Rtx 5ms

Gas chromatography-mass spectrometry (GC-MS) analysis was carried out (model; Japan, Kyoto, Shimadzu Corporation, QP2010 Ultra) on a capillary column with a 0.25 mm inner diameter and a 30 m length. The stationary phase used was of 0.25 mm film thickness (U.S.A, Restek Corporation, Rtx-5MS, Bellefonte, PA, USA). Helium (99.999%) was employed as the carrier gas, moving at a constant speed of 36.3 cm/s. A sample volume of 1 l was injected using the AOC-20i + s auto-injector. At 290 °C, the injection port was maintained in split-less mode. The GC oven was preheated to the following temperature: 5 min at 50 °C, followed by 10 min of holding at 300 °C at a rate of 2 °C/min. The m/z range of 30 to 700 was used to construct a total ion chromatogram. By comparing their mass spectra to the National Institute of Standards and Technology’s database (NIST), and with the literature, GC peaks were identified [70 (link),71 (link)]. By comparing each constituent’s peak area to the chromatogram’s overall peak area, the relative percentage quantity of each constituent was calculated [72 (link)].

GC–MS data was acquired with Single Quadrupole Mass Spectrometer and Gas Chromatograph (Trace 1310) (Thermo Fisher Scientific, USA). The GC column was 30 m long dimethyl (95%)/diphenyl polysiloxane (5%) RTX-5MS with 0.25 mm ID, film of 0.25 µm, and guard column (10 m) (Restek, PA, USA). Starting temperature of GC oven was 60 °C for 60 s and the temperature increased to 325 °C (@10 °C/min) and final hold time was 5 min. The MS ion source temperature was 230 °C. Helium acts as carrier gas.

The chromatographic separation was recorded using Shimadzu GC/MS-QP2010 (Japan) apparatus equipped with Rtx-5MS (Restek, Bellefonte, PA, USA) fused-bonded column (30 mL, 0.25 mm i.d., and 0.25 µm film thickness) that is coupled to SSQ 7000 quadrupole mass spectrometer, Thermo-Finnigan, Germany. The chromatographic procedure and conditions followed the method adopted by Mostafa (2018) [21 ]. Identification of compounds was performed according to comparing the mass spectra, calculated retention indices by those published in Adams [22 ], NIST Mass Spectral Library, co-chromatographic standards, and other literature [20 (link),23 (link),24 (link)].

100 Mg of finely freeze-dried powdered sample (for both fruits) was extracted with 5 mL 100% methanol with sonication for 30 min using Branson CPX-952-518R set at 36 °C, (Branson Ultrasonics, Carouge, SA Switzerland.) and with regular shaking, followed by centrifugation (LC-04C 80-2C regen lab prp centrifuge, Zhejiang, China) at 12,000× g for 10 min to eliminate debris. For evaluation of biological replicates, 3 independent samples for each fruit was analyzed under the same conditions. Then, 100 µL of the methanol extract was kept in opened screw-cap vials and left to evaporate under stream of nitrogen gas until full dryness. For derivatization, 150 µL of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA), previously diluted 1/1 with anhydrous pyridine, was mixed with the dried methanol extract and incubated (Yamato Scientific DGS400 Oven, QTE TECHNOLOGIES, Hanoi, Vietnam) for 45 min at 60 °C previous analysis using GC–MS. Separation of silylated derivatives was completed on a Rtx-5MS Restek, Bellefonte, PA, USA (30-m length, 0.25-mm inner diameter and 0.25-m film). Analysis of these primary metabolites followed the exact protocol detailed in Sedeek, M.S. et al., and Farag, M.A. et al. [11 (link),12 (link)].

The samples were analyzed using LECO Pegasus 4D GC×GC−TOF MS (St Joseph, MI, USA). The first dimension capillary column was Restek Rtx-5MS (30 m × 0.25 mm × 0.25 μm) and the second capillary column was Restek Rtx-200 (2 m × 0.25 mm × 0.25 μm). The GC temperature program was set as follows: injection temperature: 280 °C; oven temperature: 40 °C maintained for 1 min, and increased at a rate of 10 °C/min to 310 °C and held constant for 8 min. The helium flow rate was set at 1 mL/min. The mass spectrometry temperature was set at 320 °C. The ion source temperature was 200 °C, and the analysis mass range was 50-800 m/z. KWM-EO was ran in hexane with a dilution of 1 mg/mL. Hexadecane solution, 64.25 μg/mL, was used as an internal standard to monitor the shift of retention time. Compounds were identified by matching the mass spectra fragmentation patterns, and the results were compared with LECO/Fiehn and Wiley Registry 9th Edition mass spectral library and NIST. Linear Kovats index of n-alkanes (C₇-C₄₀, C₇-C₃₀) were calculated for each compound and compared with the literature to identify the compound ID [30 (link)].

Shimadzu GC-17A gas chromatograph (Shimadzu Corporation, Kyoto, Japan) with a DB-5 fused-bonded cap column (Phenomenex; 29 m × 0.25 mm i.d., film thickness 0.25 µm; Torrance, CA, USA) and a FID detector was used for the quantitative analysis of the samples. Shimadzu GC-2010 plus a gas chromatograph (Shimadzu Corporation, Kyoto, Japan) were employed for GC analyses and equipped with Rtx-5MS (Restek, Bellefonte, PA, USA) and a quadrupole mass spectrometer for identification of the volatile oil components. Instrumental settings were adjusted according to what was previously reported [5 (link)]. Three independent runs were used to calculate AUP (areas under the peaks) using Class GC 10^® software (Shimadzu Corporation, Kyoto, Japan) in which the total area is considered 100%. Meanwhile, for qualitative interpretation of the chromatograms, GC solution^® software ver. 2.4 (Shimadzu Corporation, Kyoto, Japan) was used. For comparing and identifying the constituents of the different essential oil samples, Wiley Registry of Mass Spectral Data 8th edition, NIST MassSpectral Library (December 2011), and previously reported techniques were employed [6 (link)].