Trace ultra

Liver samples used for fatty acid analyses were kept frozen at −80°C and then lyophilized overnight. Total lipids were extracted from the powdered samples with the method of Bligh and Dyer (30 (link)) and evaporated to dryness under N₂ flow. Separation of total lipid extracts into neutral and polar lipid fractions was performed by sequential elution with chloroform and methanol on silica gel cartridges (Bond- Elut, USA). Polar and neutral fractions were then transmethylated, and fatty acid methyl esters (FAMEs) were extracted as described in Nayak et al. (31 (link)). A GC analysis of FAMEs was carried out on a Trace Ultra gas chromatograph (Thermo Fisher Scientific, USA) equipped with a flame ionization detector, a programmable temperature vaporization injector and a capillary column (SupelcoWAX, Sigma-Aldrich, USA) as described in Nath et al. (14 (link)).

To identify major volatiles in the Strawberry Flavor Concentrate (Liquid Barn), headspace gas chromatography/mass spectrometry (GC/MS) analyses were conducted with a dynamic headspace analyzer (HT3 Automated Headspace Analyzer, Teledyne Tekmar) outfitted with a thermal desorption trap (Supelco Trap K Vocarb 3000 Thermal Desorption Trap, Sigma-Aldrich). The GC/MS (Trace Ultra, Thermo Fisher Scientific) was equipped with a single quadrupole mass spectrometer (Thermo Fisher Scientific) and a 30 m × 0.25 mm ID fused-silica capillary column (Stabiliwax-DA, Restek). The e-liquid additive solutions (0.1% in water) were subjected to dynamic headspace GC/MS analysis by placing 50 μl in a sealed 20 ml headspace vial. The vial was maintained at 30°C, swept with helium for 10 min (flow rate, 75 ml/min), and the volatiles were collected on the thermal desorption trap. Trap contents were desorbed at 260°C directly into the GC/MS using a split injection. The GC oven program had an initial temperature of 40°C (held for 3.0 min) followed by a ramp of 7.0°C/min to a final temperature of 230°C (held for 6.0 min). The MS was used in scan mode with a mass/charge ratio from 33 to 400 using a 3 min solvent delay. Mass spectral peak identifications were assigned based on the library search of the National Institute of Standards and Technology Standard Reference Database.

Urine samples collected from ASD and CTR children were frozen and stored at −20 °C before the analysis.
Evaluation of urinary 3-PBA level requires hydrolysis to convert it into free 3-PBA that might be conjugated. Then the 3-PBA was extracted from the urine samples (1 mL) pre-treated with KOH by solid-phase extraction using Oasis Strata X C cartridge. Derivatization of 3-PBA and 2-PBA (the internal standard) was achieved by the addition of DIC and HFIP. In the final phase of the procedure, a liquid-liquid extraction (LLE) was made twice with 150 μL of n-hexane, followed by a vortex mixing during 10 min. Using a micropipette, 50 μL of supernatant were withdrawn (each time) and transferred in an “insert” placed in a vial.
A volume of 1 µL of upper layer was injected on a Thermo Trace-Ultra gas chromatograph coupled to an ion trap mass detector Thermo Polaris, operated in the electron impact ionization at 70 eV. The ion source temperature and the MS transfer temperature were at 250 °C. Operating in the splitless mode, the helium was used as carrier gas at a constant flow rate of 1.3 mL/min. The injector was maintained at 240 °C.
A program was developed in the SIM mode, based on the detection of selected ions for 3-PBA and 2-PBA (134, 140, 169, 195, 197, and 364).

The sample was placed on a quartz wool inserted inside a 2 internal diameter (i.d.)X 40 mm quartz tube. The tube were placed in a CDS Pyroprobe 5150 pyrolyzer directly coupled to a Thermo Trace Ultra gas chromatograph (GC) and a quadrupolar DSQ II mass spectrometer (MS). The resistively heated coil of the apparatus heat the samples with a heating rate of 5 °C.ms⁻¹ from room temperature to the selected temperature which was maintained 30 s. The pyrolysis products were sent directly to the GC/MS in a stream of helium. Direct coupling prevents the loss of volatile compounds or possible degradation of the pyrolysis products. The GC separations were conducted in a fused silica capillary column (BPX 5 (SGE), 5% Phenyl Polysilylphenyl-siloxane, 30 m length, 0.25 mm i.d., 0.25 µm film thickness) and helium 5.5 (Messer), 99.9995% purity as carrier gas. The injector was set to 250 °C with a split of 100/1. The column temperature was programmed from 60 to 300 °C at 5 °C.min⁻¹ and held at 300 °C for 15 min. The ionization mode was electron impact (70 eV), the data were recorded in full scan mode, the source temperature was 220 °C and the transfer line was set to 280 °C. The pyrolysis or thermochemolysis products were identified on the basis of their GC retention times and by comparison of their mass spectra with analytical standards and library data (NIST).