Agilent 5973 mass spectrometer

The essential oil was characterized and quantified by GC–MS analysis on an Agilent 6890N gas chromatograph instrument equipped with an Agilent 5973 mass spectrometer and an HP-5MS capillary column (length 30 m × 0.25 mm ID, film thickness 0.25 mm; Agilent-Technologies, Palo Alto, CA, USA). The carrier gas was helium at a constant flow of 1.0 mL/min. The oven temperature programs were as follows: from 50 °C (held for 2 min) to 80 °C (2 °C/min), from 80 °C to 150 °C (5 °C/min), from 150 °C to 200 °C (10 °C/min), from 200 °C to 300 °C (20 °C/ min) and held there for 5 min. The temperature of injector was 250 °C. The samples were diluted in hexane (1:40 v/v), then 1 μL of the diluted samples were injected in splitless mode. Component identification was done based on MS library search (NIST and Wiley). The percentage composition was calculated by integrating the peak areas of the chromatograms.

The analysis of FAME was done according to Ristivojević et al.^{55 (link)}. In short, an Agilent 6890 gas chromatograph equipped with a DB-23 capillary column (30 m × 0.25 mm id;film thickness of 0.25 μm) was used (Aglient Technologies Inc., Santa Clara, CA, USA). The capillary column was directly joined to an Agilent 5973 mass spectrometer (Agilent Technologies Inc.). The sample (1 μL) was injected into the capillary column with a split ratio of 10:1. Helium (purity of 5.0) was applied as the carrier gas with a flow rate of 0.6 mL/min. The temperatures of the detector and the injector were set to 230 °C and 250 °C, respectively.
The FAME were determined through comparing their retention times with those of the FAME standards (Supelco-37 FAME mix) under the same conditions, and through comparing their mass spectra with those stored in the Mass Spectral Library of the National Institute of Standards and Technology (NIST).

GC analyses were conducted with an Agilent 6890 GC (Agilent Technologies Inc., Santa Clara, CA, USA) with DB-23 capillary column (30 m × 0.25 mm ID, 0.25 μm film thickness) (Agilent Technologies Inc.). The capillary column was connected to an Agilent 5973 mass spectrometer (Agilent Technologies Inc.) with a quadruple analyzer and an electron energy of 70 eV. The analytical conditions were as follows. In the on-column inlet, the temperature was set to 50 °C, and the injection volume was 1 µL with a split ratio of 10:1. The column temperature was increased to 175 °C for 8 min and further increased to 235 °C at a gradient of 2 °C/min. Helium was used as the carrier gas at a flow rate of 0.7 mL/min. The temperatures of the detector and the injector were set to 230 °C and 250 °C, respectively. The mass spectra were recorded in the range of 30–800 m/z with a scanning frequency of 1.95 scans s⁻¹.

Headspace solid-phase microextraction (HS-SPME) was performed using 5 mL of MF. To enhance the volatilization of the VOCs to the headspace (HS) and standardize sampling conditions, protonation of the acidic fraction of the MF was accomplished by saturating all the samples with NaCl and adjusting their pH to 3.2 using HCl [20 ]. Volatile organic compounds (VOCs) were extracted from the HS with a DVB/CAR/PDMS (divinylbenzene/Carboxen/polydimethylsiloxane, 1 cm length, 50/30 µm coating thickness) SPME fiber (Supelco, Bellefonte, PA, USA) at 60 °C for 90 min. Desorption was performed in the GC injector at 240 °C for 5 min. An HP 5890 series II Gas Chromatogram (GC) hyphenated to an Agilent 5973 Mass Spectrometer (MS, Agilent, Palo Alto, CA, USA) was used for GC–MS analysis. The GC injector was operated at 240 °C in splitless mode (2 min). The column used was a DB-FFAP (Agilent) of 30 m length × 0.25 mm i.d. × 0.25 µm film thickness with a constant carrier gas flow of helium at 1 mL/min. The GC oven program was as follows: 40 °C, hold for 2 min, ramped at 5 °C/min to 240 °C, and hold for 2 min. The MS was operated in full-scan mode (scanning from 50–350 amu at a rate of 4.6 scans/s) with ionization energy of 70 eV. The MS transferline, ion source, and quadrupole temperature were held at 240, 230, and 150 °C, respectively.

This was determined according the method of Adewuyi et al. [12 (link)]. Briefly, oil was refluxed for 1 h in 25 mL of 2 M ethanolic potassium hydroxide. The reaction mixture was later diluted to 150 mL with distilled water and transferred into a separating funnel and the unsaponifiable matters were then extracted three times with 50 mL diethyl ether. The ether extract was first washed with 100 mL aqueous solution of 0.5 M potassium hydroxide in order to remove any residual fatty acids. This was further washed with distilled water until it was free of potassium hydroxide, dried over anhydrous sodium sulphate, and concentrated using a rotary evaporator. The unsaponifiables were identified by GC-MS using Agilent (Palo Alto, USA) 6890N gas chromatography equipped with an HP-1 MS capillary column connected to an Agilent 5973 mass spectrometer operating in the EI mode (70 ev; m/z 50–550; source temperature 230°C and quadruple temperature 150°C). Structural assignments were made based on interpretation of mass spectrometric fragmentation and confirmation by comparison of retention time as well as fragmentation pattern of authentic compounds and the spectral data obtained from the Wiley and NIST libraries.