Triacylglycerols and Fatty Acid Compositions of Cucumber, Tomato, Pumpkin, and Carrot Seed Oils by Ultra-Performance Convergence Chromatography Combined with Quadrupole Time-of-Flight Mass Spectrometry

The triacylglycerol (TAG) compositions of cucumber, tomato, pumpkin, and carrot seed oils were analyzed using ultra-performance convergence chromatography (UPC2) combined with quadrupole time-of-flight mass spectrometry (Q-TOF MS). A total of 36, 42, 39, and 27 different TAGs were characterized based on their Q-TOF MS accurate molecular weight and MS2 fragment ion profiles in the cucumber, tomato, pumpkin, and carrot seed oils, respectively. Generally, different vegetable seed oils had different TAGs compositions. Among the identified fatty acids, linoleic acid was the most abundant fatty acid in cucumber, tomato, and pumpkin seed oils and the second most abundant in carrot seed oil with relative concentrations of 54.48, 48.69, 45.10, and 15.92 g/100 g total fatty acids, respectively. Oleic acid has the highest concentration in carrot seed oil and the second highest in cucumber, tomato, and pumpkin seed oils, with relative concentrations of 78.97, 18.57, 27.16, and 33.39 g/100 g total fatty acids, respectively. The chemical compositions of TAGs and fatty acids could promote understanding about the chemical profiles of certain vegetable seed oils, thus improving the potential ability to select appropriate oils with specific functions and a high nutritional value and then develop functional foods in the future.


Introduction
Cucumber (Cucumis sativus), tomato (Solanum lycopersicum), pumpkin (Curcubita pepo), and carrot (Daucus carota) are widely consumed vegetables around the world. After industrial manufacturing, these vegetables could be processed into canned food, juice, smoothies, sauce, etc., thus they became more acceptable due to their convenience, health, nutrition, and safety [1,2]. Following industrial processing, significant amounts of vegetable seeds end up as waste, even though they have been found

UPC 2 System Condition
The conditions of UPC 2 were set according to our previous manuscript [22]. A Waters Acquity ultra-performance convergence chromatography (UPC 2 ) system (Milford, MA, USA) equipped with a binary pump, an oven, an auto-sampler, a back-pressure regulator, and an Acquity UPC 2 BEH HSS C 18 column (150 mm × 3.0 mm i.d.; 1.7 µm) was utilized for the separation of TAGs. The back pressure was 1800 psi and column oven temperature was 30 • C. The mobile phase A was pure supercritical fluid CO 2 and the mobile phase B was methanol. The flow rate was 1.6 mL/min and the injection volume was 2.0 µL. The elution gradient started at 99% A, decreased linearly to 98.2% A over 5 min, decreased linearly to 98% A over the next 7 min, decreased linearly to 97% over the next 3 min, decreased linearly to 70% over the next minute, was held at 70% A for 3 min, and back to 99% A to re-equilibrate the column. Methanol with 0.1% ammonium formate as a compensated solvent was pumped at a flow rate of 0.3 mL/min by a Waters 1525 pump.

Quadrupole Time-of-Flight (Q-TOF) MS Conditions
A Waters Xevo-G2 Q-TOF MS system was utilized to characterize the chemical structures of TAGs in cucumber, tomato, pumpkin, and carrot seed oils. The electrospray ionization (ESI) in positive mode was used in a mass range from 100 to 1200 Da at a capillary voltage of 3.0 kV and a cone voltage of 50.0 V. The source offset was 80.0 V. Leucine enkephalin was used as the lock mass (m/z 556.2771 in ESI + ). The source temperature was 120 • C and the desolvation temperature was 500 • C. The desolvation gas (nitrogen) was set at a flow of 800.0 L/h and the collision gas (argon) was set at 150.0 L/h. Data were collected using Masslynx 4.1 software (Milford, MA, USA) in MS E mode. The MS acquisition method consisted of a low collision energy mode (MS 1 ) and a high collision energy mode (MS 2 ). In the MS 1 mode, the collision energy was 6 eV and parent ions were obtained. The information of fragment ions was collected in the MS 2 mode and the collision energy was 35 eV. The scan time was 0.2/s.

Free Fatty Acid Composition Analysis
Free fatty acid compositions of cucumber, tomato, pumpkin, and carrot seed oils were determined by previous lab protocol [23]. Briefly, 20 mg of each oil sample was vortex mixed thoroughly with 0.4 mL of methylbenzene and 0.4 mL of KOH-MeOH (0.5 mol/L). The mixture was sealed and heated at 60 • C for 10 min. After cooling the reaction mixture to an ambient temperature, 2 mL of boron trifluoride-MeOH (14%) was added and the mixture, which was sealed with a lid, was heated at 60 • C for 5 min. Before being vortexed, 2 mL of isooctane and 3 mL of ultrapure water were added. The supernatant was injected for GC analysis. Fatty acid compositions were determined using Agilent 7890A gas chromatograph equipped with FID detector and DB-23 silica capillary column (60 m length × 0.25 mm with a 0.25 µm film thickness) using helium as the carrier gas. The injection volume was 1 µL. The oven temperature was initially at 100 • C and increased to 184 • C by 10.5 • C/min. After holding at 184 • C for 3 min, it was increased again to 240 • C at a rate of 6 • C/min. Fatty acid methyl esters were identified by the retention time compared with those of the standard FAMEs. Relative concentrations of identified fatty acids were quantified based on the area normalization method, which is calculated using each identified individual peak area divided by the total area of all peaks identified in the extracts. Each sample was analyzed in triplicate.

Statistical Analysis
The relative concentrations of TAGs and fatty acids were reported as the mean ± standard deviation (SD) based on the peak area normalization method. The relative concentrations of each TAG and fatty acid were analyzed with one-way ANOVA and Tukey's post hoc test using SPSS 18.0 (Chicago, IL, USA), and p < 0.05 was considered a significant difference.

Identification of TAGs
A total of 36, 42, 39, and 27 different TAGs were tentatively identified in the cucumber, tomato, pumpkin, and carrot seed oils, respectively (Table 1). Representative base peak intensity (BPI) chromatograms of cucumber, tomato, pumpkin, and carrot seed oils obtained by UPC 2 -Q-TOF-MS are presented in Figure 1. The TAG compositions in the seed oils were determined using the accurate mass data of quasi-molecular ions of [M+NH 4 ] + and MS 2 fragmentation ions of [M+H] + information [21,22]. There are three typical types of TAGs: TAG that contain 3 same fatty acid side chains; or 3 different fatty acid side chains; or 2 same and 1 different fatty acid side chain with the different one at sn-2 or sn-1/sn-3 position. Three mass spectrums were presented to explain the identification of these three typical types of TAGs (Figures 2-4). A total of 36 TAGs were tentatively identified in cucumber seed oil, with SLO as the most abundant TAG, followed by LLO and LLL (Table 1). Further, 23 of the identified TAGs, namely PLPo, LnPoL, PLnLn, PoLL, PLLn, LnLnLn, SLS, SOS, LLG, LLA, OLG, LOA, OOA, SLA, OSA, LLB, OLB, LSB, LLT, OLT, LLLi, LOLi, and LLH, were reported in cucumber seed oil for the first time. By extrapolating the ratios of increments for fatty acids to the HPLC retention of component TAGs, Deineka and others found OLLn, OOO, and PPO in cucumber seed oil 20-and 30-days after fruit setting, which were not detected in the present study [26]. Different TAG compositions in cucumber seed oil may be due to different identification techniques as well as different ripening and growing conditions of the cucumber samples. X-X-Y, X-Y-X, X-X-X, and X-Y-Z represent structures of triacylglycerols, for example, S-P-O stands for the structure of 1/3-stearoyl-2-palmitoyl-1/3-oleoylglycerol. The relative concentration of each triacylglycerol is reported as grams of triacylglycerols/100 g of oil samples. Cucumber, tomato, pumpkin, and carrot seed oils were analyzed in triplicate and results reported as mean ± standard deviation (SD). Different letters represent significant differences within a column (p < 0.05).
Foods 2020, 9, 970 6 of 12 myristic acid, S: stearic acid, O: oleic acid, L: linoleic acid, Ln: linolenic acid, G: gondoic acid, A: arachidic acid, B: behenic acid, Li: lignoceric acid, E: Erucic acid, T: Tricosanoic acid, H: Hexacosanoic acid. X-X-Y, X-Y-X, X-X-X, and X-Y-Z represent structures of triacylglycerols, for example, S-P-O stands for the structure of 1/3-stearoyl-2-palmitoyl-1/3-oleoylglycerol. The relative concentration of each triacylglycerol is reported as grams of triacylglycerols/100 g of oil samples. Cucumber, tomato, pumpkin, and carrot seed oils were analyzed in triplicate and results reported as mean ± standard deviation (SD). Different letters represent significant differences within a column (p < 0.05).           A total of 42 TAGs were detected and identified in tomato seed oil, with LLL, SOL, and OLL as the three major TAGs (Table 1). Among all the identified TAGs, 33 of them including SOO, SLS, PLS, LLLn, SOS, LLA, LOA, PLP, PoLL, PLPo, MLL, LSA, LLLi, PLLn, LSB, OOA, LOLi, LLB, LLH, OSA, LnPoL, SLE, LLnLn, LLG, OLG, LnLA, PLnLn, OOB, LLT, LnLB, LnLLi, LnLH, and OLT were reported in tomato seed oil for the first time. Silva and others calculated the theoretical compositions of 5 triacylglycerols in tomato seed oil according to a computer program developed based on the fatty acid profile by Antoniosi Filho, Mendes, and Lanças [27], which were all directly detected and identified in the present study [7]. Compared with the identification strategy of TAGs based on the fatty acid compositions, UPC 2 -Q-TOF MS technology not only directly showed higher accuracy and feasibility, but also provided the actual structure of TAGs, which is not limited by the detection of fatty acid composition.
A total of 39 TAGs were identified in pumpkin seed oil and OSL, LOO, and OLL were the three major TAGs, with a relative concentration of 9.37, 9.18, and 7.97 g/100 g TAGs, respectively (Table 1) [26,[28][29][30][31][32] but not detected in this study. These differences might due to the different varieties of the seed samples and the different identification techniques that were used.
Similarly, a total of 27 TAGs were detected in carrot seed oil, with OOO, OLO, and OLL having the highest concentrations of 22.53, 15.33, and 10.27 g/100 g TAGs, respectively (Table 1). In agreement with a previous study, Giuffrè found that OOO was the highest TAG in all detected virgin and extra virgin olive oil and constituted 30-50% of the total TAGs by detecting the TAG content of 10 different cultivars grown in South West Calabria [33]. Considering that olive oil is a vegetable oil with many recognized biological properties [34], carrot seed oil may have similar bioactivates but warrants additional research to reveal its specific health effects. There are 17 TAGs, including OSL, OOG, PoOO, OLG, OLA, PPoO, PoLL, LLLn, MOL, OOB, OEO, PLP, OLE, OLB, LLnLn, LnLB, and LOLi, that were reported in carrot seed oil for the first time. Thao and others found 10 TAGs in carrot seed oil using LC-Q-TOF MS and seven of them, including OOO, OLO, OSO, POO, LLL, PoLO, and OOA, were also detected in the present study, whereas OLLn, LAA, and LnLnLn were not observed [35].

Fatty Acid Compositions
Four major fatty acids, including palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2) were identified and their relative concentrations for each seed oil sample were investigated and are listed in Table 2. Linoleic acid was the most abundant fatty acid in cucumber, tomato, and pumpkin seed oils and the second abundant in carrot seed oil, with relative concentrations of 54.48, 48.69, 45.10, and 15.92 g/100 g total fatty acids, respectively ( Table 2). The results were consistent with previous studies that linoleic acid had the highest content of 68.1%, 54.7%, and 56.4% in cucumber, tomato, and pumpkin seed oils, respectively [5,26]. Conjugated linoleic acid has been associated with several vital biological functions such as antitumor, anti-obesity, antidiabetic, anti-inflammatory, and cardioprotective activities [36][37][38], suggesting that the consumption of cucumber, tomato, and pumpkin seed oils in daily life might be good for human health, but more research is needed to verify its accuracy. In addition, oleic acid has the highest concentration in carrot seed oil and was the second abundant in cucumber, tomato, and pumpkin seed oils, with relative concentrations of 78.97, 18.57, 27.16, and 33.39 g/100 g total fatty acids, respectively ( Table 2). The content of oleic acid was in agreement with previous reports, contributing about 68.1-81.2% in carrot seed oil [13,35]. As we know, oleic acid is not only the most important monounsaturated fatty acid in the diet, but also the predominate constituent of plasma free fatty acids with many bioactivities such as anti-tumor, anti-inflammatory, and cardioprotective functions [34,[39][40][41]. For an extra virgin olive oil, oleic acid has to be in the 55-83% range of the total fatty acids [42,43], which is widely considered as a healthy Foods 2020, 9, 970 9 of 12 oil. With many properties such as anti-oxidant, anti-inflammatory, and cardiovascular protective effects [34]. Consumption of carrot seed oil might have similar health effects as olive oil, but more investigation is needed in the future. Palmitic acid was the third abundant fatty acid in cucumber, tomato, pumpkin, and carrot seed oils, with concentrations of 14.98, 16.81, 14.21, and 5.07 g/100 g total fatty acids, respectively ( Table 2). The content of stearic acid in cucumber, tomato, pumpkin, and carrot seed oils were 11.97, 7.34, 7.29, and 1.04 g/100 g total fatty acids, respectively ( Table 2). Some minor content fatty acids such as linolenic, behenic, arachidic, lignoceric, eicosenoic, palmitoleic, and myristic acids were detected in previous studies in these seed oils, however they were not detected in this study [5,13,16,35]. This might be due to the fact that the content of these fatty acids (FAs) was under a detectable level in the present study, which may be caused by different cultivars or different harvest times of these seed samples [44]. FAs stands for fatty acids; C:D represents carbon number:double bounds number. The relative concentration of each fatty acid is reported as grams of fatty acids/100 g of total fatty acids. Cucumber, pumpkin, carrot, and tomato seed oils were analyzed in triplicate and results are reported as mean ± standard deviation (SD). Different letters represent significant differences within a column (p < 0.05).
In the comparison of fatty acid compositions determined using gas chromatography directly and calculated from TAGs compositions ( Table 3), 7 of 16 relative deviations (RDs) were less than 8%, which means that most of the TAGs compositions identified in this study were consistent with their fatty acid composition results. But there was still one result with an abnormal RD value of stearic acid in carrot seed oil greater than 70%, which might be due to its low relative concentration of 1.04 g/100 g total fatty acids. The relative deviations of oleic acid and linoleic acid indicated good consistency between the determined value and calculated value in cucumber (−2.01%, 2.23%), pumpkin (−0.24%, 6.22%), and tomato (3.74%, 2.94%) seed oils. These results showed that the determined value was close to the calculated value in general. MV, mean value of fatty acid compositions determined by GC; CV, calculated value of fatty acid compositions calculated from triacylglycerols compositions reported in Table 1; RD, relative deviation.

Conclusions
In summary, a total of 36, 42, 39, and 27 different TAGs were identified in the cucumber, tomato, pumpkin, and carrot seed oils, respectively. A total of 23, 33, 22, and 17 TAGs detected in cucumber, tomato, pumpkin, and carrot seed oils were reported for the first time. Generally, different vegetable seed oils contained different TAGs compositions. SLO, LLL, OSL, and OOO were the most abundant TAGs in cucumber, tomato, pumpkin, and carrot seed oil, with relative concentrations of 11.55, 7.52, 9.37, and 22.53 g/100 g TAG, respectively. Among the identified fatty acids, linoleic acid concentration was the highest in cucumber, tomato, and pumpkin seed oils and the second highest in carrot seed oil, with relative concentrations of 54.48, 48.69, 45.10, and 15.92 g/100 g total fatty acids, respectively. Oleic acid was the most abundant in carrot seed oil and the second most abundant in cucumber, tomato, and pumpkin seed oils, with relative concentrations of 78.97, 18.57, 27.16, and 33.39 g/100 g total fatty acids, respectively. UPC 2 -Q-TOF MS technology was utilized to analyze the TAGs in four vegetable seed oils and showed higher accuracy and feasibility than the identification strategy based on the fatty acid composition. These results could provide a reference for selecting appropriate oils with specific functions and high nutritional values to develop functional foods in the future.