Slb 5ms

Additional identification
of the N-compounds present in the real samples was carried out using
a Shimadzu GC × GC-QqQ MS instrument (operated in SCAN mode),
consisting of a GC-2010 with a split/splitless injector and an AOC-20i
autosampler, coupled with a TQ8040 MS. SLB-5ms (20 m × 0.18 mm
ID × 0.18 μm) and SLB-35 (5 m × 0.32 mm ID ×
0.25 μm) columns (Merck Life Science) were used as first and
second dimension, respectively. The modulation was performed every
5 s (an accumulation time of 4.6 s and a re-injection period of 0.4
s) by using a flow modulator consisting of a 7-port wafer with an
accumulation loop with a dimension of 20 cm × 0.51 μm developed
by Chromaleont and Trajan (Trajan Scientific and Medical).^{19 (link)} The system was operated in the constant flow
mode in both the first and the second dimensions at 0.4 and 8 mL min^–1, respectively.

Volatile compounds from samples were isolated using headspace solid phase microextraction (HS-SPME) using a method detailed earlier in [53 (link)] with slight modifications. Each oil (1.5 mL) was placed in different vials (vials volume: 15 mL). The oils were heated for 5 min at 40 °C. SPME fiber divinylben-zene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS; Supelco, Bellefonte, PA, USA) was used for extraction. The fiber was firstly conditioned in the GC injection port at 270 °C for 4 h. To absorb volatiles, fiber was exposed in vials with samples for 5 min at 40 °C in split-less mode. Desorption was carried out at the GC injection port for 5 min. The volatiles were analyzed on a 7890A GC System with a triple-axis mass detector (Agilent Technologies, Santa Clara, CA, USA). Injector temperature was 280 °C. A capillary column SLB5MS (30 m × 0.25 mm × 0.5 µm; Merck, Darmstadt, Germany), with helium as a carrier gas at a flow rate of 0.8 mL/min, was used. The initial oven temperature was 40 °C for 3 min, which was then increased to 160 °C at 4 °C/min. Finally, the temperature was increased to 280 °C at a rate of 10 °C/min, and it was held for 5 min. Mass spectra were recorded in an electron impact mode (70 eV), and masses were scanned from 33 to 333 Da. Mass spectra of volatile components were compared with the mass spectra from MS library–NIST MS Search 2.0.

Hemi-pituitaries and hemi-hypothalamus were mixed with 800 µl boron trifluoride in methanol (14%) and internal standard (heptadecanoic acid, 10 mg/ml). The mixture was heated at 100°C for 40 min and cooled down to room temperature. Aliquots of 400 µl of heptane and 800 µl of H₂O were added, centrifuged at 2 000 g for 2 min and methyl esters were then extracted from the upper heptane phase. The sample was concentrated under nitrogen, 100 µl of heptane was added and submitted to gas chromatography analysis. Fatty acid methyl esters (FAME) were analyzed on GC-MS instruments. One microliter of each sample was injected into the instrument with 1/100 split, a Thermo interfaced with a Focus DSQ mass selective detector and equipped with the Xcalibur software package. The mass spectra and retention indices registered in the FAME GC-MS library were obtained using the Thermo FOCUS DSQ Single Quadrupole GC-MS. This was done using the SLB^®-5ms and the Supelcowax-10 columns made by Supelco (Sigma, France).

Volatile compounds were identified by a Shimadzu GC-17A gas chromatographer coupled to a Shimadzu QP-5050A mass spectrometry detector (Shimadzu Corporation, Kyoto, Japan). The GC-MS system was equipped with a 30 m × 0.25 mm Supelco (Supelco, Inc., Bellefonte, PA, USA) SLB-5 MS (fused silica) column with a 0.25 µm film thickness. The carrier gas used for this analysis was helium maintained at a column flow rate of 0.6 mL min⁻¹ and a total flow of 181.2 mL min⁻¹ in a division ratio of 1:300. The ramp used was: 3 °C min⁻¹ from 80 to 170 °C, and 25 °C min⁻¹ at 300 °C, maintaining this final temperature for 1 min. The detector temperature was 300 °C and 230 °C for the injector.