Elite 5ms capillary column

A total of 8 mg of EE was dissolved in 1 mL of MeOH and injected directly into the GC–MS apparatus (Clarus 680 GC, Perkin Elmer) coupled to a quadrupole mass spectrometer (Clarus SQ8 MS, Perkin Elmer, Singapore). A Perkin Elmer Elite-5MS capillary column (length 30 m × inner diameter 0.25 mm × film thickness 0.25 µm) was used. The temperature gradient was performed as follows: 12 min at 100 °C, 100–180 °C at 15 °C/min, 180 at 300 °C at 5 °C/min, and 10 min at 300 °C. The injector and detector temperatures were 280 and 250 °C, respectively, and the carrier gas flow rate (He) was 1 mL/min. The injection volume was 1 µL. Alkaloids were identified by comparing their mass spectra and retention index (RI). Mass spectra were analyzed using AMDIS 2.64 software (NIST) (Gaithersburg, MD, USA), and RI was recorded with a calibration mixture of hydrocarbon standards (C9–C36). The proportion of each alkaloid present in extracts and fractions analyzed through GC–MS was expressed as a percentage of the alkaloid peak area as a function of the total ion current (TIC).

The amount of rhynchophorol recovered was determine using a gas chromatograph (Clarus 680), coupled to a mass spectrometer detector (Clarus 600C), with an ELITE-5MS capillary column (Perkin Elmer/USA). Samples (1 µL) were injected through a CTC Combipal automatic injector (Pal System/Switzerland). The run conditions were: helium carrier gas with 1 mL min⁻¹ flow, 50 mL split, and injector temperature of 150 °C. The initial temperature of the oven was 50 °C for 3 min with a heat ramp of 10 °C min⁻¹ up to 200 °C, held for 1 min. The mass spectrometry detector was configured to operate with ionization of 70 eV in scanning mode (SCAN), in the mass range of 25–500 m/z. The temperatures were fixed at 200 °C for the ionization source and 180 °C for the quadrupole. The interface with the mass detector was kept at 200 °C.

GC–MS analyses of leaf and rhizome extracts were carried out using the Perkin-Elmer Clarus 680 system (Perkin-Elmer, Inc. U.S.A) equipped with a fused silica column, packed with Elite-5MS) capillary column (30 m in length × 250 μm in diameter × 0.25 μm in thickness). Pure helium gas (99.99%) was used as the carrier gas at a constant flow rate of 1 mL/min. For GC–MS spectral detection, an electron ionization energy method was adopted with high ionization energy of 70 eV (electron Volts) with 0.2 s of scan time and fragments ranging from 40 to 600 m/z. The injection quantity of 1 μL was used (split ratio 10:1), and the injector temperature was maintained at 250 °C (constant). The column oven temperature was set at 50 °C for 3 min, raised at 10 °C per min up to 280 °C, and final temperature was increased to 300 °C for 10 min. The contents of phytochemicals present in the test samples were identified based on comparison of their retention time (min), peak area, peak height and mass spectral patterns with those spectral database of authentic compounds stored in the National Institute of Standards and Technology (NIST) library⁶⁰ .

The phytosterol profile of SBP extracts was determined via GC-MS using the extracts dissolved in n-hexane as mentioned above. Briefly, 2 mL of extract was evaporated to dryness under a nitrogen-free oxygen stream. The residue was derivatized with 100 µL of MSTFA (Mackerey-Nagel, Dueren, Germany) at 60 °C for 45 min, and 1 µL was injected in the GC-MS system equipped with an Elite-5ms capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness of 5% phenyl-95% methylpolysiloxane, Perkin Elmer, Waltham, MA, USA), using helium as the carrier gas (flow rate 1.0 mL/min). The temperature program was: initial temperature 50 °C, hold time 1 min, increase by 20 °C/min to 230 °C, hold time 3 min, then increase by 10 °C/min to final temperature of 310 °C and hold for 3 min. The injection was performed in split mode (split ratio 10:1), and the injector temperature was set to 230 °C. The MS operating conditions were: source temperature 230 °C, transfer line temperature 240 °C, electron impact ionization EI+ at 70 eV and a solvent delay of 1.5 min. The identification of phytosterols was achieved in the full scan mode with the scanned mass range 100–600.

The extracted oil of T. serrulatus was analyzed by the Perkin Elmer gas chromatograph (Clarus 600) coupled with a Perkin Elmer (Clarus 600 T) mass selective detector. Subsequently, 1 µL of the aliquots of the extracts were injected into Elite 5-MS capillary column (30m × 250 µm I. D, 0.25 µm film thickness; Perkin Elmer, Shelton, CT, United States) in the splitless mode of 1:20. The column temperature was kept at 40°C during sample injection and was raised to 150°C for 2 min having rate of 10°C/min and further increased to 300°C held for 2 min. The injector temperature was at 280°C, inlet line temperature at 220°C, and source temperature was 220°C. The total run time was 32 min. Helium was used as the carrier gas at a flow rate of 1.0 ml per min. Detection was carried out by using MS detection in electron-ionization mode using a single quadrupole detector and full-scan monitoring mode (m/z 40–600).
Each individual phyto-constituent was recognized on the basis of its retention time and mass spectrum included in the National Institute of Standards and Technology (NIST, 2005) and Wiley 2006. Out of 90 identified compounds, most of the compounds were identified from the matrix, and some came from the column itself. The compounds that occupied a negligible percentage of the total areas were discarded. Finally, about 50 compounds with match factor 60% were added in the method.

GC analyses for detection and quantification of MCFA were carried out on a Perkin Elmer Clarus 400 instrument (Perkin Elmer, Germany) equipped with an Elite 5MS capillary column (30 m × 0.25 mm, film thickness 1.00 µm, Perkin Elmer, Germany) and a flame ionization detector (Perkin Elmer, Germany). 1 μL of the sample was analyzed after split injection (1:10) and helium was used as carrier gas (90 kPa). For quantification of FAMEs, the temperatures of the injector and detector were 250 and 300 °C, respectively. The following temperature program was used: 50 °C for 5 min; increase of 10 °C/min to 120 °C and hold for 5 min; increase of 15 °C/min to 220 °C and hold for 10 min; increase of 20 °C/min to 300 °C and hold for 5 min. Medium-chain FAMEs were identified and quantified by comparison to FAMEs in standard samples.

The EEPO was analyzed using GC-MS for the identification of various phytochemicals. The analysis was carried out using the Perkin-Elmer Clarus 680 system (Perkin-Elmer, Inc., Shelton, CT, USA) equipped with a fused silica column, packed with an Elite-5MS capillary column (30 m in length × 250 μm in diameter × 0.25 μm in thickness). Pure helium gas (99.99%) was used as the carrier gas at a constant flow rate of 1 mL/min. For GC-MS spectral detection, an electron ionization energy method was adopted with a high ionization energy of 70 eV (electron Volts), 0.2 s scan time and fragments ranging from 40 to 600 m/z. An injection quantity of 1 μL was used (split ratio 10:1), and the injector temperature was maintained at 250 °C (constant). The column oven temperature was set at 50 °C for 3 min, then raised at 10 °C per min up to 280 °C, and the final temperature was increased to 300 °C for a period of 10 min. The contents of phytochemicals present in the test samples were identified based on comparison of their retention time (min), peak area, peak height and mass spectral patterns with those spectral databases of authentic compounds stored in the National Institute of Standards and Technology (NIST) library.