5973n mass selective detector

GC-MS analysis was performed using an Agilent GC-MS system (6890N GC system and 5973N mass selective detector, CA, USA) equipped with a HP-5 capillary column (5% diphenyl and 95% dimethyl polysiloxane phase; 0.25 μm film thickness and 30 m × 0.25 mm i.d.). The electron ionization (El) source was operated at 230°C. Helium was used as the carrier gas at a constant flow rate of 0.8 mL/min. The injection volume was 1 μL, and the injection port was maintained at 250°C. The column temperature protocol was 70°C for 2 min with a ramp rate of 3°C/min to 160°C and then 20°C/min to 280°C, where the temperature was maintained for 20 min. The total run time was 58 min. Data were acquired in a full scan mode with m/z range 50–550 using ChemStation software (Hewlett-Packard, Waldbronn, Germany). Computer searches using the NIST Ver. 2.1 MS data library were employed to identify the spectrum of compounds found by the GC-MS results.

GC-MS analyses were carried out on an Agilent 6890 Series gas chromatograph equipped with a 5973 N mass selective detector (USA). A programmed temperature vaporization injector operating in the split mode (1:100) at 250 °C and a ZB-5 capillary column (30 m × 0.25 mm i.d. 0.25 μm film thickness; 5% diphenyl, 95% dimethylpolysiloxane; Phenomenex, USA) were used. The increase in the oven temperature was programmed from 80 °C (held for 1 min) to 300 °C (held for 10 min), at a rate of 20 °C/min. Helium was used as carrier gas at constant pressure (9.8 psi) and the injection volume was 1 μL. The transfer line, ion source and quadrupole temperatures were maintained at 280, 230 and 150 °C, respectively, and a solvent delay of 4 min was selected. Electron ionization was performed at 70 eV and the mass spectrometer was operated in the full scan mode in the range 35–550 Da. All results were compared with Wiley’s library reference spectral bank (G1035B; RevD.02.00). Data recording and instrument control were performed by the MSD ChemStation software (G1701 CA; ver.C.00.00; Agilent Technologies, Little Falls, DE, USA.

Serum (25 µl) collected from ed18 and nd3 broiler birds were spiked with an equal volume of stable isotope-labelled internal standards and were deproteinized with 700 µl of 70% acetonitrile. The samples were centrifuged at 13,500 rpm for 15 min at 4°C, and the supernatant was transferred to a 1 ml v-vial and dried under a stream of nitrogen gas. The metabolites were then converted to their oximes with the addition of 20 µl of 2% methoxamine hydrochloride in pyridine (W/V) and microwaving at 350 W for 90 s. The samples were then derivatized with TBDMS (Tert-butyldimethylsilyl) at 90°C for 1 h. The metabolites were separated on a HP-5MS UI column (30 m × 0.25 mm × 0.25 μm; Agilent, CA, United States) and the ion fragments determined by single ion monitoring (SIM) under electron ionization mode using a GC-MS (5973N, Mass Selective Detector coupled to a 6890 Series GC System, Agilent, CA, United States). Metabolite concentrations were determined in relation to their respective stable isotope-labelled internal standard. For primary hepatocytes, the cells were collected in 1X RIPA and were deproteinized with 700 µl of 70% acetonitrile. The samples were spiked with a known volume of stable isotope-labeled organic acid and amino acid internal standards and sonicated for 10-min to extract the cellular contents. This extract was processed similar to the serum samples for GC-MS analysis.

Free MGO from cell extracts was analyzed on an Agilent 6890 N gas chromatograph equipped with a 7683 Series auto sampler and a 30-m J&W Fisher DB-35 ms capillary column (250-μm I.D. and 0.25 μm film) coupled with a 5973 N mass selective detector (all modules and columns from Agilent Technologies, Waldbronn, Germany). Samples (2 μL) were injected at 250 °C in splitless mode with helium as carrier gas with a flow rate of 1 mL/min. Initial GC oven temperature was set to 50 °C, held for 2 min, then increased at a rate of 15 °C/ min up to 320 °C and held for 10 min. The electron impact ionization source operated at 230 °C, 70 eV and a scan range of m/z 50 to 550. For MGO quantitation, the peak area of m/z 181 of the corresponding derivative was integrated.

GC analyses were performed using an Agilent 6850 gas chromatograph oven equipped with a split/splitless injector and a SPME injector liner (0.75 mm ID) and coupled with an Agilent 5973N Mass Selective Detector (MS). The installed capillary column was a Mega-5MS 5% Phenyl Methyl (length 30.0 m, ID 0.25 mm, film thickness: 0.25 µm, MEGA S.r.l., Legnano, Italy).
For SPME analyses, a Supelco DVB/CAR/PDMS fiber assembly, length 1 cm, 50/30 µm film thickness (fused silica 24 Ga, gray) was installed in a manual holder and then used for the extraction of volatiles from samples, which were weighed directly in headspace glass vials (20 mL) and hermetically sealed before equilibration.

The fatty acid composition in blue honeysuckle seed oil was analyzed using GC–MS. To increase the volatility of the seed oil, pre-column derivatization was used. In a 10 mL screw-cap glass tube, 30 µL of seed oil was dissolved in a mixed solution of n-hexane and benzene (1:1, v/v) and gently shaken. Then, 2 mL of 0.5 mol/L KOH in methanol was added, and the mixture was allowed to stand for 30 min. Subsequently, 5 mL of distilled water was added to separate the organic phase solution (n-hexane), causing it to rise to the upper layer of the tube. The top layer solution was diluted 20 times and analyzed by GC–MS on an Agilent 6890 GC system equipped with a 5973 N mass selective detector (Agilent Technologies Inc., Wilmington, DE, USA) using a DB-5 capillary column (60 m × 0.25 mm id, 0.25 µm film thickness, J&W Scientific, CA, USA). The GC injection port temperature was 250 °C, and helium was used as the carrier gas at a flow rate of 1.0 mL/min. The injection volume was 1.0 µL, and the injection split ratio was 1:10. The oven temperature was started at 180 °C and held for 5 min, and then ramped to 240 °C at 3 °C/min and held for 8 min. The total program time was 32 min. The ion source temperature was 230 °C. The MS detection was operated at 70 eV with a scan range of 50–550 m/z.