Hp 6890 series gc system

The volatile compounds of the L. angustifolia were identified by comparing the mass spectra data with the spectrometer database of the NIST 11 Library, and by comparison of their retention index calculated against n-alkanes (C₉–C₂₀). Each chromatographic analysis was repeated three times. The average value of the relative composition of the essential oil percentage was calculated from the peak areas. The Hewlett Packard HP 6890 series GC system chromatograph was used for the study, which was coupled with the Hewlett Packard 5973 mass selective detector. The GC column used was a non-polar, high-temperature ZB-5HT (5% diphenyl- and 95% dimethylpolysiloxane) with a capillary column that was 30 m long, an inner diameter of 0.32 mm, and a film thickness of 0.25 μm (Phenomenex Inc., Torrance, CA, USA). The gas chromatograph was equipped with a split injector; the split ratio was 20:1 and 1 μm of a sample was introduced. The injector temperature was 250 °C. Helium served as the carrier gas, and its flow rate was 2 mL/min. The oven program was 40–180 °C with the heating rate 5 °C/min, and 180–280 °C with the heating rate of 10 °C/min. The essential oil sample (20 µL) was dissolved in 1 mL of dichloromethane and directly analysed. The relative amounts of the identified (and few unidentified) components represent the percentage abundance (area percent, solvent peak excluded).

The chemical analyses of all extracts were performed on an Agilent 7890 A Series gas chromatograph (Agilent Technologies, Germany) with a flame ionization detector (detector temperature: 310°C) and a DB-5 capillary column (30 m × 0.25 mm inner diameter, J&W), with the carrier gas hydrogen (constant flow 2.0 ml min⁻¹). The split valve opened 1 min after splitless injection (injector temperature 310°C) of 1 μl of the samples; the oven temperature increased by 10°C min⁻¹ from 50°C to 310°C. The identification of 76 compounds was based on the results of former gas chromatographic mass spectrometric analyses [46 (link)] and the comparison of mass spectra (gas chromatography mass spectrometry, HP 6890 Series GC System and HP 5973 Mass Selective Detector (Hewlett-Packard Company, Wilmington, USA)) and gas chromatographic retention indices with those of authentic references, of literature data from the B. terrestris semicochemical complex [24 (link),25 ], of mass spectral libraries (Nist08 database, Wiley 7 database) and of non-public internal databases. The absolute and relative amounts of these compounds were measured with Agilent ChemStation software (Agilent Technologies, Germany).

For the analysis of cellular fatty acid, polar lipids, and isoprenoid quinones, cells were obtained from 7 days of culture in a modified B2M medium. The fatty acids were methylated and analyzed using gas chromatography (HP 6890 Series GC System; Hewlett Packard, Palo Alto, CA, USA) [39 ]. Total lipids were extracted and separated by two-dimensional TLC plates (20 cm × 20 cm silica gel; Merck, Rahway, NJ, USA) [40 (link)]. Chromatography was performed by using chloroform/methanol/water (65:24:4, v/v/v) in the first dimension followed by chloroform/methanol/acetic acid/water (80:12:15:4, v/v/v/v) in the second dimension. The total polar lipids, aminolipids, glycolipids, and phospholipids were identified by spraying 10% ethanolic molybdophosphoric acid (Sigma), ninhydrin (Sigma), α-naphthol, and molybdenum blue regents onto the plates, respectively. Isoprenoid quinones were extracted from freeze-dried cells with chloroform/methanol (2:1, v/v) and purified by TLC [41 ,42 (link)]. The purified quinones were identified by HPLC equipped with a ZOBAX ODS C18 column (4.6 × 150 mm; Agilent, Santa Clara, CA, USA).

Blood plasma and brain samples were harvested immediately after decapitation of mice, frozen in liquid nitrogen and stored at− 80 °C until metabolites were measured by GC–MS. Brain homogenates were extracted using Folch’s procedure, the aqueous supernatant was dried under a stream of nitrogen, and the dry residues were derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS) (99:1). In plasma samples, proteins were precipitated by addition of methanol/water (9:1), centrifuged, and the supernatants were treated as described above.
Samples were measured on an HP-6890 Series GC-System (Hewlett Packard®, Palo Alto, California) coupled to an Agilent Mass Selective Detector 5973 (Agilent®, Waldbrunn, Germany) and an Agilent® Autosampler 7683. We used a VF-5MS capillary column (30 m × 0.25 mm inner diameter) (Varian Technologies®, Palo Alto, CA) with a silylated precolumn (5 m). After the qualitative analysis of the metabolites (spectra adjusted to N.I.S.T. database), we established single ion monitoring (SIM) parameters and used them for quantification of glucose, BHB, citrate, succinate, fumarate and malate. The calculations were done with internal and external standard methods.