Analysis of Lipids in Pitaya Seed Oil by Ultra-Performance Liquid Chromatography–Time-of-Flight Tandem Mass Spectrometry

Red pitaya (Hylocereus undatus) is an essential tropical fruit in China. To make more rational use of its processing, byproducts and fruit seeds, and the type, composition, and relative content of lipids in pitaya seed oil were analyzed by UPLC-TOF-MS/MS. The results showed that the main fatty acids in pitaya seed oil were linoleic acid 42.78%, oleic acid 27.29%, and palmitic acid 16.66%. The ratio of saturated fatty acids to unsaturated fatty acids to polyunsaturated fatty acids was close to 1:1.32:1.75. The mass spectrum behavior and fracture mechanism of four lipid components, TG 54:5|TG 18:1_18:2_18:2, were analyzed. In addition, lipids are an essential indicator for evaluating the quality of oils and fats, and 152 lipids were isolated and identified from pitaya seed oil for the first time, including 136 glycerides and 16 phospholipids. The main components of glyceride and phospholipids were triglycerides and phosphatidyl ethanol, providing essential data support for pitaya seed processing and functional product development.


Introduction
Hylocereus polyrhizus, commonly known as dragon fruit or pitaya, is native to Mexico but widely cultivated worldwide, including in southern China, Malaysia, Thailand, Vietnam, and Australia [1]. The varieties of pitaya mainly include Hylocereus undatus, Hylocereus polyrhizus, and Hylocereus megalanthus [2]. Hylocereus undatus is distributed primarily in tropical areas such as Guangdong and Hainan in China, and the planting area only in Guangdong has exceeded 500,000 mu. Pitaya is rich in nutrients and unique functions; it contains phytoalbumin and anthocyanins, which are rarely found in plants and are rich in vitamin C and water-soluble dietary fibers [3][4][5].

Determination of Fatty Acid Composition by Gas Chromatography
The fatty acid composition of pitaya seed oil was determined by the external standard method (Carbon XVII fatty acid methyl ester standard, Avanti Polar Lipids, U.S. Alabama) of potassium hydroxide methylation. Weigh 6-10 mg of pitaya seed oil accurately and 50 µL of 5 mg/mL of the internal standard carbon XVII fatty acid methyl ester, add 2 mL of 0.4 mol/L KOH methanol solution, vortex, and shake for 15 min, add 1 mL of n-hexane and 2 mL of 0.9% NaCl aqueous solution, shake for 2-3 s, centrifuge at 4500 rpm at 4 • C for 10 min, take the supernatant and transfer it to a 2 mL injection vial.
Gas chromatography-mass spectrometry (GC-MS) was equipped with a hydrogen flame ionization detector and DB-FastFAME column (30 m × 0.25 mm × 0.25 µm, 7890A gas chromatograph tandem hydrogen flame ionization detector, Agilent, Palo Alto, CA, U.S.). The measurement conditions were as follows: nitrogen as the carrier gas, injection volume of 1.0 µL, the inlet temperature of 260 • C, splitting ratio of 20:1; programmed temperature rise, and column initial temperature of 150 • C. The column was ramped up to 210 • C at 10 • C/min and held for 8 min, then ramped up to 230 • C at 20 • C/min and maintained for 6 min, and the detector temperature was 280 • C.

Determination of Lipid Composition Using UPLC-TOF-MS/MS [16]
An aliquot of 150-200 mg of pitaya seed oil was placed in a 10 mL test tube, and 10 µL of 10 µg/mL triglyceride deuterium (internal standard), 2 mL of hexane, 2 mL of methanol, and 0.2 mL of ultrapure water were added sequentially, shaken and mixed, vortexed, and centrifuged. The supernatant was removed, and the supernatant was placed in a test tube and blown dry with a nitrogen-blowing instrument (DC-24, Shanghai Ampli Experimental Technology Co., Shanghai, China). Then, 100 µL of methanol was added for re-dissolution, and the re-dissolved solution was passed through a 0.22 µm organic filter membrane and placed in a sample bottle for use.

Analytical Conditions for UPLC-MS
The analytical instrument was a Shimadzu UPLC LC-30A system (LC-30A liquid chromatograph, Shimadzu Corporation, Kyoto, Japan) equipped with a Phenomenex Kinete C18 column (100 × 2.1 mm, 2.6 µm). A sample of 1 µL was pumped onto the column at a rate of 0.4 mL/min. The column temperature was 60 • C, and the sample chamber temperature was 4 • C. Gradient elution was performed using phase A (H 2 O-methanolacetonitrile = 1:1 psi1, containing 5 mM ammonium acetate) and phase B (isopropanolacetonitrile = 5:1, containing 5 mM ammonium acetate) with elution conditions of 20% B for 0.5 min, 40% B for 1.5 min, 60% B for 3 min, 98% B for 13 min, 20% B for 13 min, and 20% B for 17 min. In addition, the mass spectrometry system (Q-TOF-6600 Mass Spectrometer, AB Sciex, Concord, Ontario, Canada) was an AB Sciex TripleTOF ® 6600 with an ESI ion source in positive and negative modes, and the mass number range for mass spectrometry was m/z 100-1200:50.00. The ion spray voltage was set to 5500 V (+) and −4500 V (−). Pressures of ion source gas 1, ion source gas 2, and curtain gas were set at 50, 50, and 35 psi, respectively, and the interface heater temperature was 600 • C. The detection limit of the instrument was 6 (peak height) and the quantification limit was 10.
Extraction methods and varieties affected the seed oil's extraction rate and fatty acid composition. The unsaturated fatty acid content of pitaya seed oil measured in this study (75.43%) was higher than that of Wang [11], which might stem from the different extraction methods and some other differences. In addition, linoleic acid in white pitaya seed oil was higher than that in red pitaya seed oil, and the content of the red pitaya seed oil obtained by chloroform-methanol extraction was greater than that obtained by the Soxhlet extraction. Compared with the cold pressing method, the Soxhlet and chloroformmethanol extraction methods yielded more fatty acid species in pitaya seed oil. The Soxhlet extraction method produced the most species. The extraction method affected pitaya seed oil's extraction rate and fatty acid composition; pitaya seed oil's composition and content varied greatly by species.
The molecular species of compounds were identified by retention time, isotopic distribution, MS mass-to-charge ratio, and MS/MS secondary mass spectrometry in positive and negative ion modes. Figure 1A shows the MS/MS spectra of TG 54:5|TG 18:1_18:2_18:2 in positive ion mode. From Figure 1A, m/z 898.7898 was the [M + NH 4 ] + parent ion of this compound, which was neutral to the loss of FA 18:1 and FA 18:2 to produce two diester fragment ions, which were 36:4DDAG + (m/z 599.5042) and 36:3DDAG + (m/z 601.5217), respectively. Figure
As shown in Table 2  phospholipids was 34-42, and the double bond number was 1-4. The number of carbon atoms of PEtOH was 34-36, and the number of double bonds was 1-4. The number of carbon atoms of PG was 29-42, and the number of double bonds was 0-4. The number of carbon atoms of PMeOH was 16-34, and the double bond number was zero.

Analysis of the Lipid Content of Pitaya Seed Oil
Under the same conditions, the mass spectra of the same class of lipids should be similar and comparable. In this experiment, the peak areas of the extracted ion chromatographic peaks in the primary mass spectra of pitaya seed oil were used for the quantitative calculation of the same class of lipids, as shown in Figure 3. As seen in Figure 3, the lipid composition of pitaya seed oil mainly consisted of glycerides and phospholipids with contents of 505.69 ± 18.79 mg/g and 2.08 ± 0.24 mg/g, respectively. Glycerides were mostly TG, DG, and OxTG, with contents of (482.80 ± 17.0) mg/g, (7.41 ± 0.64) mg/g, and (14.12 ± 0.98) mg/g, accounting for 95.47, 2.79, and 1.46% of the glycerides, respectively. Thus, these three compounds accounted for 99.73% of the total glycerides. Dietary triglycerides are the main component of oils, and their main functions are to supply and store energy, fix and protect internal organs, participate in the energy supply in several aspects of maternal and intrauterine fetal growth and development during pregnancy, and play a key role in lipid metabolism [32,33]. Accordingly, pitaya seed oil could be used in oil and fat dietary supplements. tion of pitaya seed oil mainly consisted of glycerides and phospholipids with contents of 505.69 ± 18.79 mg/g and 2.08 ± 0.24 mg/g, respectively. Glycerides were mostly TG, DG, and OxTG, with contents of (482.80 ± 17.0) mg/g, (7.41 ± 0.64) mg/g, and (14.12 ± 0.98) mg/g, accounting for 95.47, 2.79, and 1.46% of the glycerides, respectively. Thus, these three compounds accounted for 99.73% of the total glycerides. Dietary triglycerides are the main component of oils, and their main functions are to supply and store energy, fix and protect internal organs, participate in the energy supply in several aspects of maternal and intrauterine fetal growth and development during pregnancy, and play a key role in lipid metabolism [32,33]. Accordingly, pitaya seed oil could be used in oil and fat dietary supplements. Phospholipids were mainly PEtOH (1.04 ± 0.14 mg/g), accounting for 49.74% of the total phospholipid content. In oilseeds, phospholipids are primarily present in the colloidal phase in a complex state with molecules such as proteins and sugars [34]. In general, phospholipids in plant oilseeds consist mainly of PC, PE, and PI, and the content of phospholipids varies from one oilseed to another or from one variety and growing region of the same oilseed [35]. While PEtOH and PMeOH are the main components of dragon fruit seed oil, there are significant differences in lipid composition among soybean, sesame, peanut, and rapeseed [36]. The phospholipids in food are transformed into choline through a series of chemical reactions under the body's digestive action, which can Phospholipids were mainly PEtOH (1.04 ± 0.14 mg/g), accounting for 49.74% of the total phospholipid content. In oilseeds, phospholipids are primarily present in the colloidal phase in a complex state with molecules such as proteins and sugars [34]. In general, phospholipids in plant oilseeds consist mainly of PC, PE, and PI, and the content of phospholipids varies from one oilseed to another or from one variety and growing region of the same oilseed [35]. While PEtOH and PMeOH are the main components of dragon fruit seed oil, there are significant differences in lipid composition among soybean, sesame, peanut, and rapeseed [36]. The phospholipids in food are transformed into choline through a series of chemical reactions under the body's digestive action, which can promote the speed of information transmission between nerve cells in the brain and enhance memory function. In addition, phospholipids not only improved arterial vascular composition, but also maintained esterase activity, improved lipid metabolism in the body, emulsified neutral esters and cholesterol deposited in the vascular wall, promoted the absorption of fats and fat-soluble vitamins, and enhanced intelligence and cellular activity [37,38]. Therefore, pitaya seed oil has broad application prospects in functional product development.

Conclusions
In this study, the lipid profile of pitaya seed oil was first profiled by UPLC-TOF-MS/MS, and 11 fatty acid components were identified from pitaya seed oil, mainly linoleic acid, oleic acid, and palmitic acid, with an unsaturated fatty acid content of more than 75%, which are highly unsaturated fatty acid oils. In addition, 152 lipid components were identified in pitaya seed oil, mainly composed of 136 triglycerides and 16 phospholipids, with triglyceride content (505.69 ± 18.79) mg/g, which provided basic data support for the later separation of pitaya seed components and in-depth functional research.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.