5973 mass selective detector

Analyses using gas chromatography-mass spectrometry were carried out with an HP 6890 GC System (Hewlett Packard, Böblingen, Germany) coupled to a 5973 Mass Selective Detector (Hewlett Packard, Böblingen, Germany). The mass detector worked in the electron impact ion-isolation mode at 70 eV, the mass range was 10–600 units, and the ion source temperature was 230 °C. A total of 1 µL of extract was injected into the GC equipped with a 30 m × 0.25 mm i.d. capillary column with an HP-5MS ((5%-phenyl)-methylpolysiloxane, Agilent J and W GC column) with a coating thickness of 0.25 µm. Helium, at a constant flow rate of 2 mL/min, was used as the carrier gas, while chromatographic conditions were set as follows. Oven temperature was initially held at 40 °C for 3 min, and then increased to 180 °C at a rate of 5 °C/min, and, finally, increased to 250 °C at a rate of 10 °C/min. The equilibration time was 0.5 min. The components were identified by comparison of their mass spectra with those of the spectrometer database using the NIST library (National Institute of Standards and Technology, Gaithersburg, MD, USA), with probabilities higher than 80%. Each sample was analyzed three times.

Deuterated citric acid (2,2,4,4, d4 citric acid) from Sigma Aldrich, Poole Dorset, UK was added to each sample to a final concentration of 0.1 mM as an internal standard. Metabolites were then extracted using cold methanol before being dried under vacuum desiccation. The samples were re-suspended in anhydrous pyridine containing the derivatisation agents methoxyamine hydrochloride, followed by N-Methyl-N-trimethylsilyltrifluoroacetamide, with 1% 2,2,2-Trifluoro-N-methyl-N-(trimethylsilyl)-acetamide, and Chlorotrimethylsilane (MSTFA + 1%TMCS). GCMS was performed in pulsed splitless mode on a Hewlett Packard HP6890 series GC system with Agilent 6890 series injector, a 30 m long 250 µm diameter capillary column (Agilent, Stockport, Cheshire, UK) model number 19091s-433HP5MS) using a flow rate of 1 mL/min, and a Hewlett Packard 5973 Mass selective detector. The acquisition was conducted in selective ion monitoring mode, the ion masses detected for citrate were: 273, 347, 375, and 465 and the corresponding heavy ions were 276, 350, 378, and 469. The dwell time for all these ions was 50 ms. Data were normalised for cell number following subtraction of the medium blank value, and expressed as mM citrate per 10⁵ cells per mL.

The initial screening for determination of differences and/or similarities in the cuticle profiles of Bactrocera spp. was performed with a Hewlett Packard HP 6890 GC System connected to a Hewlett Packard 5973 Mass Selective Detector. For analyses, an HP-5MS capillary column (30 m × 250 μm i.d. × 0.25 μm film; Agilent Technologies, Santa Clara, CA, USA) was used. Temperature was programmed from 150°C to 300°C at a rate of 5°C/min with 10 min final hold at 320°C. Samples (1 μl) were injected using a splitless mode with He as the carrier gas (constant pressure, 1 ml/min). Electron ionization at 70 eV was used in the range from 25 to 600 m/z, with ion source temperature 250°C and quadrupole temperature 150°C.
Detailed identification and quantification of the components was performed by GC×GC/MS, using a LECO Pegasus 4D instrument (LECO Corp., St. Joseph, MI, USA) equipped with a non-moving quad-jet cryomodulator connecting the 1st and the 2nd columns. The methodology has been described in detail elsewhere [23 ,25 (link),26 (link)]. A series of n-alkanes (C₁₂–C₄₀; Sigma-Aldrich) was used to determine the retention indices for the analytes. The compounds were identified by a comparison of their MS fragmentation patterns and retention indices with values published previously [22 (link),23 ,26 (link),29 ,32 ,33 (link)].

Deuterated citrate (d4Citrate) was added to each sample to a final concentration of 0.1 mM as an internal standard. Metabolites were then extracted using cold methanol before being dried under vacuum desiccation. The samples were re-suspended in anhydrous pyridine containing the derivitisation agents methoxyamine hydrochloride followed by N-Methyl-N-trimethylsilyltrifluoroacetamide with 1% 2,2,2-Trifluoro-N-methyl-N-(trimethylsilyl)-acetamide, Chlorotrimethylsilane (MSTFA + 1%TMCS). GCMS was performed in pulsed splitless mode on a Hewlett Packard HP6890 series GC system with Agilent 6890 series injector and a 30 m long 250 µm diameter capillary column (Agilent, model number 19091s-433HP5MS) using a flow rate of 1 mL/minute, and a Hewlett Packard 5973 Mass selective detector. The acquisition was conducted in selective ion monitoring mode, the ion masses detected for citrate were: 273, 347, 375 and 465 and the corresponding heavy ions were 276, 350, 378 and 469. The dwell time for all of these ions was 50 ms.

A Hewlett Packard 6890 Plus gas chromatograph with an Agilent 7683 series injector and a 5973 mass selective detector (Hewlett Packard, Palo Alto, CA, USA) were used for qualitative analyses. The compounds were separated on an HP-5MS capillary column (5%-phenyl-methylpolysiloxane, 30 m × 0.25 mm i.d., 0.25 μm film thickness). Helium was used as a carrier gas at a constant flow of 1 mL/min. The GC inlet temperature was changed from 110 °C to 250 °C by increments of 20 °C and finally to 275 °C, which is the temperature used in our routine general unknown screening. The inlet liner was Agilent splitless single taper, deactivated liner with glass wool (Agilent, Palo Alto, CA, USA, P/N: 5062-3587). The initial oven temperature of 60 °C was held for 2 min and then ramped at 20 °C/min to 300 °C with a final hold time of 15 min. The temperature of the transfer line was 280 °C. The ion source and quadrupole temperatures were 230 °C and 150 °C, respectively. The mass selective detector was used in EI scan mode, the ionization energy was 70 eV, and the electron multiplier voltage was set 200 V above autotune value. Data were collected for a splitless injection of 1.0 µL of each tropane in methanol, ethyl acetate and n-hexane in the range from 50 to 550 m/z at a rate of 2.9 scans/s. The software used was MSD Chemstation D.01.02.16.

The volatile compounds of the S. montana L. herb were identified by comparing the mass spectra data with 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 (Hewlett Packard, WALDBRONN, Germany) was used for the study, which was coupled with the Hewlett Packard 5973 mass selective detector (Hewlett Packard, Waldbronn, Germany). The chromatograph was equipped with the non-polar, high-temperature ZB-5HT (5% diphenyl- and 95% dimethylpolysiloxane) capillary column of length of 30 m, inner diameter of 0.32 mm, 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. Helium served as the carrier gas, and its flow rate was 2 mL/min. Analyses were performed at the temperature range of 40–280 °C and the heating rate was 10 °C/min. Injector temperature was 250 °C.