Sacha inchi (Plukenetia volubilis L.) oil composition varies with changes in temperature and pressure in subcritical extraction with n-propane

Industrial Crops and Products 87 (2016) 64–70

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Sacha inchi (Plukenetia volubilis L.) oil composition varies with changes in temperature and pressure in subcritical extraction with n-propane Ana Beatriz Zanqui a , Claudia Marques da Silva a , Damila Rodrigues de Morais c , Jandyson Machado Santos c , Suellen Andressa Oenning Ribeiro a , Marcos Nogueira Eberlin c , Lúcio Cardozo-Filho b , Jesuí Vergílio Visentainer a , Sandra Terezinha Marques Gomes a , Makoto Matsushita a,∗ a b c

Department of Chemistry, State University of Maringá, UEM, Maringá, PR 87020-900, Brazil Department of Chemistry Engineering, State University of Maringá, UEM, Maringá, PR 87020-900, Brazil ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of Campinas—UNICAMP, Campinas, SP 13083-970, Brazil

a r t i c l e

i n f o

Article history: Received 22 January 2016 Received in revised form 6 April 2016 Accepted 7 April 2016 Chemical compounds studied in this article: Stigmasterol (PubChem CID: 5280794) Sitosterol (PubChem CID: 222284) Cholestane (PubChem CID: 6857534) ␣-Linolenic acid (PubChem CID: 5280934) Linoleic acid (PubChem CID: 5280450) Oleic acid (PubChem CID: 445639) Palmitic acid (PubChem CID: 985) Stearic acid (PubChem CID: 5281) Vaccenic acid (PubChem CID: 5281127)

a b s t r a c t Sacha inchi is an oilseed crop that shows high oil content and it is an excellent source of polyunsaturated fatty acids. Temperature and pressure can influence in lipid composition on the subcritical extraction with n-propane. The highest extraction yield obtained was 30% under the conditions of 60 ◦ C and 12 MPa. The extracted oil presented 442 mg of fatty acid g−1 in terms of alpha linolenic acid. Analysis by easy ambient sonic-spray ionization mass spectrometry showed a typical TAG (Triacylglycerols) profile for Sacha inchi oil with major ions of m/z (mass-to-charge ratio) 895 (ALA-ALA-ALA), 897 (ALA-ALA-LA), 899 (ALA-LA-LA) and 901 (LA-LA-LA or OL-LA-ALA) in a form of adduct of [TAG + Na]+ , in which ALA is alfa linoleic acid, LA is linoleic acid and OL is oleic acid. The phytosterols content (86.39–101.92 mg 100 g−1 of total lipids) varied according to the conditions applied for the n-propane extraction. In general, the subcritical fluid extraction with n-propane was found to preserve the important Sacha inchi oil bioactive constituents, that is, fatty acids, triacylglycerols and phytosterols. Regression analysis also showed that higher temperatures and pressures result in higher yields of phytosterols. © 2016 Elsevier B.V. All rights reserved.

Keywords: Plukenetia volubilis L. Subcritical fluid extraction Alpha linoleic acid Easy ambient sonic-spray ionization Omega 3 Phytosterols

1. Introduction Sacha inchi (Plukenetia volubilis L.) is an oilseed crop mainly originated from the Amazon Rainforest in Peru. This crop shows a high oil content which varies from 40 to 60% in weight (Follegatti-

Abbreviations: ALA, ␣-linolenic acid; FA, fatty acids; HDL, high density lipoprotein; LA, linoleic acid; MUFA, monounsaturated fatty acids; OL, oleic acid; PCA, principal components analysis; PUFA, polyunsaturated fatty acids; S, stearic acid; SE, soxhlet method; SFA, saturated fatty acids; SubFE, subcritical fluid extraction method; TAG, triacylglycerols; TL, total lipids. ∗ Corresponding author. E-mail address: [email protected] (M. Matsushita). http://dx.doi.org/10.1016/j.indcrop.2016.04.029 0926-6690/© 2016 Elsevier B.V. All rights reserved.

Romero et al., 2009). The oil obtained from P. volubilis L. is an excellent source of polyunsaturated fatty acids (PUFA) such as ALA (18:3n-3, ␣-linolenic acid) and LA (18:2n-6, linoleic acid), which are precursors of the omega-3 (n-3) and omega-6 (n-6) series, respectively. Since our metabolism is incapable to synthesize both LA and ALA, they are also essential fatty acids (FA) which should be present in the human diet. In the last years, these FA have also received significant emphasis since their health benefits have been firmly confirmed (Gutiérrez et al., 2011; Maurer et al., 2012; Souza et al., 2013). According to Chirinos et al. (2013), Sacha inchi grain is an excellent source of bioactive molecules such as phytosterols which decrease the risk of certain types of cancer and reduce cholesterol in the blood (Lagarda et al., 2006; Moreau et al., 2002).

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The ␣-linolenic acid is also known to prevent cardiovascular diseases, decrease the risk of heart and other chronic diseases, increase HDL (high density lipoprotein) blood quantities, prevent cancer and relief the effects of autoimmune diseases, rheumatoid arthritis and depression (Calder, 2006; Capurso et al., 2014; Jump, 2002; Ruxton et al., 2005; Scoditti et al., 2014; Simopoulos, 2011). Oleic acid (18:1n-9, OL) is not an essential FA to humans but its consumption has also been associated with numerous healthy benefits such as antithrombotic effect (Capurso et al., 2014; Scoditti et al., 2014). To extract triacylglycerols (TAG) from seeds, the food industry has mainly used extractions by hexane or others organic solvents such as petroleum ether, ethylic ether, methanol and chloroform. However, the use of hexane for food processing is facing restrictions, due to the large amounts of toxic solvents normally required as well as the need of demanding operations for solvent removal. The presence of solvent residues in the final product is also a concern. Therefore, studies have been performed to develop alternative methods of lipids extraction. Supercritical CO2 extraction is very beneficial for oil processing, particularly when dealing with functional food, for which the demand for “natural” products obtained through clean solvent-free technology has increased significantly in recent years (Martínez and Aguiar, 2014). Carbon dioxide supercritical fluid oil extraction results in nearly pure oils and relative high yields but is quite timeconsuming. Subcritical oil extraction using propane is faster as compared to CO2 due to the higher solubility of lipids in n-propane. n-propane has also been reported as an efficient and non-toxic oil solvent. But the purity and yields of n-propane subcritical extraction as well as the final composition of the oil has been found to considerably vary depending on the extraction conditions (Silva et al., 2015; Follegatti-Romero et al., 2009; Nimet et al., 2011; Pederssetti et al., 2011; Zanqui et al., 2015a,b). The aim of this study was evaluate the influence of pressure and temperature in the composition of the Sacha inchi oil (fatty acids, phytosterols and TAG) obtained via subcritical extraction with n-propane. The oils obtained either by n-propane subcritical extraction or the conventional Soxhlet method was also compared via analysis performed by gas chromatography with flame ionization detection (GC-FID), GC coupled to mass spectrometry (GC–MS) and easy ambient sonic-spray ionization coupled to mass spectrometry (EASI-MS).

2. Material and methods

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Fig. 1. Scheme of the experimental unit for lipid extraction by subcritical fluid, where: A—n-propane cylinder; B—pump-type syringe; C—puller; D and E—thermostated baths; F—micrometric valve; G—expansion valve; H—oil outlet; I—bottle collector; J—thermoregulator.

Table 1 Factors and levels evaluated in the experimental design full 22 with central point for the subcritical fluid extraction. Factors/Test

Temperature (◦ C)

Pressure (MPa)

Density (g mL−1 )

A B C D E

45 30 30 60 60

10 8 12 8 12

0.489 0.504 0.513 0.460 0.474

temperature-controlled thermostatic bath at 10 ◦ C (Nimet et al., 2011). The extraction was carried out with 1 mL min−1 of n-propane flow during 60 min, controlled by an expansion valve (Autoclave Engineers) maintained at 80 ◦ C using a thermoregulator (Tholz, model CTM-04E). The data analysis was performed according to the 22 factorial design (Table 1), with three replications of the central point and the responses were the extraction yield, quantity of some fatty acids and phytosterols. In Table 1 is also reported the densities of n-propane in its conditions of temperature and pressure (NIST web Book).

2.1. Sample preparation

2.3. Soxhlet extraction method (SE)

Three kilograms (3 kg) of Sacha inchi (P. volubilis L.) obtained from regional market of Bogota (Colombia) were used as representative samples. The Sacha peanut peel and grain fractions were carefully separated. The grains were ground in a Wiley mill (Tecnal, model TE 631/3, Brazil) to obtain a fine flour that was then sieved, using the fraction that passed through a 6 mesh and not that passed through a 9 mesh Tyler series sieve (WS Tyler, USA). Later, the sample was thoroughly mixed and vacuum packed in polyethylene bags and frozen at −18 ◦ C.

Approximately 4.0 g of Sacha inchi were extracted in a Soxhlet extractor (Nova Etica, Brazil) using a mixture of ethyl etherpetroleum ether (1:1 v−1 ) for 16 h at boiling point. Later, the solvent was evaporated with aid heated water at 65 ◦ C and total lipids content was determined gravimetrically (Soxhlet, 1879).

2.2. Subcritical fluid extraction method For lipid extraction with pressurized n-propane in a laboratory scale (Fig. 1), 15.0 g of Sacha inchi and porcelain beads were introduced into the extractor with an internal volume of 53.4 cm3 . The porcelain beads were inserted so that the whole volume of the extractor was filled. The solvent n-propane (White Martins, 99.5% purity) was pressurized via a pump-type syringe with a

2.4. Fatty acid composition Fatty acid methyl esters (FAME) were prepared by the methylation of total lipid (TL) according to Hartman and Lago (1973) and analyses were performed in triplicate. Methyl esters were separated by GC-FID (Trace Ultra 3300 model-Thermo Scientific) equipped with a flame ionization detector and a cyanopropyl capillary column (100 m × 0.25 i.d., 0.25 ␮m film thickness, CP-7420 Varian, EUA). The injector, detector and gases conditions, and the main operational parameters were according to Zanqui et al. (2015b). The peak areas were determined by the ChromQuest 5.0 software. For FA identification, retention times were compared with those of standard methyl esters. Quantification (in mg FA g−1

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Fig. 2. EASI(+)-MS (left) and expanded EASI(+)-MS in the m/z 800–1000 TAG ions range (right) for Sacha inchi oils obtained by n-propane subcritical extraction in different conditions (A–E) or via Soxhlet extraction (F).

of TL) was performed against tricosanoic acid methyl ester as an internal standard (23:0) (Joseph and Ackman, 1992).

analyses were performed in triplicate. Data were acquired and processed using the LCMS solution v.3.70 software (Shimadzu, Japan).

2.6. Phytosterols and tocopherols composition 2.5. Triacylglycerol composition The EASI-MS fingerprintings of the Sacha inchi oil were obtained using a single quadrupole mass spectrometer LCMS-2010 EV (Shimadzu, Japan) with a home-made EASI source (Haddad et al., 2008; Haddad et al., 2006) operating in the positive ion mode—EASI(+). The EASI(+) source was operated with a methanol flow rate of 20 ␮L min−1 and 2 L min−1 for the nebulizing gas (N2 ). The surfaceentrance angle was fixed in 30◦ . A single droplet of the Sacha inchi oil (1 ␮L) was deposited on a paper surface (brown Kraft envelope paper) and EASI(+)-MS data were collected for over 30 s, scanning over the m/z (mass-to-charge ratio) 100–1200 range. The data

Phytosterols and tocopherols were simultaneously evaluated by GC–MS (Du and Ahn, 2002). The extracted oils were previously derivatized (Beveridge et al., 2002), using N-O-bis (trimethylsilyl) trifluoracetamide (BSTFA) (Sigma-Aldrich Co., Brazil) as the derivatizing reagent. The analysis were performed using a GC–MS Agilent 7890A (Agilent-Technologies, USA) using a DB-5 capillary column (5% phenil, 95% methilpolisiloxane, 30 m × 0.25 mm × 0.25 ␮m) (J & W Scientific). The MS was an Agilent 5975C instrument (VL MSD) and used 70 eV EI and a quadrupole mass analyzer. Data analysis used database NIST MS Search version 2.0 2014. The GC–MS were those previously described by Zanqui et al. (2015a). Quantitation

A.B. Zanqui et al. / Industrial Crops and Products 87 (2016) 64–70 Table 2 Yields of total lipids extracted by subcritical and Soxhlet extraction. Test

Temperature (◦ C)

Pressure (MPa)

Extraction Yield (%)

A B C D E SE

45 30 30 60 60 –

10 8 12 8 12 –

29.2b ± 0.5 28.0b ± 0.5 26.7b ± 0.5 28.9b ± 0.5 29.7b ± 0.5 42.0a ± 1.0

sizes ranging from 3 to 6 mesh, and an oil yield of only 18.8% was obtained. Therefore was decided to reduce the size of the particles to a range of 6–9 mesh, obtaining statistically different yields for the Soxhlet and SubFE extractions. That is, the extraction yield for the SubFE method was 30% lower than that for SE extraction. 3.2. Composition Table 3 shows the FA compositions of the SubFE and SE oils. The predominant FA in the lipid composition of Sacha inchi oil (442 mg g−1 ) was found to be the alpha-linolenic acid (18:3n-3). This%FA closely matches that obtained by Maurer et al. (2012). Souza et al. (2013) also reported approximately 438.77 mg g−1 of this FA in the oil. The same lipids as those reported in Table 3 were extracted with solvents and by SubFE extraction, which shows that these extraction methods provide similar FA compositions. Follegatti-Romero et al. (2009) also studied the lipid composition of the Sacha inchi oil extracted by supercritical CO2 , and found a composition similar to that reported in Table 3. This similarity indicates that n-propane also extracts the same FAs as those extracted by the conventional SE methodology. Statistical analysis indicates significant differences in FA levels for the SubFE and SE methods (Tables 3 and 4). In relation to the summations and n-3/n-6 ratios of the extracted Sacha inchi oil (Table 4), no significant differences in total saturated (SFA) FA, monounsaturated (MUFA) and polyunsaturated (PUFA) n3 and n-6 were observed using both methods. A relatively higher concentration of SFA and lower concentration of PUFAs in SE lipid composition were however observed. The level of saturated FA was 33% higher for the SE oil as compared to that using SubFE, showing a bias against saturated FA for the SubFE method. The n-3/n-6 ratio was 1.32 for TL. According to the World Health Organization (WHO, 1995), balanced ingestion of omega-3 and 6 series FAs are recommended since these compounds act in chronic diseases and in cardiovascular and hypertension prevention.

Mean values. Means followed by different letters in the same column demonstrated significant difference by Tukey’s test (p < 0.05). A–E: letters representing the testing of extraction with subcritical fluid. SE = Soxhlet method.

was performed using 5␣-cholestane (Sigma-Aldrich Co., Brazil) (Li et al., 2007) as the internal standard. 2.7. Statistical analysis Fatty acids, tocopherols and phytosterols analyses were performed in triplicate. Means and standard deviations of the analytical error propagation were calculated and the results were submitted to Tukey’s test at 5% probability, using the Statistica software, version 8.0 (StatSoft, 2007). Data was also submitted to principal component analysis (PCA). Analysis of variance was used to evaluate the effect of independent variables on the responses using mathematical model expressed by equation 1 (Ri = response, ␤0 = constant, and ␤1, ˇ2 and ␤12 = regression terms) where T refers to temperature, P refers to pressure, using the Design Expert software, version 7.1.3 (StatEase, 2008). Ri = ˇ0 + ˇ1 T + ˇ2 P + ˇ12 TP

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(1)

3. Results and discussions 3.1. Subcritical fluid extraction method (SubFE) and soxhlet method (SE)

3.3. Typification of Sacha inchi oil via EASI-MS fingerprinting Table 2 shows the yields for both the SubFE with n-propane and SE using different temperatures and pressures. For the SubFE extraction, preliminary tests were made with larger particles, with

The EASI-MS technique has been widely applied for vegetable oil typification and quality control (Bataglion et al., 2015; Simas

Table 3 Quantification of fatty acids of Sacha inchi oil. FA (mg g−1 of TL)

A

B

C

D

E

SE

16:0 18:0 18:1n-9 c 18:1n-7 18:2n-6 18:3n-3

40.05b ± 0.6 28.43b ± 1.8 81.40a ± 2.2 4.76a ± 0.2 336.38a ± 0.9 440.04a ± 4.9

40.28b ± 0.6 27.80b ± 1.4 80.82a ± 1.2 4.64a ± 0.3 335.74a ± 1.0 443.44a ± 2.9

40.19b ± 0.2 27.95b ± 0.7 80.57a ± 0.2 4.76a ± 0.1 335.51a ± 0.6 443.29a ± 1.7

40.04b ± 0.3 27.46b ± 1.0 81.22a ± 1.4 4.75a ± 0.1 334.22a ± 2.2 443.06a ± 2.1

39.73b ± 0.6 28.83b ± 1.1 81.11a ± 1.4 4.72a ± 0.1 334.23a ± 1.0 441.28a ± 4.1

56.66a ± 1.0 34.63a ± 0.6 82.38a ± 1.9 4.92a ± 0.1 325.08b ± 0.9 425.30b ± 2.0

Mean values ± standard deviation; Means followed by different letters in the same line demonstrated significant difference by Tukey’s test (p < 0.05). A–E: letters representing the testing of extraction with subcritical fluid, where A = 45 ◦ C/10 MPa; B = 30 ◦ C/8 MPa; C = 30 ◦ C/12 MPa; D = 60 ◦ C/8 MPa; E = 60 ◦ C/12 MPa; SE = Soxhlet method. Detection limit = 0.015 mg g−1 .

Table 4 Summations and n-3/n-6 ratio of Sacha inchi oil extracted by subcritical fluid method using n-propane. Summations (mg g−1 of TL)

A

B

C

D

E

SE

SFA MUFA PUFA n-3 n-6 Ratio n-3/n-6

68.48b ± 1.9 86.16a ± 2.2 776.42a ± 4.9 440.04a ± 4.9 336.38a ± 0.9 A 1.32a ± 0.1

68.09b ± 1.6 85.46a ± 1.3 779.19a ± 3.0 443.44a ± 2.9 335.74a ± 1.0 B 1.32a ± 0.1

68.14b ± 0.7 85.33a ± 0.2 778.81a ± 1.8 443.30a ± 1.7 335.51a ± 0.6 C 1.32a ± 0.1

67.51b ± 1.0 85.97a ± 1.4 777.28a ± 3.0 443.06a ± 2.2 334.22a ± 2.2 D 1.33a ± 0.1

68.56b ± 1.3 85.82a ± 1.4 775.52a ± 4.2 441.28a ± 4.1 334.23a ± 1.0 E 1.32a ± 0.1

91.30a ± 1.2 87.31a ± 2.0 750.38b ± 2.2 425.30b ± 1.9 325.08b ± 0.9 SE 1.31a ± 0.1

Mean values ± standard deviation; Means followed by different letters in the same line demonstrated significant difference by Tukey’s test (p < 0.05). A–E: letters representing the testing of extraction with subcritical fluid, where A = 45 ◦ C/10 MPa; B = 30 ◦ C/8 MPa; C = 30 ◦ C/12 MPa; D = 60 ◦ C/8 MPa; E = 60 ◦ C/12 MPa; SE = Soxhlet method. SFA = total saturated fatty acids; MUFA = total monounsaturated fatty acids; PUFA = total polyunsaturated fatty acids; n–6 = total n-6 fatty acids; n–3 = total n-3 fatty acids.

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Table 5 Attribution of ions detected by EASI(+)-MS for the Sacha inchi oil. FA Composition

[DAG + Na]+ m/z

[DAG + K]+ m/z

LA-LA TAG Composition LA-LA-P LA-LA-P/ALA-OL-P ALA-ALA-ALA LA-ALA-ALA LA-LA-ALA LA-LA-LA/OL-LA-ALA LA-LA-OL LA-LA-S

639 [TAG + Na]+ m/z 875 877 895 897 899 901 903 905

655 [TAG + K]+ m/z 901 903 911 913 915 917 919 921

[Monohydroperoxide + Na]+ m/z

Monohydroperoxide + K]+ m/z

[Monohydroperoxide + Na]+ m/z

Monohydroperoxide + K]+ m/z

927 929 931 933

943 945 947 949

P = Palmitic Acid; S = Stearic Acid; OL = Oleic Acid; LA = Linoleic Acid; ALA = Linolenic Acid. Table 6 Quantification of phytosterols and tocopherols in Sacha inchi oil extracted by subcritical fluid method using n-propane (mg 100 g−1 of TL). Point

(␥ + ␦) Tocopherols

Stigmasterol

Sitosterol

Sum of phytosterols

A B C D E

104.88a ± 2.4 105.44a ± 0.3 102.58a ± 0.1 104.32a ± 4.6 104.46a ± 3.6

36.27b ± 0.6 36.11b ± 0.5 34.61b ± 1.8 45.63a ± 0.2 41.87a ± 2.8

50.13b ± 0.1 50.67b ± 0.1 43.46c ± 1.9 56.29a ± 0.7 50.11b ± 2.7

86.39b ± 0.7 86.78b ± 0.6 78.07c ± 2.3 101.92a ± 0.8 91.98b ± 3.3

Mean values ± standard deviation; Means followed by different letters in the same column demonstrated significant difference by Tukey’s test (p < 0.05). A–E: letters representing the testing of extraction with subcritical fluid, where A = 45 ◦ C/10 MPa; B = 30 ◦ C/8 MPa; C = 30 ◦ C/12 MPa; D = 60 ◦ C/8 MPa; E = 60 ◦ C/12 MPa.

et al., 2010; Zanqui et al., 2015a) due the various advantages of this ambient MS method. It provides undisturbed sampling and highspeed of data acquisition, ease of analysis, low cost, direct analysis, voltage-free ionization and no need for pre-separation (Alberici et al., 2010). Major oil components are identified via the specific m/z values of typically [M + Na]+ ions (Cabral et al., 2013; Haddad et al., 2006). Fig. 2 shows the EASI(+)-MS data of the Sacha inchi oil obtained either by the SE and SubFE in different conditions. Note that indeed unique and contrasting triacylglycerols (TAG) profiles are obtained for both extraction methods. TAG ions were detected mainly as adduct of [TAG + Na]+ , but minor as adduct of [TAG + K]+ series separated by 16 m/z units was also observed. The TAG ions were detected along the ca. m/z 800–950 range (Fig. 2) whereas oxidized TAG ions appear on the ca. m/z 940–970 range. But oils obtained either by n-propane SubFE in different conditions or by SE showed nearly identical EASI(+) spectra (Fig. 2). This similarity demonstrates both the reliability of EASI(+)-MS as a typification protocol for such oils regardless the extraction protocol as well as that very similar TAG compositions are also obtained regardless the temperature and pressure used. The EASI(+)-MS data for the SE (Fig. 2-F) also showed a set of unique ions mainly separated by 44 Da detected along the m/z 400–800 range, but these ions are probably from polyethyleneglycol (PEG) present as contamination from the SE method. Table 5 shows the ions assignments from Fig. 2. Note that, fortunately, the FA composition from the GC-FID analysis (Table 3) closely matches the FA composition inferred from the intact TAG composition revealed by EASI(+)-MS (Table 5), which was obtained in a much simpler and faster way. For instance, LA and ALA are determined to be predominant by both techniques. Chasquibol et al. (2014) used RP-HPLC to characterize Sacha inchi oil extracted by hydraulic pressing and found the same predominant TAG, that is, mainly those composed by three chains from linolenic acid (ALA), two chains from linolenic acid (ALA) and one of Linoleic acid (LA), one chain from linolenic acid (ALA) and two of Linoleic acid (LA), and three chains from linoleic acid (LA), respectively for the TAG ALA-ALA-ALA, ALA-ALA-LA, ALA-LALA and LA-LA-LA. A similar profile was detected by the EASI(+)-MS of Fig. 2, via the [TAG + Na]+ ions of m/z m/z 895 (ALA-ALA-ALA), 897

Table 7 Analysis of variance for the response sum of phytosterols of Sacha inchi oil extracted by SubFE method. Source

Sum of squares

Degrees of freedom

Mean square

F value

p-value

Model T P T.P Curvature Pure Error Total

888.39 440.77 34.89 412.73 32.62 62.99 984.00

3 1 1 1 1 10 14

296.13 440.77 34.89 412.73 32.62 6.30 –

47.01 69.98 5.54 65.53 5.18 – –

<0.0001 <0.0001 0.0404 <0.0001 0.0461 – –

T = temperature; P = pressure.

(ALA-ALA-LA), 899 (ALA-LA-LA) and 901 (LA-LA–LA or OL-LA-ALA) (Fig. 2). The authors also used HPLC to monitor hydrolysis and oxidation of Sacha inchi oils using prior selective solid phase extraction (SPE). Note therefore that the same information can be obtained by EASI(+)-MS in seconds without any sample preparation at all since [DAG + Na]+ (diacylglycerol) ions are detected in the m/z 400–700 range, as example the ion of m/z 639 with two chains from Linoleic acid (LA-LA). For oxidation, hydroperoxide TAG ions were detected in the m/z 929 to m/z 947 range. Note also that both the DAG (diacylglycerols) and hydroperoxide TAG ions were detected in quite low abundances indicative an extracted oil of high quality. Interestingly, when compared to other Amazon oils such as Ac¸ai, Buriti (Simas et al., 2010) and Andiroba oils (Cabral et al., 2013), the extracted Sacha inchi oil investigated herein show extraordinarily much high contents of bioactive FA such as ALA and LA with great predominance of TAG from the combination of theses acids. Indeed the faster and simpler EASI(+)-MS technique was able to display profiles of oil composition in regard to TAG, DAG and oxidized TAG quite similar to both GC-FID and HPLC data. 3.4. Phytosterols and tocopherols composition Table 6 shows tocopherols and phytosterols composition in Sacha inchi lipids extracted by the SubFE method. There was no significant difference on tocopherols levels. Point D stood out with higher values for all phytosterols.

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extraction process with contributions of 44.8 and 42.0%, respectively. Via Eq. (2), it was possible to conclude that all studied factors contribute to the increase of the extraction of phytosterols in Sacha inchi oil, and the greatest yield was obtained at 60 ◦ C and 12 MPa. The ANOVA also indicates that the model was significant and confirms that factors such as temperature and pressure in the SubFE with n-propane may influence selectively in the lipophilic bioactive composition of Sacha inchi oil (Fig. 3). Ri = 88.29 + 6.06T +1.71P + 5.86TP

(2)

R2 = 0.934

Fig. 3. Response surface for the sums of phytosterols of Sacha inchi oil extracted by subcritical fluid extraction with n-propane.

Chirinos et al. (2013) studied 16 different cultivars of Sacha inchi and reported 21.2–26.9 mg 100 g−1 of stigmasterol extracted by the SE method. They also found 45.2–53.2 mg 100 g−1 of sitosterol. These values are similar to those obtained herein (Table 6), but the sum of phytosterols shows that our SubFE protocol was able to extract 25% more phytosterols than that used by Chirinos et al. (2013). They also reported tocopherol levels, and the sum of tocopherols was close to those obtained herein.

Principal components analysis (PCA) (Fig. 4) was applied to find correlations for the FA summations in relation to both SE and SubFE. PC1 (Principal component 1) and PC2 (Principal component 2) accounted for 46.89% and 11.99% of data variance, respectively. The remaining PCs yielded progressively smaller eigenvalues (P < 1), therefore PC1 and PC2 were indeed the PCs that better describe the data. In the PC1 x PC2 plot, two groups related to both extraction methods were formed, showing that they provided significantly different FA compositions for the Sacha inchi oils. The extraction with subcritical fluid approximated the maximum values of n-6, n-3 and PUFA, showing a good correlation with these summations. The solvent extraction method showed the highest composition of SFA and MUFA. So PCA confirmed that the Sacha oil extracted using n-propane as a subcritical fluid is a selective method. 4. Conclusions

3.5. Statistics and multivariate analyses Table 7 shows the results obtained by ANOVA for the responses of phytosterols of Sacha inchi oil extracted by the SubFE method. The ANOVA for the sum of phytosterols (Ri ) of Sacha inchi oil extracted by SubFE process showed that both temperature and pressure factors were significant for the model. The analysis of the effects indicated that the temperature and the temperature x pressure interaction effect were the most significant factors of the

In subcritical extraction using n-propane, the reduction of particle size contributed to higher yields of lipids. The extraction yield obtained by SubFe method was about 28%. As rapidly and easily determined by EASI(+)-MS directly from the undisturbed sample, and also via comparison with GC-FID results, the subcritical fluid extraction with n-propane can provide a Sacha inchi oil with a rich composition of bioactive compounds (fatty acid, triacylglycerol, phytosterols and tocopherols). The temperature and pressure

Fig. 4. PCA of sums de FA for Sacha inchi oil. A–E: letters representing the testing of extraction with subcritical fluid. SE = Soxhlet extraction method. MUFA = total monounsaturated fatty acids; PUFA = total polyunsaturated fatty acids; n–3 = total n-3 fatty acids; n–6 = total n-6 fatty acids.

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