Tsq 9000

GC-MS analysis was performed with a gas chromatograph (TRACE 1310, Thermo Scientific), coupled to a triple quadrupole mass spectrometer (TSQ 9000, Thermo Scientific). 1 μl of each sample was injected in split mode (ratio: 10) with about 9% of injected samples being transported by a carrier gas into a non-polar column (TG-5MS, 30 m, 0.25 mm ID, 0.25 μm film thickness, Thermo Scientific). The temperature of injector was 280°C. Helium at constant flow (1 mL/min) was used as the carrier gas. The GC used the following program: 50°C for 2 min; ramp 10°C min⁻¹ to 200 °C; hold 5 min; ramp 5°C min⁻¹ to 290 °C; hold 5 min; ramp 3°C min⁻¹ to 320°C; hold 14 min. MS operating parameters were as follows: electron impact ionization, electron energy 70 eV, scan range 45–650 amu. Compounds were tentatively identified by mass spectrometry analysis: i.e., matching mass spectrum of samples with database in NIST 2017 mass spectral library. Single compounds were quantified against the internal standard and the amount was normalized to the surface area (μg/cm²). The amount of each compound in each sample was listed in Supplementary Table 1. The total amount of wax was calculated by summing up all fractions. The analysis was performed in triplicate.

Fifty mg of fresh stool samples were homogenised in 500 μL of saturated NaCl solution and acidified with 40 μL of 10% sulphuric acid. Added to the samples was 1 mL diethyl ether to extract SCFAs, after which the samples were centrifuged at 14,000× g for 15 min at 4 °C. One microlitre of each supernatant was injected into an Rtx-WAX capillary column for gas chromatography-mass spectrometry (GC-MS) analysis (TSQ 9000, Thermo Scientific). The initial oven temperature (100 °C) was elevated to 140 °C at a rate of 7.5 °C/min. Then, the temperature was further elevated to 200 °C at a rate of 60 °C/min and held for 3 min. Helium was used as the carrier gas (flow rate: 0.89 mL/min, column head pressure: 62.7 kPa. The injector temperature was set at 240 °C. The mass spectrometer was set at an ion source temperature of 220 °C, an interface temperature of 250 °C and a scan range of 2–100 m/z.

The remaining extract solutions, after determination by the HPLC-FLD technique, were subjected to GC-MS analysis to confirm the presence of azaarenes in food samples. A gas chromatograph, Trace 1310 Thermo Scientific (Milano, Italy), connected to a triple quadrupole mass spectrometer, TSQ 9000 (San Jose, CA, USA), was used. The samples were analyzed by means of split-less injection (2 min) into a TG-5MS GC capillary column (30 m × 0.25 mm; film thickness: 0.25 µm) (Thermo Scientific, Waltham, MA, USA). Conditions for the analysis of azaarenes were as follows: electron ionization (EI) 70 eV; helium flow rate of 1 mL/min; temperatures: injector 270 °C, interface 320 °C, ion source 320 °C; GC temperature program: 70 °C (2 min), 5 °C/min to 320 °C (5 min). One-microliter samples were injected using a TriPlus RSH auto sampler (Thermo Fisher Scientific, San Jose, CA, USA). The samples were analyzed in the full scan mode (mass range 50–550 Da). The mass spectra of azaarene standards are dominated by their molecular ion [M]⁺. The identification of azaarenes consisted of comparing retention times and the appropriate mass spectra of standards and compounds identified in meat samples. Identification was performed using the Chromeleon software (version 7.2.10) and mass spectral databases Mainlib and NIST (National Institute of Standards and Technology in USA).

Serum sample collection was referred to Yan et al. (19 (link)). Shanghai Lu-Ming Biotech Company Limited (Shanghai, China) provided an experimental platform and assistance for the targeting amino acid metabolomics analysis. Briefly, a mixture of methanol/water (4:1 by volume) was used to collect 2 × 10⁷ per sample. The sample was quickly stored in liquid nitrogen. Before testing on the machine, the sample was equilibrated to ambient temperature for 30 min. The sample was dispersed using the ultrasonic lysis method. It was then concentrated and centrifuged and then freeze-dried. Finally, a mixture of BSTFA and n-hexane (4:1 by volume) was added to the sample, vortexed vigorously for 2 min, and derivatized at 70°C for 60 min. These samples were analyzed by a gas chromatography system (Thermo Fisher Scientific TSQ 9000, USA). UPLC–ESI–MS/MS was utilized as the analytical method for the quantitative detection of targeted amino acid metabolites.

A Thermo Electron chromatograph GC Trace 1310 equipped with a mass detector TSQ 9000 was used for the study. The quantification of the chemicals of interest, CBMand o-chlorobenzaldehide, was based on a 10-point calibration curve (for CBM) and a 10-point calibration curve (for o-chlorobenzaldehide) obtained by plotting the area ratio of the target compounds and concentration of the calibration standards.
The range of the calibration curve used for CBM identification was located in the domain of 2000–2 µg/mL and that used for o-chlorobenzaldehide identification was located in the domain of 189–2.7 µg/mL.
Excellent linearity of response was observed over the concentration range from 2 to 300 µg/mL for CBM-C3 (calibration curve equation: y = 3e + 07x + 2e + 08, R² = 0.9998) and 2.7 to 189 µg/mL for o-chlorobenzaldehide (calibration curve equation: y = 3e + 07x + 5e + 08, R² = 0.9994).
The method for quantitative determination of CBM and o-chlorobenzaldehide was pre-validated within the laboratory procedure “Quantitative determination of irritant-lachrymatory agents from various types of samples”, in accordance with ISO/IEC 17025 and Recommended Operating Procedures for Analysis in the Verification of Chemical Disarmament. The Ministry of Foreign Affairs of Finland, University of Helsinki (2017) [38 ].

The major volatile compounds of the lyophilized hydroalcoholic extract of Achyrocline satureioides were analysed using a Trace™ 1310 gas chromatograph with the TSQ 9000 mass detector (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the Supelcowax capillary column (30 m × 0.25 mm; I.D. 0.25 μm, Supelco, Bellefonte, PA, USA). Helium was used as carrying gas at a flow rate of 1 mL·min⁻¹.
The lyophilized extract material was extracted in amounts of 5, 10 and 25 mg using ethyl acetate for 10 min, and then filtered through a nylon filter (0.22 μm). One microliter of the sample was injected onto the column (splitless mode, 1 min). The temperature of the injector was 250 °C, and the temperature of the transfer line was 220 °C. The conditions of the analysis were as follows: incubation at 70 °C for 1 min, followed by temperature increase up to 135 °C (10 °C/min), and the temperature was maintained at 135 °C for 2 min. The temperature was increased up to 170 °C (3 °C/min) and maintained for 1 min. This was followed by an increase in temperature to 250 °C (10 °C/min.), where the temperature was maintained for 20 min. The TSQ 9000 MS detector was used in electron ionization mode and set at 70 eV, ion source temperature: 250 °C, scanning range: 35–350 m/z and scanning speed: 0.2 s.