Combined Analysis of Stable Isotope, 1H NMR, and Fatty Acid To

Sep 23, 2015 - The aim of this study was to verify the authenticity of sesame oils using combined analysis of stable isotope ratio, 1H NMR spectroscop...
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Combined Analysis of Stable Isotope, 1H NMR, and Fatty Acid To Verify Sesame Oil Authenticity Jeongeun Kim,†,|| Gyungsu Jin,§,|| Yunhee Lee,§ Hyang Sook Chun,† Sangdoo Ahn,*,§ and Byung Hee Kim*,† †

Department of Food Science and Technology, Chung-Ang University, Anseong 456-756, Korea Department of Chemistry, Chung-Ang University, Seoul 156-756, Korea

§

S Supporting Information *

ABSTRACT: The aim of this study was to verify the authenticity of sesame oils using combined analysis of stable isotope ratio, 1 H NMR spectroscopy, and fatty acid profiles of the oils. Analytical data were obtained from 35 samples of authentic sesame oils and 29 samples of adulterated sesame oils currently distributed in Korea. The orthogonal projection to latent structure discriminant analysis technique was used to select variables that most effectively verify the sesame oil authenticity. The variables include δ13C value, integration values of NMR peaks that signify the CH3 of n-3 fatty acids, CH2 between two CC, protons from sesamin/sesamolin, and 18:1n-9, 18:3n-3, 18:2t, and 18:3t content values. The authenticity of 65 of 70 blind samples was correctly verified by applying the range of the eight variables found in the authentic sesame oil samples, suggesting that triple analysis is a useful approach to verify sesame oil authenticity. KEYWORDS: 1H NMR, authenticity, discriminant analysis, fatty acid, sesame oil, stable isotope ratio

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INTRODUCTION Roasted sesame oil is an unrefined oil obtained from sesame seeds (Sesamum indicum L.) that have been roasted before pressing. This oil is frequently used to impart a specific flavor to a variety of Korean dishes and foods due to the characteristic flavor that develops during roasting.1 Because the domestic production of sesame seeds (∼11000 t per year) does not typically sustain the national need, ∼77000 t of sesame seeds were imported to Korea annually during 2009−2011.2 Indian and Chinese sesame seeds made up ∼48 and ∼39% of Korea’s sesame imports, respectively.3 In Korea, the retail price of sesame oil is as low as 5 (for imported products) to as high as 25 (for Korean products) times that of other edible oils because sesame seeds cost more to cultivate and have a lower production per unit area than other oilseed crops. Intentional adulteration of sesame oil products has been an issue of public concern in Korea for decades in that it is not only an economic fraud but also threatens consumer health. Adulterated sesame oils have been manufactured by blending with low-cost edible oils (e.g., corn and soybean oils) and lowquality ingredients (e.g., extracts from feed sesame cake) in unsanitary conditions. This has created the need for accurate analytical methods to verify the authenticity of sesame oil. Several different analytical techniques have been employed to verify the adulteration of sesame oil by analyzing different oil constituents. These methods include spectrophotometry for sesame lignan analysis,4 gas chromatography for fatty acid,5,6 and phytosterol analyses,6 liquid chromatography for triacylglycerol4,7 and tocopherol analyses,6 isotope ratio mass spectrometry for stable isotope ratio analysis,5 electronic nose for volatile pattern analysis,8,9 and Fourier transform infrared spectroscopy for functional group analysis.10 However, to the best of our knowledge, few published studies have verified the © 2015 American Chemical Society

authenticity of sesame oil using nuclear magnetic resonance (NMR) spectroscopy, although several European researchers have applied this analytical technique to verify the adulteration of olive oil, which is one of the most important and expensive foodstuffs in those countries.11−13 Some of these published studies have used a combination of methods to analyze more than one type of sesame oil constituent5,6 because in many cases, the analysis of only one type of oil constituent is not adequate to distinguish sesame oils adulterated with a particular type of or more than one kind of foreign edible oil from authentic sesame oils. However, a major problem in the methodology used in most of these published studies is that sesame oils blended with only one other edible oil were used as a model of adulterated sesame oil. Many adulterated sesame oils distributed in Korea are known to contain more than one kind of foreign edible oil. Sesame-flavored oil is a legal product that is widely used as a substitute for roasted sesame oil in many restaurants in Korea due to its much cheaper price. Sesame-flavored oil is commonly manufactured by blending sesame oil with several different kinds of other edible oils such as corn, soybean, sunflower, perilla, cottonseed, and canola oils. In the present study, we employed commercial sesame-flavored oil as the main model of adulterated sesame oil because many adulterated sesame oils are manufactured in the same way as sesame-flavored oil. The aim of this study was to verify the authenticity of roasted sesame oils distributed in Korea using the combined analysis of stable isotope ratio, 1H NMR spectroscopy, and fatty acid Received: Revised: Accepted: Published: 8955

May 19, 2015 September 21, 2015 September 23, 2015 September 23, 2015 DOI: 10.1021/acs.jafc.5b04082 J. Agric. Food Chem. 2015, 63, 8955−8965

Article

Journal of Agricultural and Food Chemistry

Table 1. Ingredient Composition (w/w %) of Sesame-Flavored Oil Samples Used as the Model of Adulterated Sesame Oils in the Present Study ingredients

a

sample

sesame oil

corn oil

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

44 42 40 38 36 35 32 30 27 24 22 22 20 14 10 10 9 8 5 5 undefc

53 50 50 32 48 52 40 30 31 30 76 56 75 37 89 69 90 26 95 52 70 91 90 80 64

soybean oil

sunflower oil

5 10 30 14 10 20 10 32 45 20 5 48 20 63

perilla oil

cottonseed oil

canola oil

othersa

3 3

trb tr

2 2

tr tr tr

2 1 2 2 1 1 1 1 1

1 8 29 8

1 tr tr

1

43 28 7 10 10 36

10

tr tr tr tr 1 tr tr tr 2 tr tr

Other ingredients include flavoring agents or imported sesame flavored oil with unknown ingredients. bTrace, 0.5 w/w %) were obtained from local grocery stores. Samples of adulterated sesame oils (n = 29) consisted of commercial sesame-flavored oils and commercial roasted sesame oils. The above samples of authentic and adulterated sesame oils were used to select variables that best characterize authentic sesame oils using an OPLS-DA technique. Blind samples of sesame oils (n = 70) were provided by the National Institute of Food and Drug Safety Evaluation (NIFDS) of the Ministry 8956

DOI: 10.1021/acs.jafc.5b04082 J. Agric. Food Chem. 2015, 63, 8955−8965

Article

Journal of Agricultural and Food Chemistry

Table 2. Stable Isotope Ratio Values of Carbon, Hydrogen, snd Oxygen in the Authentic and Adulterated Sesame Oil Samples variable δ C (‰ vs VPDB) δ2H (‰ vs VSMOW) δ18O (‰ vs VSMOW) 13

authentic sesame oila (n = 35)

adulterated sesame oilb (n = 29)

−32.4 to −28.0 −29.8 −205.6 to −137.9 −181.4 14.5−27.6 18.8

−31.1 to −16.7 −27.0c −202.1 to −151.6 −172.5c 15.1−22.5 19.2

range mean range mean range mean

a Prepared in the laboratory by extraction from sesame seeds after roasting. bConsists of 25 samples of commercial sesame flavored oils and 4 samples of commercial roasted sesame oils in which the 18:3n-3 content deviated from the standards of roasted sesame oil in the Korea Food Code. c Significantly different from authentic sesame oil, P < 0.05.

analysis. Each sample (1 μL) was injected in split mode (split ratio of 200:1). The carrier gas was helium at a flow rate of 1.0 mL/min. The injector and detector temperatures were maintained at 225 and 285 °C, respectively. The oven temperature was initially held at 100 °C for 4 min. It was then programmed to increase to 240 °C at a rate of 3 °C/min and held at 240 °C for 17 min. The FAMEs were identified by comparing their retention times with those of reference standards (Supelco 37 component FAME Mix), and their relative contents were calculated as w/w %. Statistical Analysis. A two-tailed Student’s t test was performed to determine the differences between the authentic and adulterated sesame oil samples (P < 0.05). The differences among oil samples including corn, soybean, sunflower, perilla, cottonseed, and canola oil samples as well as authentic and adulterated sesame oil samples were determined using one-way ANOVA followed by Duncan’s multiplerange test (P < 0.05). Pearson’s correlation test was performed to determine whether a significant linear relationship existed between the two variables (P < 0.01). The above three statistical analyses were conducted using IBM SPSS Statistics (version 20) software (SPSS Inc., Chicago, IL, USA). Using the stable isotope ratio, 1H NMR spectrum, and fatty acid composition data, OPLS-DA was performed in SIMCAP+ (version 12.0) software (Umetrics, Umeda, Sweden). Pareto scaling was applied to each of the three data sets prior to OPLS-DA. The OPLS-DA model was validated using cross-validated ANOVA. An S-plot from OPLS-DA was used to identify significant variables that effectively discriminated between the authentic and adulterated sesame oil samples.

convert the hydrogen and oxygen in the oil samples into H2 and CO gases, respectively. The H2 and CO were subsequently separated in a GC column (PTFE, 2 m; 6 × 5 mm, Agilent Technologies), and their isotopic compositions were analyzed on the IRMS. The stable isotope ratio was expressed as the delta (δ) value in per mille (‰) deviation from the respective international standard, that is, Vienna Pee Dee Belemnite (VPDB, CaCO3) for carbon (i.e., δ13C) and Vienna Standard Mean Ocean Water (VSMOW, H2O) for hydrogen (i.e., δ2H) and oxygen (i.e., δ18O), according to the equation

δ (‰) = [(R s − R std)/R std] × 1000

(1,)

where R represents the ratio of heavy to light isotopes and Rs and Rstd are the isotope ratios of the oil sample and the international standard, respectively. The δ13C value of each oil sample was determined from the standard calibration curve of fuel oil NBS 22 (IAEA-International Atomic Energy Agency, Vienna, Austria), cellulose IAEA-CH-3 (IAEA), and sucrose IAEA-CH-6 (IAEA) with certified δ13C values of −30.0, −24.7, and −10.4‰ on the PDB scale, respectively. The δ2H value was calibrated with the standard curve of ice core water USGS46 (U.S. Geological Survey (USGS)), NBS 22 (IAEA), and VSMOW (IAEA) with certified δ2H values of −235.8, −119.6, and 0‰ on the VSMOW scale, respectively. The δ18O value of the oil samples was determined using the standard calibration curves of VSMOW (IAEA), water UC04 (USGS), and benzoic acid IAEA-602 (IAEA), which had certified δ18O values of 0, 38.9, and 71.4‰ on the VSMOW scale, respectively. 1 H NMR Analysis. Each oil sample (100 μL) was dissolved in 600 μL of deuterated chloroform (99.8% atom D, with 0.03 v/v % tetramethylsilane (TMS)) from Sigma-Aldrich and injected into 5 mm NMR tubes (Wilmad-Lab Glass, Buena, NJ, USA). The chloroform and TMS provided a field frequency lock and a chemical shift reference, respectively. 1H NMR spectra were obtained using a 600 MHz NMR spectrometer (VNS-600, Varian Inc., Palo Alto, CA, USA). Prior to analysis, a phase correction was applied to all spectra using VnmrJ software (Agilent Technologies Inc., Santa Clara, CA, USA); then, MestReNova software (Mestrelab Research, Santiago de Compostela, Spain) was used as the analytical software for the spectra. The baseline correction of all of the spectra was performed with the Whittaker smoother method, and some dislocated spectra were aligned using the align spectra method. The integration value of each peak was determined and normalized on the basis of the first peak (0.7446− 1.0445 ppm), which was derived from the terminal methyl group of fatty acids to avoid dilution effects on the samples. Fatty Acid Analysis. Each oil sample (20 mg) was saponified with 0.5 M methanolic sodium hydroxide (3 mL) at 85 °C for 10 min, cooled to room temperature, and then methylated with 14% boron trifluoride in methanol (3 mL) at 85 °C for 10 min. After the samples had cooled to room temperature, isooctane (3 mL) and saturated sodium chloride solution (5 mL) were added, and the mixture was vortexed. The upper isooctane layer containing fatty acid methyl esters (FAMEs) was collected and passed through an anhydrous sodium sulfate column. The FAMEs were analyzed using gas−liquid chromatography. An Agilent Technologies 7890 N gas chromatograph (Palo Alto, CA, USA), equipped with a flame ionization detector and a fused silica capillary column (SP-2560, 100 m × 0.25 mm i.d. × 0.2 mm film thickness, Supelco, Bellefonte, PA, USA) was used for the

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RESULTS AND DISCUSSION Stable Isotope Ratio. The carbon, hydrogen, and oxygen stable isotope ratios of adulterated sesame oils were compared to those of authentic sesame oils (Table 2). The δ13C value of adulterated sesame oil samples was significantly (P < 0.05) greater than that of authentic sesame oil samples. This arose from the fact that corn oil, the δ13C value of which is known to be greater than that of sesame oil,5 is most frequently added to adulterated sesame oils. In the present study, we also analyzed the carbon stable isotope ratio of six different kinds of edible oils commonly used for the adulteration of sesame oil, that is, corn, soybean, sunflower, perilla, cottonseed, and canola oils. Our analysis showed that the δ13C value was significantly greater in corn oil samples (range, −16.9 to −15.7‰; mean, −16.2‰) than in authentic sesame oil samples, whereas the δ13C values of the soybean (range, −31.0 to −30.4‰; mean, −30.6‰), sunflower (range, −29.2 to −28.1‰; mean, −28.6‰), perilla (range, −30.0 to −29.1‰; mean, −29.5‰), cottonseed (range, −29.4 to −29.0‰; mean, −29.2‰), and canola oil samples (range, −31.3 to −28.2‰; mean, −29.6‰) were not significantly different from that of the authentic sesame oil samples. The δ2H value was significantly (P < 0.05) greater in adulterated sesame oil samples compared to authentic sesame oil samples. However, the difference in δ2H values between the 8957

DOI: 10.1021/acs.jafc.5b04082 J. Agric. Food Chem. 2015, 63, 8955−8965

Article

Journal of Agricultural and Food Chemistry

Figure 1. Typical 1H NMR spectra of sesame oil samples used in the present study. Labeled peaks are assigned in Table 3.

Table 3. Chemical Shift and Peak Assignments in the 1H NMR pectra (Figure 3) of Sesame Oil Samples According to Previously Published Studies15−17 peak

chemical shift range (ppm)

chemical formula moiety

2 5 7 8 9 10 11 12 15 17 18 19 20 21 22 23 24 25 26

0.9406−1.0030 1.9146−2.1659 2.7208−2.8533 2.9811−3.1151 3.2495−3.3649 3.5914−3.6819 3.8254−3.8970 3.9256−3.9914 4.6679−4.7513 5.2904−5.4201 5.9028−5.9244 5.9318−5.9471 5.9471−5.9625 6.4788−6.5214 6.6017−6.6369 6.6763−6.7230 6.8321−6.8598 6.8605−6.8910 6.7429−6.8997

CH3CH2CHCHCH2CHCHCH2CHCH(CH2)7COOH −CH2CHCHCH2CHCHCH2− −CH2CHCHCH2CHCHCH2− sesamolin H1 sesamolin H5 sesamolin H4a sesamin H4, H8 sesamolin H8a sesamin H2, H6 −CH2CHCHCH2CHCHCH2− sesamolin H5′ sesamin H5′, H5″ sesamolin H5″ sesamolin H9″ sesamolin H2″ sesamolin H8″ sesamin H2′, H2″ sesamolin H2′ rest of the aromatic Hs in sesamin and sesamolin

The δ18O value of the adulterated sesame oil samples was not significantly different from that of the authentic sesame oil samples. Our analysis found that the δ18O value of the authentic sesame oil samples was also not significantly different from that of the corn (range, 19.7−23.1‰; mean, 20.6‰) soybean (range, 20.0−21.8‰; mean, 20.7‰), sunflower (range, 20.1− 23.4‰; mean, 22.0‰), perilla (range, 16.4−20.3‰; mean, 17.4‰), or cottonseed oil samples (range, 19.8−22.7‰; mean, 20.8‰). However, the δ18O value of the canola oil samples (range, 20.8−40.5‰; mean, 27.0‰) was significantly (P < 0.05) greater than that of the authentic sesame oil samples. 1 H NMR Spectrum. Figure 1 shows a typical 1H NMR spectrum of the authentic sesame oils obtained in the present study. The spectrum was composed of 26 main peaks, which have been assigned previously.15−17 The chemical shift ranges

authentic and adulterated sesame oil samples was not pronounced compared to that in the δ13C values. This occurs because, unlike the Student’s t test results, the ANOVA results showed that the δ2H value of the authentic sesame oil samples was not significantly different from that of the adulterated sesame oil samples or that of the corn (range, −180.7 to −150.7‰; mean, −170.2‰) soybean (range, −174.7 to −165.3‰; mean, −170.8‰), sunflower (range, −180.6 to −150.2‰; mean, −168.4‰), perilla (range, −192.3 to −187.9‰; mean, −190.2‰), or canola oil samples (range, −230.7 to −137.1‰; mean, −197.5‰). However, the δ2H value of the cottonseed oil samples (range, −143.6 to −139.9‰; mean, −142.1‰) was significantly (P < 0.05) greater than that of the authentic sesame oil samples. 8958

DOI: 10.1021/acs.jafc.5b04082 J. Agric. Food Chem. 2015, 63, 8955−8965

Article

Journal of Agricultural and Food Chemistry

Table 4. Integration Values of 1H NMR Peaks in Authentic and Adulterated Sesame Oil Samples

of the 19 peaks attributed to the unsaturated acyl moieties in the triacylglycerols and lignans present in the sesame oils and their assignments are summarized in Table 3. Peak 2 signifies the terminal methyl group of n-3 fatty acids including 18:3n-3. Peaks 5 and 7 signify a methylene group next to a CC double bond and a methylene group between two CC double bonds, respectively. Peak 17 signifies a vinyl proton (i.e., a proton directly attached to a CC double bond). The other 15 peaks (i.e., peaks 8−12, 15, and 18−26) are attributed to the protons of sesamin and sesamolin, which are two major sesame lignans that are unique phenolic compounds possessing a methylene dioxy fragment (−OCH2O−), as shown in Figure 2. The carbon numbering system used in this study is also shown in the figure.

variablea I2 I5 I7 I8 I9 I10 I11 I12 I15 I17

Figure 2. Carbon numbering in sesamin (A) and sesamolin (B).

I18

Table 4 compares the integration values of the 19 peaks mentioned above for authentic and adulterated sesame oils. The integration value of peak 2 (I2 value) was significantly (P < 0.05) greater in the adulterated sesame oil samples than in the authentic sesame oil samples. This is attributed to the fact that the edible oils rich in 18:3n-3 such as soybean, perilla, and canola oils are commonly used for the adulteration of sesame oil. Our analysis demonstrated that the I2 value of the soybean (range, 0.0649−0.0752; mean, 0.0712), perilla (range, 0.5699− 0.6049; mean, 0.5871), and canola oil samples (range, 0.0865− 0.1057; mean, 0.0942) was significantly (P < 0.05) greater than that of the authentic sesame oil samples. However, the I2 values of corn (range, 0.0156−0.0203; mean, 0.0177), sunflower (range, 0.0051−0.0136; mean, 0.0098), and cottonseed oil samples (range, 0.0028−0.0150; mean, 0.0109) were not significantly different from that of the authentic sesame oil samples. The integration value of peak 7 (I7 value) of the adulterated sesame oil samples was significantly (P < 0.05) greater than that of the authentic sesame oil samples. This result indicates that adulterated sesame oil contains greater amounts of polyunsaturated fatty acids, such as linoleic acid (18:2n-6), having methylene-interrupted double bonds in their structures. Our fatty acid analysis also demonstrated that the 18:2n-6 content was higher in the adulterated sesame oil samples than in the authentic sesame oil samples, as described below. Adulterated sesame oil samples had significantly (P < 0.05) smaller integration values for all of the peaks derived from sesamin and sesamolin compared to those in the authentic sesame oil samples. For example, the adulterated and authentic sesame oil samples had mean integration values of 0.0027 and 0.0135 for peak 26 (I26 value), respectively. These results were caused by the much higher levels of lignan compounds including sesamolin and sesamin in sesame oil than in most other edible oils. In the present study, peak 26 was not detected in the 1H NMR spectra of corn, soybean, sunflower, perilla, cottonseed, or canola oil.

I19 I20 I21 I22 I23 I24 I25 I26

range mean range mean range mean range mean range mean range mean range mean range mean range mean range mean range mean range mean range mean range mean range mean range mean range mean range mean range mean

authentic sesame oilb (n = 35)

adulterated sesame oilc (n = 29)

0.0061−0.0165 0.0122 1.0543−1.1466 1.0901 0.2517−0.3152 0.2806 0.0012−0.0077 0.0033 0.0002−0.0017 0.0010 0.0002−0.0018 0.0008 0.0016−0.0085 0.0035 0.0003−0.0019 0.0008 0.0017−0.0087 0.0036 0.7800−0.8656 0.8192 0.0007−0.0047 0.0019 0.0033−0.0178 0.0071 0.0008−0.0044 0.0019 0.0004−0.0020 0.0010 0.0004−0.0022 0.0009 0.0003−0.0023 0.0009 0.0015−0.0086 0.0035 0.0003−0.0022 0.0009 0.0056−0.0329 0.0135

0.0175−0.1517 0.0698d 1.0342−1.1133 1.0871 0.2850−0.4492 0.3775d 0.0000−0.0038 0.0006d 0.0000−0.0015 0.0002d Traced−0.0011 0.0002d 0.0000−0.0040 0.0007d 0.0000−0.0010 0.0001d 0.0000−0.0040 0.0007d 0.8174−0.9798 0.9102d 0.0000−0.0024 0.0003d Trace−0.0081 0.0016d Trace−0.0024 0.0004d 0.0000−0.0012 0.0001d 0.0000−0.0012 0.0001d 0.0000−0.0012 0.0001d 0.000−0.0040 0.0007d 0.0000−0.0012 0.0001d 0.0000−0.0147 0.0027d

a

Integration values of 1H NMR peaks. Numbers represent the peak numbers shown in Figure 1. bPrepared in the laboratory by extraction from sesame seeds after roasting. cConsists of 25 samples of commercial sesame flavored oils and 4 samples of commercial roasted sesame oils in which the 18:3n-3 content deviated from the standards of roasted sesame oil in the Korea Food Code. dSignificantly different from authentic sesame oil, P < 0.05.

Fatty Acid Composition. The total fatty acid compositions of the authentic sesame oils were compared to those of the adulterated sesame oils (Table 5). The two major fatty acids of the authentic sesame oil samples were 18:2n-6 and oleic acid (18:1n-9). The adulterated sesame oil samples had significantly (P < 0.05) greater 18:2n-6 content than the authentic sesame oil samples. However, the 18:1n-9 content was significantly (P < 0.05) lower in the adulterated sesame oil samples than in the authentic sesame oil samples. This is attributed to the fact that most of the edible oils used for adulteration of sesame oil contain more 18:2n-6 and less 18:1n-9 than sesame oil. Our analysis found that the 18:2n-6 contents of corn (range, 48.5− 53.8 w/w %; mean, 52.0 w/w %), soybean (range, 51.1−52.9 8959

DOI: 10.1021/acs.jafc.5b04082 J. Agric. Food Chem. 2015, 63, 8955−8965

Article

Journal of Agricultural and Food Chemistry

than the authentic sesame oil samples. The mean 18:2t contents of the authentic and adulterated sesame oil samples were 0.1 and 1.0 w/w %, respectively. The mean 18:3t content of the adulterated sesame oil samples was 0.7 w/w %, whereas authentic sesame oil samples contained little 18:3t. Our analysis found that the 18:2t and 18:3t contents of most edible oils (i.e., corn, soybean, sunflower, perilla, and canola oils for 18:2t content; corn, soybean, sunflower, perilla, and cottonseed oils for 18:3t content) used as the main ingredients in adulterated sesame oils were not significantly different from those of authentic sesame oil (data not shown). These results suggest the possibility that low-quality ingredients containing a considerable amount of trans fatty acids were added to the adulterated sesame oil samples (particularly, sesame-flavored oils) used in the present study. Variables To Verify Sesame Oil Authenticity. OPLSDA, a supervised multivariate statistical method, was performed to obtain information on differences in the stable isotope ratio, 1 H NMR spectrum, and fatty acid composition data between the authentic and adulterated sesame oils. An OPLS-DA model was generated from each of the three data sets, as well as by including all three types of analytical data. The cross-validated ANOVA P value was 4.92 × 10−2 for the model from the stable isotope ratio data; 3.24 × 10−17 for the model from the 1H NMR spectral data; 1.05 × 10−19 for the model from the fatty acid composition data; and 3.79 × 10−16 for the model from all three types of data. The model fit parameters were R2Y = 0.316 and Q2Y = 0.193 for the model from the stable isotope ratio data; R2Y = 0.769 and Q2Y = 0.752 for the model from the 1H NMR spectral data; R2Y = 0.854 and Q2Y = 0.823 for the model from the fatty acid composition data; and R2Y = 0.853 and Q2Y = 0.792 for the model from all three types of data. These results suggest that the model from the stable isotope ratio data had low goodness-of-fit compared to the other three models. However, these models were not employed to verify the authenticity of sesame oils in the present study because they did not clearly discriminate between the authentic and adulterated sesame oil samples when displayed as score plots (data available in Figure S1 of the Supporting Information). Instead, an S-plot constructed by the OPLS-DA model, in which the X-axis and Yaxis represent the contribution and confidence of the variables, respectively, was used to identify statistically significant variables that can be used to discriminate between the authentic and adulterated sesame oil samples (Figure 3). In the S-plot, the closer a variable is to the upper right or lower left corner of the graph, the more strongly the variable contributes to the difference between the two groups and the more significant is its contribution. Accordingly, the δ13C value in the upper right corner in Figure 3A was selected as a variable to effectively discriminate between the authentic and adulterated sesame oil samples. Among the integration values of 19 different 1H NMR peaks, I2 and I7 values in the upper right corner and the I26 value in the lower left corner were selected (Figure 3B). However, the I17 value, which was very close to the I7 value, was not selected as a variable to verify the authenticity of sesame oil samples because it showed strong positive correlation to the I7 value (Pearson’s R = 0.991). In Figure 3C, among the content values of 12 different fatty acids, the 18:1n-9 content in the upper right corner and the 18:3n-3, 18:2t, and 18:3t contents in the lower left corner were regarded as the variables that contribute the most to the difference between the authentic and adulterated sesame oil samples.

Table 5. Content Values (w/w %) of Fatty Acids in the Authentic and Adulterated Roasted Sesame Oil Samples variable saturated fatty acid 16:0 range mean 18:0 range mean 20:0 range mean unsaturated fatty acid 16:1 range mean 18:1n-9 range mean 18:1n-7 range mean 18:2n-6 range mean 18:3n-3 range mean 20:1 range mean trans fatty acid 18:1t range mean 18:2t range mean 18:3t range mean

authentic sesame oila (n = 35)

adulterated sesame oilb (n = 29)

8.2−10.1 9.1 4.3−6.6 5.3 0.5−0.8 0.6

8.4−11.7 10.3c 2.2−5.9 3.9c 0.4−0.6 0.5c

0.1−0.2 0.1 35.3−43.0 39.3 0.8−1.0 0.9 38.3−48.7 44.0 0.3−0.4 0.3 0.1−0.2 0.2

nad 0.1 23.3−38.5 28.4c 0.6−1.5 1.1c 38.6−52.7 47.8c 0.5−11.4 5.1c 0.2−4.2 0.9c