Hp 5ms column

Table 1 details the origin of the 19 EOs samples used in this study. Industrial-scale extractions by dragging water vapor were applied to obtain the EOs, which were selected based on their commercial availability. All samples were standardized according to the methods and quality indicators described in the Brazilian Pharmacopoeia (≥ 90% purity).
The chemical composition of the EOs was assessed by gas chromatography/mass spectrum (GC/MS) performed with a Shimadzu 2030 equipment coupled to a sequential Shimadzu TQ8040 mass detector, using an HP-5MS column (30 m × 0.25 mm × 0.25 μm). Analytical conditions were established at 250 and 260 °C injector and transfer line temperatures, respectively; the oven temperature was programmed from 60 to 240 °C, at a rate of 3 °C min⁻¹ and maintained at 240 °C for 10 min; helium gas at 1.0 mL.min⁻¹; 0.1 μL injection volume (5% HPLC grade n-hexane solution); and a 1:30 split ratio.
The identification of the components was performed by comparing the mass spectra with those of commercial libraries [55 (link)], and by their linear retention rates [56 (link)] after injection of a homologous series of alkanes (C₈–C₂₆) under the same experimental conditions, and compared with data from the literature [57 ].

The samples were ground into powder using liquid nitrogen with mortars and pestles. Then, 0.5 g of powder was transferred into a tube containing 1 mL of MTBE with 0.01 ng/mL tetradecane as an internal standard. The tube was vortexed for 3 min at maximum speed, incubated at 24 °C with rotation at a speed of 50 rpm for 1 h, and then dried using Na₂SO₄ and filtered through a 0.22 mm-mesh filter.
GC/MS analysis was performed using GC/MS-QP2010Ultra apparatus (Shimadzu Corporation, Kyoto, Japan) with an HP-5 MS column. The column temperature was programmed as follows: holding at 40 °C for 3 min, 40 °C to 280 °C at 10 °C/min, and holding for 2 min. The ion source and transfer line temperatures were 230 °C and 280 °C, respectively; the electron ionization was 70 eV; and the mass scanning was performed from 45 to 450 m/z with 5 scans/s. The results were qualitatively and quantitatively analyzed using the GC Solutions software (Shimadzu Corporation, Kyoto, Japan) with NIST (2017) and the PESTEI3 library.

After the catalyzed reaction was stopped (interrupted), the reaction mixture was extracted with four-fold volume of hexane. The organic phase was collected by centrifugation at 8000 × g, then dried by N₂ and resolved in 300 μL of isooctane. After that, the samples were analyzed by gas chromatography (GC).
Squalene and hopene were assayed qualitatively and quantitatively by using an Agilent 6890A GC apparatus equipped with a HP-5 column (30 m × 0.25 mm × 0.25 μm) and a flame ionization detector. Two microliters of the solvent extract was applied onto the column (splitless, helium flow rate: 0.5 mL/min). The injector temperature was 250°C. The oven temperature was initially 60°C, kept constant for 3 min, then raised to 220°C at a rate of 20°C/min, kept constant for 3 min, further increased to 310°C at a rate of 6°C/min, and kept constant for 10 min. The retention time of squalene and hopene were 24.50 min and 29.70 min, respectively. Alternatively, a Shimadzu GC-MS QP 2010 system with an HP-5MS column (30 m × 0.25 mm × 0.25 μm) was used for coupled GC and mass spectrometry (MS) analysis. The condition of MS is: ion source temperature, 230°C, interface temperature, 280°C, and the temperature programming was the same as that in GC (Wu et al., 2017 (link)).

The solvents were distilled, dried and stored according to standard procedures.²⁰ Unless otherwise stated, reagents were obtained from commercial sources and were used as received. The imidazolium salts were synthesized according to our previously reported method.^{14 (link)1}H, ¹³C NMR, and 2D-HSQC spectra were recorded with a Bruker Advance 300 spectrometer. Chemical shifts (δ) are reported in ppm with the residual solvent resonance signal: δ H/C 7.27 : 77.2 for CDCl₃; and δ H 4.79 for D₂O. Melting points were determined on a Reichert–Kofler hot-stage microscope and were uncorrected. Microanalytical data were obtained using an Exeter Analytical Inc. CE-440 microanalyzer. Infrared spectra were collected on an FTIR spectrometer Nicolet Nexus-470. Stille coupling and Suzuki–Miyaura reactions mixture were analyzed by gas–liquid chromatography using a Shimadzu GC-14B instrument equipped with a flame-ionization detector and a HP-5MS column (30 m × 0.25 mm × 0.25 mm), using nitrogen as carrier gas. Mass spectra (EI) were obtained at 70 eV on a Hewlett Packard HP-5890 GC/MS instrument equipped with a HP-5972 selective mass detector.

3-HP was determined by HPLC with an LC-20AT instrument (Shimadzu, Kyoto, Japan) using an Aminex HPX-87H column (Bio-Rad, Hercules, USA). The column temperature was maintained at 60 °C using 5 mM H₂SO₄ as the mobile phase at a flow rate of 0.6 ml/min, and the signal was detected using a RI-101 refractive index detector.
Mesaconate was quantified using a GC/MS-QP2010 Ultra (Shimadzu, Kyoto, Japan) equipped with a HP-5 MS column (0.25 mm ID × 0.25 μm Film Thickness × 15 m Length). The column temperature was programmed at an initial temperature of 55 °C for 1 min, then increased to 115 °C at a rate of 30 °C/min and held for 15 min, then increased to 160 °C at a rate of 5 °C/min, then increased to 300 °C at a rate of 30 °C/min, and finally held at 300 °C for 15 min. The temperature of the injector was set at 270 °C. Helium was used as the carrier gas at a flow rate of 1 ml/min. The MS system was operated in full scan mode within the m/z range of 50–600.

Shimadzu 17A GC with Head Space Sampling System HSS-4A and an HP-5MS column (30 m 0.25 mm 0.1 m) were used to perform the analysis of the active fractions of the methanolic extract using gas chromatography-mass spectrometry (GCMS-S80) (Attimarad et al., 2011 (link)) (The sample was injected in split mode at 220°C, and the transfer line was set to 240°C. Helium was used as the carrier gas at a constant flow rate (1 mL/min). The temperature was ramped up from 60°C to 260°C at the rate of 3°C/min and then exposed at 260°C for 25 min in full scan mode, m/z (mass-to-charge ratio) 20–600. Electron impact ionisation was employed (70 eV), and the MS (mass spectrometry) ion source was maintained at 240°C. Compounds were identified by their gas chromatography retention times and mass spectra using standard compounds for comparison with methanolic fraction’s GC-MS graph (Pratap et al., 2020a (link); Pratap et al., 2020b ).

Gas chromatography–mass spectrometry was performed on a GCMS-QP2010 Ultra gas chromatography mass spectrometer (Shimadzu, Kyoto, Japan) equipped with an HP-5MS column (the internal diameter was 0.32 mm, and the length was 30 m). The parameters were as follows: Helium 99.995% purity was the carrier gas, the temperature of the injector was 250 °C, the flow rate through the column was 2 mL/min, and the thermostat temperature program was a gradient temperature increase from 70 to 250 °C with a step of 10 °C/min. The range of the scanned masses was m/z 35–400. The internal standard method using dodecane was used for the quantitative analysis.