Fid detector

Distributions of enantiomeric compounds in G. squarrosa essential oils were analyzed by using two different chiral columns, which were appropriate for each enantiomer, and an Agilent 5973 Network Mass Selective Detector on a 6890N GC system that also had an FID detector (Agilent Technologies, Santa Clara, CA, USA) (see further GC system details above). The chiral columns were Rt-βDEXse (2,3-di-O-ethyl-6-O-tert-butyl dimethylsilyl-β-cyclodextrin added into 14% cyanopropylphenyl/86% dimethyl polysiloxane, 30 m × 0.32 mm ID, 0.25 μm film thickness, USA) and Lipodex G (6-methyl-2,3-pentyl-γ-cyclodextrin added into 60% polysiloxane, 25 m × 0.25 mm ID, 0.125 µm film thickness, Germany). For separation of α-pinene, the Lipodex G chiral column was used, while separation of borneol, camphor, and limonene was performed on the Rt-βDEXse column. Samples were injected (10% prepared in hexane) with a 10:1 split ratio. Injection port and detector temperatures were 250 °C. Detailed analysis parameters are provided in Supplementary Materials.

Percentages of total fatty acids from placental lipids (including glycerolipids, phospholipids, sphingolipids, sterol lipids, and free fatty acids) were analyzed as fatty acid methyl ester derivatives (FAMEs) by gas chromatography (GC) as previously described [57 (link)]. Briefly, lipids from 500 mg of placental samples were extracted with chloroform/methanol (2:1, v/v) in the presence of 0.01% butylated hydroxytoluene. The fatty acids were trans-esterified and the resulting fatty acid methyl esters were extracted. A total amount of 4 μL were used for GC analysis. Separation was performed with a DBWAX capillary column (30 m × 0.25 mm × 0.20 μm) in a GC System 7890A with a Series Injector 7683B and an FID detector (Agilent Technologies, Barcelona, Spain). Identification of fatty acid methyl esters was made by comparison with authentic standards (Larodan Fine Chemicals, Malmö, Sweden). Results are expressed as mol percentage (mol %) [56 (link)].
The following fatty acids groups were determined: saturated fatty acids (SFA); monounsaturated fatty acids (MUFA); polyunsaturated fatty acids (PUFA) from n-6 and n-3 series (PUFAn-6 and PUFAn-3, respectively); and the following ratios were also calculated: LA/ALA, AA/EPA, AA/DHA, AA/EPA + DHA, and the n-6/n-3 PUFA ratio as the quotient between all the PUFA n-6s divided by all the PUFA n-3s.

Sebum fat was extracted with ether using the Soxhlet method. Fatty acid methyl esters were obtained using 2M KOH solution in methanol. The profile of the fatty acids in the obtained fat samples was determined using an Agilent 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) with a flame ionization detector (FID) (Agilent Technologies, Santa Clara, CA, USA).
Fat present in serum and erythrocytes was extracted using the Folch method. Fatty acid methyl esters were obtained using 2M KOH solution in methanol. The fatty acid profile in the obtained fat samples was determined using an Agilent 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) with an FID detector (Agilent Technologies, Santa Clara, CA, USA). The determinations were made in the following conditions: HP-88 capillary column (Agilent)—100 m long and 0.25 mm diameter at an initial temperature of 50 °C and with temperature increments of 3 °C/min to 220 °C; temperature of the dispenser—270 °C.
The identification of the obtained fatty acid peaks was performed via comparison with the retention times of the Sulpeco 37 fatty acid methyl ester standards from Sigma.
Blood count and biochemical indices were determined by the commercial veterinary laboratory VetLab sp. z o.o (Wroclaw, Poland).

The analysis of volatile organic compounds isolated from T.minuta was performed using a complete HP/Agilent 6890/5973 GC-MS system with a FID detector (Palo Alto, USA). The GC column specified for the methods was a capillary HP-5MS column (5% phenylmethylsiloxane, 30 m X 0.25 mm i.d., coating thickness 0.25 μm). The injector temperature was set at 250 °C and helium carrier gas flow was adjusted to 1 ml/min. The samples pass through the interface heated to 200 °C into the mass spectrometer operated at 70 eV, which is scanned from 30-300 amu. The electron multiplier voltage was increased to near 3000 V, and the injection volume was 1 µl. GC oven temperature ranged from 70º to 240 °C at l0 °C /min. Qualitative identification for separated constituents was achieved using MS data libraries (Wiley7n.1 and NIST 2008), by comparison of their retention time (RT) obtained on HP-5MS column and then verified by (RI, HP-5MS) with those published in the literature.²²

Briefly, samples were incubated for lipid extraction and FAs transesterification in 2 ml of 5% methanolic HCL at 75 °C for 90 min. FAs methyl esters were extracted by adding 2 ml of n‐pentane and 1 ml of saturated NaCl solution. Samples were separated and evaporated under N2 gas n‐pentane phase and finally dissolved in 80 µl of carbon disulphide. Gas chromatography (GC) analysis was then performed.
The GC method was used for separation with a DBWAX capillary column (30 m × 0.25 mm ×0.20 μm) in a GC System 7890 A with a Series Injector 7683B and an FID detector (Agilent Technologies, Barcelona, Spain). The temperature of the injector was 220 °C using the splitless mode. A constant rate (1.8 ml/min) of helium (99.99%) was maintained. The column temperature was held at 145°C for 5 min; subsequently, the column temperature was increased by 2°C/min to 245°C for 50 min, and held at 245°C for 10 min, with a post‐run of 250°C for 10 min as previously described [59, 60, 61]. Based on FA composition, different indexes were calculated, and elongase and desaturase activity was estimated from specific product/substrate ratios [61, 62].

SCFAs including acetate, propionate, and butyrate were analyzed using an external standard method described by Zhu et al. [37 (link)] with minor modifications. Briefly, 0.2 g of colon contents were suspended in 2 mL of saturated NaCl solution. The mixtures were vortexed uniformly for 30 min and then centrifuged at 12,000× g for 10 min. The supernatant was acidified with 100 μL of 80% H₃PO₄ and extracted with 2 mL of ethyl ether. The contents of SCFAs were determined using an 8890N gas chromatograph with an FID detector (Agilent Technologies, Santa Clara, CA, USA). Separation was achieved using an HP-innowax capillary column (30 m × 0.25 mm × 0.25 µm film thickness, Agilent Technologies Inc.). The injector and detector temperature were both 250 °C. The flow rate of nitrogen carrier gas was kept at 1.5 mL/min. Then, 1 µL of derivatized sample was injected at a split ratio of 10:1. The initial column temperature was 100 °C, ramped to 150 °C at the rate of 8 °C /min, increased to 170 °C at 5 °C /min, and then finally increased to 230 °C at the rate of 30 °C/min, before being kept at this temperature for 2 min.

Fatty acids from mitochondrial membranes were analyzed as methyl ester derivatives by gas chromatography (GC) as previously described [25 (link)]. Separation was performed by a DBWAX capillary column (30 m × 0.25 mm × 0.20 μm) in a GC System 7890A with a Series Injector 7683B and an FID detector (Agilent Technologies, Barcelona, Spain). Fatty acid methyl esters were identified by comparison with authentic standards (Larodan Fine Chemicals, Malmö, Sweden). Results are expressed as mol%. The following fatty acyl indices were also calculated: saturated fatty acids (SFA); unsaturated fatty acids (UFA); monounsaturated fatty acids (MUFA); polyunsaturated fatty acids (PUFA) from n-3 and n-6 series (PUFAn-3 and PUFAn-6); and average chain length (ACL) = [(Σ%Total14 × 14) + (Σ% Total16 × 16) + (Σ% Total18 × 18) + (Σ% Total 20 × 20) + (Σ% Total 22 × 22) + (Σ% Total 24 × 24)]/100. Finally, the density of double bonds in the membrane was calculated by the Double Bond Index, DBI = [(1 × Σmol% monoenoic) + (2 × Σmol% dienoic) + (3 × Σmol% trienoic) + (4 × Σmol% tetraenoic) + (5 × Σmol% pentaenoic) + (6 × Σmol% hexaenoic)].