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Chemical Characterization of Sacha Inchi (Plukenetia volubilis L.) Oil

Chiara Fanali,*, Laura Dugo, Francesco Cacciola,, Marco Beccaria, Simone Grasso, Marina Dach, a Paola Dugo,, and Luigi Mondello,
 University Campus Bio-Medico, Via A lvaro del Portillo 21, 00128 Rome, Italy Chromaleont Srl, A Spin-o of the University of Messina, via Industriale 143, 98124 Messina, Italy Dipartimento Farmaco-chimico, University of Messina, viale Annunziata, 98168 Messina, Italy

ABSTRACT: A chemical characterization of the major components, namely, triacylglycerols (TAGs), polyphenols, and tocopherols in a Sacha inchi oil derived from cold pressing of the seed, is hereby reported. To tackle such a task, highperformance liquid chromatography in combination with photodiode array (PDA), uorescence (RF), and mass spectrometry (MS) detection was employed. The latter was interfaced with atmospheric pressure chemical ionization and with electrospray ionization for the analysis of TAGs and polyphenols, respectively, whereas RF detection was tested for the determination of tocopherol content. Furthermore, fatty acid methyl esters (FAMEs) were evaluated by gas chromatographyame ionization detector. A 93% amount of total fatty acids was represented by unsaturated FAMEs with the greatest percentage represented by linoleic (L) and linolenic (Ln) accounting for approximately 50 and 36%, respectively. The main TAGs (>10%) were represented by LLnL, LnLnLn, and LnLLn; the latter was present in the oil sample at the highest percentage (22.2%). Among tocopherols, tocopherol was detected to be the most abundant component (over 50%). The polyphenolic composition was also investigated, and a total of 15 compounds were positively identied, through the complementary analytical information coming from PDA and MS data. To the best of our knowledge, this is the rst report providing a thorough chemical characterization of a Plukenetia volubilis L. oil. KEYWORDS: Sacha inchi, triacylglycerols, tocopherols, polyphenols, mass spectrometry

INTRODUCTION Sacha inchi (Plukentia volubilis L.), also known as the Inca peanut, is a perennial, oleaginous plant of the Euphorbiaceae family, native to the rain forest of the Andean region of South America. The plant was known by the natives of the area for thousands of years as witnessed by several representations reported on vessels found in Incan tombs.1 The Sacha inchi plant produces star-shaped green fruits, which yield edible dark brown seeds, slightly enlarged in the center and squashed toward the edges. The seeds are rich in oil (3560%) and proteins (27%) and contain heat-labile substances with a bitter taste.2 Chancas Indians and other tribal groups of the region extract oil from the seeds, which is used for the preparation of various meals. Roasted seeds and cooked leaves are also an important component of their diets. To our knowledge, only a few papers have reported the composition of Sacha inchi oil and seeds.1,37 According to these works, such an oil is rich in unsaturated fatty acids, about 93% of the total. In particular, high levels of essential fatty acids were found, namely, C18:3 3 (-Ln, cis,cis,cis-9,12,15-octadecatrienoic acid; -linolenic) and C18:2 6 (L, cis,cis-9,12-octadecadienoic acid; -linoleic) fatty acids, accounting for approximately 47 and 37%, respectively. Essential fatty acids are intermediate in the biosynthesis of important compounds in the human body, such as prostaglandin E1 and its derivates.8,9 Several studies reported that -6 and especially -3 unsaturated fatty acids have benecial eects on human health by preventing several diseases like cancer, coronary heart disease, and hypertension; furthermore, a hypocholesterolemic eect was observed when used as food supplements.10,11 Growing evidence shows how a proper balance in the diet between 3 and 6 fatty acids plays a key role
r 2011 American Chemical Society

in the prevention of chronic diseases. The optimal ratio between linoleic acid and -linolenic acid in the diet is, in most cases, reported in the literature with values ranging between 4:1 to 5:1, without exceeding 10:1.12 These values are very often shifted to the advantage of linoleic acid in the common western diet, reaching values as high as 20:1. Such a disproportion is considered one of the main risk factors leading to the development of chronic diseases such as obesity, cancer, and cardiovascular disease. 6 linoleic acid is a precursor of arachidonic acid, which leads to the production of essential compound such as prostaglandins and leukotriens, essential for the immune function and platelet aggregation. However, an excess of arachidonic acid formation could lead to an abnormal increase of pro-inammatory mediators, translating in the development of an inammatory process. On the other hand, leukotriens, originating from 3 fatty acids, show a much smaller proinammatory eect. Linoleic and -linolenic acids compete for the same enzymes elongase and desaturase for the syntesis of fatty acids belonging, respectively, to 6 and 3 series. If 6 consumption in the diet is excessive, a signicant inhibition of the synthesis of EPA and DHA can occur. As a consequence, great attention has been so far devoted to food supplements containing essential fatty acids and to the proportion between 3 and 6 fatty acids.13 The potential use of Sacha inchi products as ingredients for food supplements is not only related to the fatty acid prole but also to the amino acid composition of seeds,
Received: August 8, 2011 Accepted: November 4, 2011 Revised: October 24, 2011 Published: November 04, 2011
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Journal of Agricultural and Food Chemistry showing relatively high levels of cysteine, tyrosine, threonine, and tryptophan.3 An albumin protein, representing 31% of the total seed proteins, was also isolated and characterized. It is a glycoprotein containing all of the essential amino acids in adequate amounts.14 Moreover, the oil is rich in tocopherols and -carotene.3,5 For its composition, especially for the essential fatty acids content, Sacha inchi oil could be considered in the elaboration of food supplements. However, very recently, Bueso et al. reported the rst case of occupational allergy induced by P. volubilis seed. This allergy was probably due to a particular protein allergen not yet identied.15 The aim of this work was to characterize the composition of a Sacha inchi oil sample by analyzing the main constituents occurring in plant oils: triacylglycerols (TAGs), fatty acid methyl esters (FAMEs), tocopherols, and polyphenols. TAGs were analyzed by nonaqueous reversed-phase high-performance liquid chromatography (NARP HPLC). Detection was performed by atmospheric pressure chemical ionization (APCI), which is the most frequently used ionization technique for TAG analysis.1618 FAMEs were derivatized to methyl esters and analyzed by gas chromatographyame ionization detector (GC-FID).19 Tocopherols were separated by normal-phase liquid chromatography (NPLC) coupled to a uorescence detector (RF). Quantitative analysis of total phenolic compounds was performed by the FolinCiocalteu method,20 while a polyphenolic ngerprint of the major polyphenols was attained by HPLC-photodiode array (PDA)/electrospray ionization (ESI)-MS analysis.

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MATERIALS AND METHODS

Materials and Reagents. All solvents were of HPLC grade and were used as received. Methanol (MeOH) and formic acid were purchased from Farmitalia-Carlo Erba (Milan, Italy). Isopropanol (IPA), acetonitrile (ACN), n-hexane (Hex), acetone, -tocopherol, -tocopherol, and -tocopherol were provided by Sigma-Aldrich (Milan, Italy). Samples. Sacha inchi oil sample, guaranteed as nonadulterated, was obtained by a Peruvian producer. Fatty Acid Analysis by GC-FID. FAMEs were converted in their methyl esters by adding 1 mL of sodium methylate in methanol (5% w/v) to 100 L of oil and incubating the mixture at 100 C for 15 min. After the mixture was cooled, 1 mL of boron trifluoridemethanol (20% BF3) reagent (Merck, Milan, Italy) was added, and the solution was heated at 100 C for 15 min. The solution was first cooled and afterward mixed with 1 mL of n-hexane and 1 mL of water prior to being centrifuged for 2 min at 1254g. The upper layer containing the FAMEs was subsequently transferred to a 2 mL vial and stored at +4 C prior to analysis.21 GC-FID analyses were carried out on a GC2010 (Shimadzu, Milan, Italy) equipped with an AOC-20i autoinjector, a splitsplitless injector (280 C), and a FID detector. FAMEs separation was performed on a Supelcowax column (Sigma-Aldrich/Supelco, Bellefonte, PA; 30 m 0.25 mm i.d. 0.25 m df). GC operational parameters were as follows: initial pressure of carrier gas (He at constant linear velocity, 30 cm/s), 99.5 kPa; temperature program 50280 at 3 C/min; injection volume, 1 L; and split ratio, 1:100. FID parameters were as follows: H2 ow rate, 50 mL/min; air ow rate, 400 mL/min; makeup gas (N2) ow rate, 50 mL/min; sampling frequency, 80 ms; and lter time constant, 200 ms. Data acquisition was performed using the GCsolution software (version 2.3). TAG Analysis by HPLC-APCI-MS. HPLC-MS analyses of TAGs were carried out on a Shimadzu HPLC system (Kyoto, Japan) equipped with a CBM-20 A controller, two LC-20AB pumps, a DGU-20A5 degasser, a SIL-20AC autosampler, and a LC-MS 2020 mass spectrometer equipped with an APCI interface (Shimadzu, Kyoto, Japan). As a chromatographic column, an Ascentis Express C18 column (150 mm 4.6 mm,

2.7 m d.p.; Supelco) was used. The mobile phase flow rate was 1 mL/min; ACN (A) and IPA (B) were used, respectively, as the mobile phase in the following linear gradient mode: 0 min, 0% B; 50 min, 70% B; 55 min, 70% B; and 56 min, 0% B. The injection volume was 2 L, and analyses were run at room temperature. Applied were the following APCI-MS parameters: APCI mode positive; mass spectral range, 2501200 m/z; interval, 0.5 s; scan speed, 2143 amu/s; nebulizing gas (N2) ow, 4.0 mL/min; APCI temperature, 400 C; heat block, 230 C; desolvation line (DL) temperature, 250 C; DL voltage, 34 V; probe voltage, +4.5 kV; Qarray voltage, 1.0 V; RF voltage, 90 V; and detection gain, 1.05 kV. Data acquisition was performed using the LabSolution software (version 5.10.153). Tocopherol Analysis by NPLC-RF. The analyses of tocopherols were carried out by using a Shimadzu HPLC system (Kyoto, Japan) equipped with a LC 10 AD Vp high pressure isocratic pump, an SCL10A Vp controller, and an RF-10 AXL fluorescence detector (programmed for excitation at 290 nm and emission at 330 nm). Data acquisition was performed using the LCsolution software (ver. 1.12). All of the analyses were performed at ambient temperature (25 C) using a microsilica column (Supelco Ascentis SI 250 mm 1.0 mm, 5 m particle size). The mobile phase was a mixture of Hex:IPA (99:1), the ow rate was 0.050 mL/min, while the injection volume was 2 L. The sample was prepared by dissolving 0.01 g of oil in 1 mL of n-hexane. The method was validated following the EURACHEM guideline for each component, namely, -tocopherol, -tocopherol, and -tocopherol.22 Linearity was tested at ve dierent concentrations for each analyte, performing three replicates per level. Regression lines were built using the square method. The linearity and the goodness of the curves used were conrmed using Mandel's23 tting tests. The signicance of the intercept was established running a t test (signicant level, 5%). Accuracy, in terms of trueness and precision, was assessed at two dierent levels, repeating the analysis ve times. The ShapiroWilk test, to check the normality of the distribution, and the Dixon and Grubbs tests, to verify the presence of outliers, were performed before calculating precision in terms of coecient of variation (CV %) and limit of repeatability (r). The limit of detection (LOD) and limit of quantication (LOQ) were calculated performing 10 times the analysis of a blank sample and applying the following formulas: LOD : yd b 2t b LOQ : yq b 10 b where yd and yq is the signal at the LOD and LOQ, respectively, b is the average signal of the blank sample, b is the blank standard deviation, and t is the constant of the t Student distribution depending on the condence level (95%) and degrees of freedom. LOD and LOQ values were, nally, obtained by plotting yd and yq in the calibration line. Total Phenolic Compounds Quantitative Analysis. The total soluble oil phenolics analysis was performed by using FolinCiocalteu reagent, according to the procedure of Montedoro et al.20 Briefly, the methanolic extract of Sacha inchi oil was obtained as follows: 10 g of oil were dissolved in a solution of Tween 20 (2% v/w), added to 10 mL of a methanol/water (80:20 v/v) solution, and mixed with an Ultra-Turrax T18 homogenizer (model B003UTSVCW, IKA WERKE GmbH & Co. KG, Staufen im Breisgau, Germany) at 24000 rpm. The mixture was centrifuged at 5000g for 10 min, and the methanolic phase was recovered. The extraction procedure was repeated two times; the two methanolic fractions were pooled and stored at 20 C to eliminate residual oil droplets. One milliliter of Sacha inchi oil extract was mixed with 70 mL of water and 5 mL of FolinCiocalteu reagent. Five minutes later, 15 mL of sodium carbonate solution (25% w/v) was added; the mixture was vortexed and diluted with water to a final volume of 100 mL. The mixture was then incubated for 2 h at room temperature after which
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the absorbance at 765 nm was measured; results were expressed as gallic acid equivalents (GAE) in milligrams per 100 g of oil (mg GA/100 g oil). The concentration of polyphenols in sample was derived from a standard curve of absorbance of gallic acid concentrations ranging from 50 to 500 g/mL. Polyphenol Analysis by HPLC-PDA/ESI-MS. Polyphenols were extracted from Sacha inchi oil by using a slightly modified procedure already reported in the literature.20 Briefly, 50 g of oil was extracted three times with 10 mL each of a methanol/water (80:20, v/v) mixture. The three fractions were pooled, and the solution was centrifuged at 5000g for 10 min. Afterward, the surnatant was recovered, and the solvent was completely removed in vacuo by using a Speed Vac Plus concentrator (model SC110A, Savant Instrument, Holbrook, NY). The obtained residue was suspended in 5 mL of acetonitrile and washed with 10 mL of hexane to remove lipid components. The hexane extract was discarded, and the ACN solution was concentrated under vacuum. The dried residue, containing the polyphenols, was suspended in 200 L of a mixture methanol/water (50:50 v/v). HPLC-PDA/ESI-MS analyses of polyphenols were performed on a Prominence LC system (Shimadzu, Milan, Italy) equipped with a CBM 20A controller, two LC-20AD pumps, a CTO-20AC column oven, a DGU-20A3 degasser, a SIL-20AC autosampler, a SPD-20AD, and LCMS-2020 mass spectrometer equipped with an ESI interface. Data acquisition was performed by the LabSolution software (Shimadzu, Version 5.10.346). Polyphenols were separated on a Supelco Ascentis Express C18 column (150 mm 4.6 mm, 2.7 m particle size). A gradient employing water (pH = 3; solvent A) and acetonitrile (solvent B) was employed under the following conditions: 060 min, 0100% B; 5055 min, 100% B. The column was reconditioned with the initial eluent composition in 5 min. The mobile phase ow rate was 1 mL/min (split to 0.2 mL/min prior to MS detection), the column temperature was set at 25 C, while the

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injection volume was 2 L. The wavelength range was 210400 nm, and the chromatograms were extracted at 280 nm. The sampling frequency was 6.25 Hz, while the time constant was 0.16 s. ESI-MS acquisition was performed in negative mode under the following conditions: mass spectral range, 100700 m/z; interval, 0.5 s; scan speed, 1250 amu/s; nebulizing gas (N2) ow, 1.5 L/min; heat block, 300 C; DL temperature, 300 C; DL voltage, 34 V; probe voltage, 4.5 kV; Qarray voltage, 1.0 V; RF voltage, 60 V; and detection gain, 1.0 kV.

RESULTS AND DISCUSSION

FAMEs Composition in a Sacha Inchi Oil Sample by GCFID. Evaluation of Sacha inchi fatty acids profile was performed

by GC-FID analysis of FAMEs. Peak identification was obtained by means of coinjection of pure standard FAMEs; the determination of the distribution of single FAMEs was expressed as a mass fraction of the total FAME content (%), calculated from Ax =ATOT 100 where Ax refers to the peak area of the FAME considered and ATOT refers to the total peak area of the FAMEs contained in the sample. The names of the identified FAMEs along with the relative percentage are reported in Table 1. These results show that approximately 93% of total fatty acids is represented by unsaturated FAMEs, the greatest percentage is for -Ln (3) and L (6) acids, approximately 50 and 36%, respectively (Table 1). Our results are in good agreement with those reported by other groups.1,35 Because of the high content Table 2. Fatty Acid Composition of Dierent Edible Vegetable Oils
fatty acid % ref vegetable oil 8:0 10:0 12:0 14:0 16:0 18:0 18:1 18:2 18:3 20:1 24 palm oil 24 sunower oil 24 soybean oil 24 linseed oil 24 canola oil 24 coconut oil 25 peanut oil Sacha inchi oil 7 7 48 18 5 36 7 11 6 4 9 9 4 2 5 4 2 2 3 3 3 50 19 23 19 60 6 52 9 8 68 54 24 21 2 32 36 47 8 47 10 1

Table 1. FAMEs Identied in Sacha Inchi Oil Sample by GCFID Analysis Together with Their Peak Area Ratio Percentage
FAMEs name palmitic stearic vaccenic oleic linoleic linolenic
a

symbol P S V O L Ln

CN:DBa 16:0 18:0 1118:1 918:1c 9,1218:2 9,12,1518:3

peak area ratio % 4.3 3.0 0.6 9.0 36.2 46.8

CN, carbon number; DB, double bond.

Figure 1. HPLC-APCI-MS total ion current chromatogram of TAGs in a Sacha inchi oil sample.
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Journal of Agricultural and Food Chemistry Table 3. TAGs Identied in the Sacha Inchi Oil Sample by Means of HPLC-APCI-MSa
TAG MW DB PN [M + H]+ 1 2 3 4 5 6 7 8 9 10 11 12 LnLnLn 873 LnLLn 875 LLnL 877 LnOLn 877 PLnLn 851 LLL 879 OLLn 879 PLLn 853 SLnLn 879 LOL 881 OLnO 881 LLP 855 SLLn 881 POLn 855 13 14 OLO 883 SLL 883 OLP 857 SOLn 883 15 16 OOO 885 SLO 885 SLP 859
a

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DG+ LnLn 596 LnL 598 LnL 598 OLn 600 LnLn 596 LL 600 OLn 600 PLn 574 LnLn 596 LL 600 OLn 600 LP 576 SL 604 OLn 600 OO 604 LL 600 PL 576 OLn 600 OO 604 LO 602 SL 604

DG+

DG+

area % 12.3

9 8 7 7 6 6 6 5 6 5 5 4 5 4 4 4 3 4 3 3 2

36 38 40 40 40 42 42 42 42 44 44 44 44 44 46 46 46 46 48 48 48

874 876 878 878 852 880 880 854 880 882 882 856 882 856 884 884 858 884 886 886 860

LnLn 596 LL 600 LnLn 596 PLn 574

22.2 18.2 7.3 3.2 5.3

LLn 598 LP 576 SLn 602 LO 602 OO 604 LL 600 LLn 598 PLn 574 OL 602 SL 604 PO 578 SLn 602

OL 602

9.3 4.4 2.1 3.0 1.8 6.9

SLn 602 PO 578 0.8 2.7 LO 602 SO 606 0.1

SL 604 LP 576

SO 606 SP 580

0.4

PNs, DBs, pseudomolecular ([M + H]+), and diglyceride ions (DG+ [M + H RCOOH]+) corresponding to each identied TAG along with the relative concentration expressed as percentage area (area %) are reported.

of polyunsaturated essential fatty acids, especially 3 and 6, Sacha inchi oil is increasingly advertised as a nutritional supplement. In fact, it provides more useful nutraceuticals than other common seed oils that contain only remarkable quantities of 6 fatty acids. Specically, edible plant oils contain dierent percentages of the ve common, nutritionally important free fatty acids (FAs) (P, S, O, L, and -Ln). Table 2 shows the fatty acid

composition of some common vegetable edible oils as compared to that of Sacha inchi oil.24,25 Among plant oils reported in Table 2, only linseed oil contains a percentage of linolenic acid comparable to that of Sacha inchi oil, together with low percentages of saturated fatty acids such as palmitic and stearic. Among edible plant oils, linseed oil, because of its predominant composition in 3 fatty acids, has been widely employed as a food supplement showing a particularly favorable nutritional prole.26 It is also worth noting that the balance between 3, 6, and 9 fatty acids is an important nutritional property of plant oils. For this reason, plant oils that are rich in polyunsaturated fatty acids could be of great interest for a balanced diet and represent a good alternative to sh-based foods and supplements that so far represent the main food sources of 3 fatty acids. Triacilglycerol (TAG) Composition in a Sacha Inchi Oil Sample by HPLC-APCI-MS. The fatty acid composition can be used to evaluate the stability and nutritional quality of fats and oils. However, to understand their physical and functional properties, the determination of the types and amounts of TAG species present in a lipidic sample is also essential. Figure 1 shows the total ion current (TIC) chromatogram of a Sacha inchi oil sample. As reported in Table 3, 21 different TAGs were detected and identified; for each TAG, the relative abundance was calculated as the ratio of the TIC area of the peak with respect to the sum of TIC areas of all identified TAGs. The latter were identified according to their HPLC-APCI-MS mass spectra considering m/z values of pseudomolecular (M + H)+ and diacylglycerol fragment (M + H RCOOH)+ ions. As can be seen from Table 3, some coelutions were observed (12, LLP + SLLn + POLn; 14, SLL + OLP + SOLn). These results may be explained considering that TAG retention times in NARP-HPLC increase with increasing partition number (PN); the latter is defined as the total carbon number (CN) in all acyl chains minus two times the number of double bonds (DBs), viz. PN = CN 2DB. As a consequence, TAGs with the same PN are, usually, very difficult to resolve. In addition, the retention behavior of TAGs with the same CN is strongly influenced by the FAs composition of the individual TAG, mainly by the unsaturation degree and acyl chain length.16 In agreement with the GC-FID analysis, identified TAGs contained five different FAs (P, L, Ln, O, and S). The predominant components (>50%) were detected at m/z 873, 875, and 877 and were identified as dilinolenoyl-linoleoylglycerol (LnLLn), dilinoleoyl-linolenoylglycerol (LLnL), and trilinolenin (LnLnLn). Most of the TAGs in Sacha inchi oil (>80%) contained at least one residue of linolenic acid. The high percentage of linolenic acid in TAGs is very important because unsaturated fatty acids deriving primarily from vegetable oils reduce the levels of total and low density lipoprotein cholesterol, holding promise against the risk of cardiovascular diseases.27 Moreover, the position of fatty acid in the glycerol skeleton is important from a nutritional and metabolic point of view. The stereospecical position of fatty acids plays a key role in tryglicerol absorption and digestion because FAs at the sn-1 and sn-3 positions are digested rst by lipases, yielding sn-2 monoacylglycerols and free FAs.28 To our knowledge, this work reports for the rst time a thorough characterization of the TAG fraction in Sacha inchi oil. Tochopherol Composition in a Sacha Inchi Oil Sample by NPLC-RF. Tochopherols were analyzed by NPLC coupled with a fluorimetric detector. Peak identification was obtained by comparing the retention time of the sample with a standard solution (Table 4). The method was validated in terms of linearity, LOD, LOQ , and accuracy (precision and trueness). Mandel's fitting
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Figure 2. HPLC-PDA chromatogram (extracted at 280 nm) of the polyphenolic prole of a Sacha inchi oil sample.

Table 4. Linearity, LOD, LOQ , Accuracy, and Concentration Values for -, -, and -Tocopherols Contained in the Sacha Inchi Oil Sample Tested
g/kg linear range compds -tocopherol -tocopherol -tocopherol (mg/kg) 0.02613 0.04824 0.03216 tocopherol content (g/kg) 0.004 1.257 0.869 LOD 0.047 0.098 0.101 LOQ 0.053 0.104 0.107 precision (CV %) 9.42 7.03 6.67 min level (n = 5) trueness (relative error %) 14.47 7.17 5.07 precision (CV %) 4.67 4.19 4.95 max level (n = 5) trueness (relative error %) 0.30 0.28 0.31

test was widely satisfactory (Fcalc < Ftab), and R2 was 0.996 for -, -, and -tocopherol, respectively. LOD and LOQ were as follows: -tocopherol, 4.7 and 5.3 g/kg; -tocopherol, 9.8 and 10.4 g/kg; and -tocopherol, 10.1 and 10.7 g/kg, respectively, equivalent to 0.047 and 0.053 g/kg of oil for -tocopherol, 0.098 and 0.104 g/kg of oil for -tocopherol, and 0.101 and 0.107 g/kg of oil, respectively (Table 4). No outliers were highlighted by Dixon and Grubbs tests. At both concentrations assessed, intraday precision results were satisfactory, less than 10% at the lower level and less than 5% at the higher level, for all considered compounds. Trueness results were lower than 15 and 0.35% in terms of relative error, at the lower and higher levels, respectively. -Tocopherol accounted for more than 50% of the entire tocopherol content. -Tocopherol is considered the most representative antioxidant in olive oil, while - and -tocopherols are found principally in seed oils like soybean and sunower, and in particular, a large amount of -tocopherol is present in soybean oil. It has been reported that the antioxidant activity decreases in the order > > > tocopherol. As already stated, the greater amounts of and -tocopherols with respect to could be attributed to their greater antioxidant capability. This makes the oil more stable to oxidation.4 From a nutritional point of view, intestinal absorption of -tocopherol is similar to that of -tocopherol, and it could play a specic role in preventive side eects of some radicals like peroxynitrite and NOx.29 Polyphenolic Composition in a Sacha Inchi Oil Sample by RPLC-ESI-MS. The total phenolic content of Sacha inchi oil was determined by the FolinCiocalteu technique.22 The concentration of phenolics was 6.20 mg/100 g of oil expressed as GAEs. Thanks to its antioxidant properties, the polyphenolic content

ensures the oxidative stability of polyunsaturated fatty acids imparting the characteristic flavor to the oil. Moreover, the effect of many of them on some common diseases such as hypertension, atherosclerosis, prevention of certain cancers, and modification of immune and inflammatory responses is notorious.30 Among edible oils, olive oil has been extensively characterized, and several studies have been published on the quantitative analysis of total phenolics and on their qualitative characterization in extra virgin olive oil (EVOO).3133 On the other hand, no data on the polyphenolic fraction of Sacha inchi have been reported so far. A qualitative characterization of Sacha inchi oil phenolics was performed by RPLCPDA/ESI-MS (Figure 2). The partially porous C18 column allowed good baseline separation for most compounds to be attained within 50 min of analysis time under gradient conditions. The advantages of shell-packed stationary phases have previously demonstrated by a number of studies for highefficient separations of complex samples.3438 Polyphenols were identified through the complementary information coming from PDA and MS spectra along with data already reported in the literature (Table 5).3133 Twenty-one phenolic compounds were detected; among them, a number of 15, belonging to phenyl alcohol, flavonoid, seicoridoid, and lignan classes, were positively identified. Concluding, in this study, a characterization of the composition of a Sacha inchi oil sample through the analysis of the main constituents, namely, TAGs, FAs, tochopherols, and polyphenols, has been carried out for the rst time. Apart from the FAs prole, the elucidation of TAG and polyphenol content has been reported for the rst time. LnLLn was detected as the major compound, whereas among tocopherols, -tocopherol turned out to be the most abundant. Finally, the polyphenolic composition was
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Journal of Agricultural and Food Chemistry Table 5. Polyphenols Identied in a Sacha Inchi Oil Sample by HPLC-PDA/ESI-MS
molecular peak 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 tR (min) 10.3 19.4 20.0 26.9 27.5 28.3 29.5 31.6 31.8 33.1 34.6 36.5 37.4 39.2 39.5 39.9 40.3 40.6 41.9 44.6 45.2 C20H22O7 C15H10O5 C19H21O8 C22H26O8 formula C8H10O3 C8H10O2 C14H16O9 C26H25O4 C21H24O10 C18H21O6 C22H21O9 C18H21O6 C10H15O3 C9H15O4 C15H9O6 [M H] (fragment ions) 153 137 327 (311) 401 (369) 435 333 357 315 183 187 285 596 (586, 550) 541 479 (431) 479 (431) 479 (431) 417 334 373 269 377 max UV/vis (nm) 271 270 278, 325 268, 350 278, 350 270, 309 272 369 270, 309 260 272, 346 278 278 278 275 278 275 275 272 268, 339 270, 309 hydroxy-pinoresinol apigenin oleuropein aglycon lignan avonoid secoiridoid hydroxytyrosol tyrosol bergenin alpinumisoavone phloretin-glucoside methyl decarboxymethyl oleuropein aglycon pinoresinol isorhamnetin-glucoside oleuropeic acid azaleic acid luteolin unknown unknown unknown unknown unknown syringaresinol lignan compd compd class phenyl alcohol phenyl alcohol isocoumarin avonoid avonoid secoiridoid lignan avonoid secoiridoid organic acid avonoid

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identication [ref] UV, MS [31, 32] UV, MS [31, 32] UV, MS UV, MS [31] UV, MS UV, MS [32] UV, MS [31, 32] UV, MS UV, MS [31] UV, MS [31] UV, MS [32]

UV, MS [31, 32] UV, MS [33] UV, MS [32] UV, MS [31 32]

investigated, and a total of 15 compounds were positively identied through the complementary information of PDA and MS data. The present study demonstrates that Sacha inchi oil is a good source of linoleic and linolenic acids with considerable amounts of tocopherols. In addition, the avonoid composition in the extracted oil suggests its potential use as dietary source of natural antioxidants.

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AUTHOR INFORMATION
Corresponding Author

*Tel: +39-06-225419123. Fax: +39-06-225411972. E-mail: c.fanali@ unicampus.it.

ACKNOWLEDGMENT We gratefully acknowledge Shimadzu and Sigma-Aldrich/ Supelco Corporations for their continuous support. ABBREVIATIONS USED P, palmitic; L, linoleic; Ln, linolenic; O, oleic; S, stearic; V, vaccenic; TAG, triacylglycerol; FAs, free fatty acids; FAME, fatty acid methyl ester; HPLC, high-performance liquid chromatography; PDA, photodiode array; RF, uorescence; MS, mass spectrometry; APCI, atmospheric pressure chemical ionization; ESI, electrospray ionization; NARP HPLC, nonaqueous reversed-phase high-performance liquid chromatography; NPLC, normal-phase liquid chromatography; GC-FID, gas chromatographyame ionization detector; LOD, limit of detection; LOQ, limit of quantication; PN, partition number; CN, total carbon number; DBs, double bonds; GAE, gallic acid equivalents

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