.00 nut oil 1 Com. pecan 1.12 ND ND 8.51 1.22 2.19 1.02 2.19 0.12 0.17 0.12 1.67 0.17 0.0 0.01 0.01 0.16 3.35 0.32 8.33 5.57 2.58 0.03 0.02 0.58 0.70 5.99 0.38 0.02 0.07 0.28 0.08 0.06 21.11 17.97 42.89 70.96 0 8 — — — 0.11 0.14 0.01 0.16 0.16 0.01 0.01 0.10 0.03 0.03 0.27 0.2 0.22 0.05 0.02 0.05 0.07 0.20 0.07 0.08 0.06 0.09 0.00 0.04 0.00 0.00 0.00 0.00 0.04 0.00 0.00 0.0 0.00 0.00 nut oil Extr. pecan 1.31 ND ND ND 1.74 8.12 5.61 0.12 0.19 0.14 9.71 0.16 0.01 0.01 2.63 2.87 6.03 0.27 0.27 0.02 0.03 0.50 4.07 0.07 0.36 0.29 2.60 0.07 0.80 0.68 0.04 0.99 0.06 67.55 38.75 23.24 20.67 3 — — — 0.12 0.01 0.01 0.10 0.01 0.01 0.10 0.01 0.3 0.02 0.03 0.02 0.05 0.26 0.06 0.06 0.00 0.00 0.04 0.00 0.00 0.04 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.04 0.00 0.00 0.00 d; S: stearic acid; O: oleic acid; L: linoleic acid; Ln: linolenic acid. Com. hazelnut oil ND ND ND 1.85 0.15 0.81 0.13 0.01 0.10 0.01 1.80 0.01 5.55 0.33 0.32 0.32 4.55 0.55 0.57 0.57 0.03 0.03 0.23 0.02 0.03 0.05 0.07 3.60 3.66 0.08 0.04 0.09 11.53 12.05 18.89 53.63 78.34 — — — — 0.17 0.12 0.12 0.10 0.01 0.01 0.32 2.22 0.02 0.03 0.02 0.65 0.88 0.02 0.79 0.54 0.08 0.20 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.06 0.00 0.04 0.00 0.00 0.00 Ext. 3 hazelnut oil ND ND ND ND ND 0.11 0.13 0.17 0.21 1.08 0.01 0.01 0.19 0.01 3.55 1.44 0.5 5.57 3.42 8.76 0.57 0.03 0.03 0.05 0.05 0.03 0.63 0.42 5.68 0.08 0.08 2.48 0.90 13.41 14.04 55.68 80.64 0 — — 0.17 0.13 0.14 0.14 0.01 0.16 0.23 0.03 0.02 0.02 0.03 0.02 0.02 0.26 0.45 0.26 0.20 0.20 0.00 0.00 0.00 0.00 0.0 0.04 0.00 0.00 0.00 0.00 0.00 0.06 0.00 0.00 0.00 0.00 0.00 Fatty acid composition % area Triacylglycerol composition % area almond oil Com. sweet ND ND ND 1.53 1.33 0.12 1.89 0.01 1.69 0.01 0.10 2.93 0.23 2.29 0.27 0.02 0.05 0.03 0.02 0.34 0.29 0.07 0.38 6.62 0.02 0.43 0.20 0.04 0.04 6.96 0.06 27.21 10.78 18.04 59.70 29.54 26.76 — — — 0.13 0.17 0.17 0.01 0.01 0.03 0.05 0.03 0.59 0.05 0.02 0.29 0.03 0.29 0.07 0.07 0.08 0.86 0.08 0.06 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Ext. sweet almond oil ND 1.17 ND ND 0.11 0.17 0.12 1.34 0.01 7.0 0 1.96 1.40 0.16 0.25 0.02 0.05 0.02 0.03 0.02 0.34 2.88 0.07 2.49 0.07 0.38 0.02 0.43 0.49 0.04 0.04 6.60 10.75 27.39 18.05 29.58 26.34 60.22 ECN 52 ECN 50 ECN 42 ECN 48 ECN 44 ECN 46 Table 2: Fatty acid and triacylglycerol composition of sweet almond, hazelnut, and pecan nut oil laboratory and commercial samples. 𝜔 11 𝜔 9 𝜔 7 𝜔 3 𝜔 11 𝜔 9 𝜔 7 𝜔 6 𝜔 9 - 16:1 𝜔 7 - 16:1 𝜔 11 - 16:1 𝜔 7 - 18:1 𝜔 9 - 18:1 𝜔 11 - 18:1 rgaric - 17:0 a Linoleic isomer - 18:2 PoOO+SLL+PLO SOL PLP + OOO + PoPP Linoleic - 18:2 POO PoOP + SPoL + POLn + SPoPo Sample Lauric - 12:0 LLL Linolenic - 18:3 SOO POP OLLn + PoLL Arachidic - 20:0 PLLn Miristic - 14:0 Margaroleic - 17:1 POS + SSL Gondoic - 20:1 OLL Stearic - 18:0 Pentadecanoic - 15:0 Behenic - 22:0 Oleic Oleic POLn + PPoPo + PPoL PLL + PoPoO OOLn + PoOL Palmitic - 16:0 Lignoceric - 24:0 OOL + LnPP Oleic Linoleic trans - 18:2 t Palmitoleic Palmitoleic Palmitoleic M Bold numbers: mean; normal numbers: standard deviation; ND: not detected; Ext.: extracted; Com.: commercial; P: palmitic acid; Po: palmitoleic aci Journal of Chemistry 7 — — 1.15 1.15 0.11 3.21 0.31 2.81 1.63 0.15 1.28 0.12 0.01 0.01 0.01 0.01 0.01 0.01 0.03 0.03 0.02 0.07 0.56 0.08 0.00 0.00 0.09 0.06 0.00 0.04 Com. pecan nut oil ND ND 1.55 1.27 0.12 4.16 0.16 1.06 3.62 0.65 0.30 0.36 3.06 0.56 8.98 0.40 0.86 6.48 15.81 15.64 75.58 74.86 55.40 84.54 36.64 44.60 111.09 192.51 320.81 1746.01 4 — — 1.01 1.01 5.10 0.15 0.17 0.17 0.01 0.01 0.01 2.76 0.25 0.03 0.02 0.02 0.03 0.05 0.65 0.24 0.05 11.51 0.00 0.09 0.00 0.00 0.00 0.06 0.0 14.58 nut oil 9 Extr. pecan ND ND 2.15 1.07 0.17 6.15 3.76 0.35 0.57 0.24 6.82 4.45 0.29 0.69 0.8 9.08 0.09 0.90 75.51 79.31 13.88 14.94 29.32 78.69 66.57 70.68 110.78 214.69 298.83 1791.44 — — — 1.85 1.43 0.12 0.01 0.81 0.10 0.01 0.01 1.04 1.04 0.55 5.28 5.34 0.03 0.03 0.02 0.03 0.03 0.03 0.02 2.42 0.24 0.67 0.68 0.04 0.46 0.40 Com. hazelnut oil ND ND ND 1.32 1.02 5.61 0.81 8.19 0.18 2.55 5.83 4.85 9.02 3.04 5.44 0.26 0.89 0.88 26.11 81.79 14.39 28.21 39.85 20.37 79.78 55.66 45.94 44.34 431.72 1807.72 — — — 1.11 1.39 1.28 0.01 0.01 0.01 0.10 0.01 7. 0 4 7. 0 4 0.41 0.32 0.39 0.03 0.02 0.02 0.45 0.45 2.94 0.30 0.20 0.09 0.04 0.48 0.04 0.04 4.06 Ext. hazelnut oil ND ND ND 5.13 5.21 1.45 0.91 7.49 0.73 4.37 3.54 2.05 0.83 6.72 0.22 0.39 9.38 0.96 18.53 81.39 74.75 29.37 50.42 26.54 29.20 49.58 43.09 46.58 340.05 1787.82 Methylsterols % area Dimethylsterols % area Desmethylsterols % area Terpenic alcohols % area — 1.41 1.22 1.22 0.13 0.17 1.26 1.26 0.01 0.01 0.01 1.48 5.37 2.55 0.58 0.63 0.03 0.02 0.65 0.02 0.62 0.68 0.00 0.00 0.00 0.04 0.00 0.04 0.04 21.85 almond oil Com. sweet ND 1.52 1.37 0.13 1.44 9.61 1.64 2.53 2.30 6.37 0.83 0.03 9.93 3.90 6.89 0.09 0.86 0.06 12.18 28.81 77.28 18.67 27.00 32.57 64.18 113.11 59.02 33.86 73.00 2804.31 — — 1.01 0.16 4.16 0.35 0.37 0.03 0.57 0.03 0.02 0.05 0.59 0.03 0.02 0.24 0.47 0.30 0.36 0.42 0.09 0.04 0.09 0.00 0.09 0.00 0.04 0.00 0.66 29.24 Ext. sweet almond oil ND ND 1.91 1.39 0.13 1.94 0.91 1.40 1.48 7.4 4 4.55 9.74 0.25 0.78 9.89 2.46 0.04 11.91 51.13 71.65 77.42 13.46 29.73 28.35 28.35 26.57 24.97 59.96 96.43 2870.48 −1 ⋅ kg −1 −1 −1 −1 ⋅ kg ⋅ kg ⋅ kg ⋅ kg Amyrin Δ 7-Avenasterol Δ 7-Stigamasterol Δ 5,23-Stigmastadienol + Clerosterol Sitostanol Δ 5,24-Stigmastadienol Stigmasterol Campestanol Δ 5-Avenasterol Campesterol Δ 7-Campesterol 𝛽 -Sitosterol 24-Methylene-cholesterol Taraxerol 𝛼+𝛽 Butyrospermol Cycloartenol Sample Cholesterol Total mg Obtusifoliol Total mg Dammaradienol Total mg Squalene mg Phytol Total mg Gramisterol Citrostadienol Geranylgeraniol 24-Methylencycloartanol Table 3: Minor compounds of sweet almond, hazelnut, and pecan nut oil samples: desmethyl-, methyl-, and dimethylsterols, terpenic alcohols, squale ne, aliphatic alcohols, and tocopherols. 8 Journal of Chemistry — — — 1.75 1.77 0.13 0.18 2.87 0.76 0.03 2.08 0.29 0.60 Com. pecan nut oil ND ND ND 1.57 5.07 6.25 11.37 15.91 12.85 24.47 39.60 197.09 203.73 — — — 0.31 0.17 0.14 2.83 2.70 2.29 0.95 0.24 0.00 0.04 nut oil Extr. pecan ND ND ND 3.36 2.38 5.08 8.56 13.16 21.30 55.22 24.88 313.80 306.34 — 1.58 1.00 0.33 0.70 0.67 0.50 0.42 0.08 2.64 0.00 13.22 14.42 Com. hazelnut oil 7.1 2 ND 3.42 0.78 8.30 8.26 0.00 41.31 16.43 12.40 33.93 221.51 205.35 −1 — 1.91 9.81 0.13 0.61 0.16 9.23 0.57 0.22 0.05 0.05 0.20 0.48 Ext. Table 3: Continued. Tocopherols⋅ kg mg Aliphatic alcohols % area hazelnut oil ND 5.31 3.71 4.47 3.96 9.38 9.97 31.70 55.10 20.21 227.95 102.00 345.22 — — 3.11 0.15 4.71 1.69 5.53 0.37 2.05 0.30 6.26 0.34 18.03 almond oil Com. sweet ND ND 7.37 0.91 1.46 8.52 6.05 11.29 10.12 30.81 19.29 255.60 236.06 — — 1.83 1.03 0.10 0.27 0.62 0.24 0.45 0.24 0.07 0.90 0.40 Ext. sweet almond oil ND ND 7.70 5.55 2.56 0.87 8.45 10.10 23.13 40.31 29.66 252.83 233.40 −1 −1 ⋅ kg ⋅ kg Sample C22-OH Total mg 𝛼 -Tocopherol Total mg 𝛾 -Tocopherol 𝛿 -Tocopherol C28-OH C23-OH C24-OH C25-OH C26-OH 𝛽 -Tocopherol C27-OH Bold numbers: mean; normal numbers: standard deviation; ND: not detected; Ext.: extracted; Com.: commercial. Journal of Chemistry 9

3 3

2 2 Commercial Extracted Hazelnut hazelnut hazelnut

1 1

0 Sweet almond 0 Commercial Extracted PC 2 (35.8%)

PC 2 (35.8%) sweet almond −1 sweet almond −1

−2 Pecan nut −2 Commercial −3 Extracted pecan nut pecan nut −3

−3 −2 −1 210 −1.0 −0.5 0 0.5 1.0 PC 1 (47.3%) PC 3 (6.4%) (a) (b)

Figure 1: Principal component analysis score plot for all analyzed samples. PC1 × PC2 (a); PC2 × PC3 (b).

means of principal components PC1 versus PC2 (Figure 1(a)) distinctive profile could be defined about sweet almond oil, and PC2 versus PC3 (Figure 1(b)). PC1 explains 47.3% of in a way that this may help to differentiate it from hazelnut the sample variances while PC2 and PC3 explain 35.8% and and pecan nut oils. 6.4%, respectively. According to the results in Figure 1(a), 𝛽-Sitosterol (desmethylsterols) was the main minor com- PC1 clearly isolates sweet almond samples (PC1 < 0zone) pound found, and the amount of Δ5-avenasterol was one from hazelnut and pecan nut oils (PC1 > 0 zone), while PC2 ofthemostdistinguishingfeaturesofpecannutoil.The separates hazelnut (PC2 > 0 zone) from pecan nut samples high concentrations of total methyl- and dimethylsterols were (PC2 < 0 zone). Figure 1(b) reflects the important influence important particularities of pecan nut oils, as well as the of PC3, which disconnects the extracted (PC3 > 0 zone) from higher quantity of 𝛾-tocopherol since in hazelnut and sweet the commercial (PC3 < 0zone)samples. almond oils the main tocopherol was 𝛼-tocopherol. Cluster hierarchical analysis results (Figure 2) reaffirm This work also describes for the first time the presence in the close relationship between hazelnut and pecan nut oils, nut oils of other minor compounds like terpenic and aliphatic while sweet almond oil is clearly isolated. Observing the alcohols. heatmap in Figure 2, there is a reflex of previous results Using multivariate statistical analysis, it was possible to and discussion, once it highlights the 15 main compounds establish relationships among samples and carry out sample responsible for the distinction of the samples, considering the grouping.Inthissense,hazelnutoilsresultedtobechemically relationship of all samples and features. In this way, for sweet closer to pecan nut oils than to sweet almond oils. From these almond samples, obtusifoliol, damaradienol, C16:1𝜔-7 acid, data analyses, it was also possible to differentiate commercial OLL + LnPP, C18:2t acid, and Δ5,24-stigmastadienol were from extracted oils. Heatmap highlighted the components themainfeaturesselectedthroughrandomforestanalysis. that are more important for the distinction of the samples Forpecannutoil,C20:1acid,cycloartenol,Δ5-avenasterol, considering the relationship among all samples and features. OLL + PoLL, PLLn, and C17:0 acid were the most important The full characterization of the samples was the main characteristics, whereas hazelnut oil, 𝛽-sitosterol, C18:1𝜔-9 noveltyofthiswork,whichbringsanewapproachtothechar- acid, and campesterol were the main ones. acterization of nut oils. In this way, it performs a complete characterization of minor and major identity parameters, and 4. Conclusion establishes the relationship among samples and features. All this information may be taken into account for giving Oils from sweet almond, hazelnut, and pecan nut are regulatory recommendations and laws. monounsaturated fats whose fatty acid composition is dom- inated by oleic acid. For the first time, the presence of 𝜔- Conflicts of Interest 7, 𝜔-9, and 𝜔-11 isomers of palmitoleic and oleic acids has been described in these kinds of oils. In general, when TAG The authors declare that there are no conflicts of interest are formed, those with oleic acids prevail, even though a regarding the publication of this paper. 10 Journal of Chemistry

1.5 Obtusifoliol 1.0

Damaradienol 0.5

Palmitoleic acid 0 -7 −0.5 OLL + LnPP −1.0 Linoleic acid trans −1.5

∆5,24-stigmastadienol

Gondoic acid

Cycloartenol

∆5-avenasterol

OLLn + PoLL

PLLn

Margaric acid

-Sitosterol

Campesterol

Oleic acid -9 Sweet almond Pecan nut Hazelnut Figure 2: Cluster hierarchical analysis and heatmap of the fifteen more important features for sample grouping, selected by random forest statistical tool.

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