Slb 5ms capillary column

An Agilent 6890N/5973N GC/MSD (Agilent, US) system equipped with a Supelco (Sigma-Aldrich, US) SLB-5MS capillary column (30 m × 250 μm × 0.25 μm) was used for the measurements. According to the program that was used, the temperature of the GC oven increased from 60°C (3 min isothermal) to 250°C at 8°C/min (1 min isothermal). The carrier gas (high purity helium at 6) was applied at 1 ml/min (37 cm/s) in constant flow mode. Detection was obtained using a mass selective detector (MSD) equipped with a quadrupole mass analyzer. The detector was operated in electron ionization mode at 70 eV in full scan mode (41–500 amu at 3.2 scan/s). MSD ChemStation D 0.02.00.275 software (Agilent, US) was used for data analysis. Compounds were identified by comparing retention data and recorded spectra with literature data, and the NIST 2 library (NIST, US). Zone averaged data were used for percentage analyses. Standard errors are calculated from three or five independent biological replicates as indicated, measured in the same period of the year on the same clone.

The analyses were carried out with an Agilent 6890N/5973N GC-MSD (Santa Clara, CA, USA) system equipped with a Supelco (Sigma-Aldrich, Philadelphia, PA, USA) SLB-5MS capillary column (30 m × 250 µm × 0.25 µm). The GC oven temperature was programmed to increase from 60 °C (3 min isothermal) to 250 °C at 8 °C/min (1 min isothermal). High purity helium (6.0) was used as a carrier gas at 1.0 mL/min (37 cm/s) in constant flow mode. The mass selective detector (MSD) was equipped with a quadrupole mass analyser and was operated in electron ionization mode at 70 eV in full scan mode (41–500 amu at 3.2 scan/s). The data were evaluated using MSD ChemStation D.02.00.275 software (Agilent, Santa Clara, CA, USA). The identification of the compounds was carried out by comparing retention data and recorded spectra with the literatury data, and the NIST 2.0 library was also consulted. The percentage evaluation was carried out by area normalization.

Breath VOCs were analysed with a thermal desorption system (TD20; Shimadzu Corporation) combined with a QP2010 GC-MS instrument (Shimadzu Corporations), having an SLB-5ms capillary column (with 5% phenyl methyl siloxane; 30 m length; 0.25 mm internal diameter; 0.5 μm thicknesses; from Sigma-Aldrich). Helium (99.999%), with additional Agilent triple filter for oxygen and humidity for further purification, served as the carrier gas. The oven profile was programmed to: (a) 5 min at 35°C; (b) increasing the temperature at 5°C/min until 180°C had been reached; (c) increasing the temperature at 13.5°C/min until 290°C had been reached; and (d) holding at 290°C for 1 min. Molecular structure of breath VOCs was tentatively determined through spectral library matches (compounds library of the National Institute of Standards and Technology, USA) using the GC-MS post-run analysis program (version 2.53; Shimadzu Corporation, Duisburg, Germany) (Figure 1C). None of the VOCs had a Gaussian distribution, thus the non-parametric Wilcoxon/Kruskal-Wallis test was used to distinguish significant compounds (p < 0.05) expressing differential values between molecularly different breast cancer lesions using SAS JMP (version 8.0, SAS Institute Inc., Cary, NC). Sensitivities and specificities were also determined.

An Agilent 6890/5975 GC/MS (Agilent Technologies Inc., Palo Alto, CA, United States) was used to analyze all samples. GC separations were carried out using a Supelco SLB 5 ms capillary column (30 m × 0.25 mm and film thickness 0.25 μm). Sample volumes of 1 μl were injected with a split ratio of 15:1. Injection temperature was set at 230°C, and the interface was set to 250°C. The carrier gas used was He at a constant flow rate of 1 ml/min. The GC temperature program was held isothermically at 70°C for 5 min, ramped from 70 to 310°C at a rate of 5°C/min, and finally held at 310°C for 7 min (analysis time: 60 min). The MS source was adjusted to 230°C, and a mass range of m/z 70–700 was recorded. All mass spectra were acquired in electron impact ionization (EI) mode (70 eV). Upon visual inspection of GC/MS chromatograms using Agilent ChemStation software (Agilent Technologies, Waldbronn, Germany), raw data was subjected to peak detection, baseline correction, alignment of mass signals, and peak height integration using the data alignment software MetAlign (Wageningen UR, Netherlands). Metabolomics raw data have been deposited to the GNPS database (Global Natural Products Social Molecular Networking, Wang et al., 2016 (link)) with the identifier: MassIVE MSV000086585. The complete dataset can be accessed here— doi: 10.25345/C5Z19Z.

The band that showed the highest inhibitory effect was scraped from the chromatographic plate and mixed with 1 mL of acetonitrile (LiChrosolv, Merck, Darmstadt, Germany). This solution was separated and its components were identified by a gas chromatograph/mass spectrometry (GCMS-QP2010 Ultra, Shimadzu Corp., Kyoto, Japan). The conditions of the technique were: SLB-5ms capillary column 30 m × 0.25 mm × 0.25 µm (Supelco, Milan, Italy); helium carrier gas flow at 1 mL min⁻¹; injection temperature at 250 °C; oven temperature program: 40 °C for 4 min, 10 °C min⁻¹ to 270 °C and hold for 10 min and, finally, 10 °C min⁻¹ to 290 °C and hold for 10 min; splitless injection at a volume of 1 µL min⁻¹ using a Shimadzu AOC-20i auto injector. The compounds of the extract were identified by comparison with the NIST 2014 database (applying > 80% match as acceptance requirement).

To determine the amount of unreacted 2-naphthol, the 2-naphthol remaining in the pretreatment liquor was extracted and analyzed by gas chromatography/mass spectrometry (GC/MS). 5 ml water was added to 10 ml pretreatment liquor and the mixture was extracted three times with 3 ml CHCl ${}_3$ . To determine remaining 2-naphthol in the biomass, 5 ml water was added to 10 g washed or filtered biomass and extracted three times with 10 ml CHCl ${}_3$ .
Two hundred and fifty microlitre of a syringaldehyde solution as internal standard was mixed with 750 μl of 2-naphthol extract. The concentration of the syringaldehyde solution was 0.5 g/l or 1 g/L for pretreament liquor extracts and biomass extracts, respectively. An autosampler (Thermo Scientific, AI 3000, Waltham, MA, USA) was used to inject the samples (5 μl for the extracts of the liquor and 1 μl for the extract from the biomass) into the GC/MS system (Thermo Scientific, Trace GC Ultra/Polaris ITQ ion trap, EI mode). The split ratio for the injections was 10:1. The GC system was equipped with a Supelco SLB 5 ms capillary column (30 m × 0.25 mm × 0.25 μm). Helium was used as the carrier gas with a flow of 1 ml/min. The following temperature program was used for the GC oven: 80 °C for 5 min, heating by 10 K/min to 280 °C, and 280 °C kept for 5 min.

Separations of the obtained extracts were performed by gas chromatography. A GC2010 gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a CombiPAL autosampler (CTC Analytics, AG, Zwingen, Switzerland) and an SLB-5MS capillary column (length 30 m, inner diameter 0.25 mm, film thickness 0.25 μm; Supelco, Bellefonte, PA, USA) was coupled to a QP 2010 Plus mass spectrometer (MS) and a flame ionization detector (FID), both from Shimadzu (Kyoto, Japan). Helium 4.6 was used as the carrier gas at a constant linear velocity of 30 cm·s⁻¹. The split ratio was set to 1:5. The temperature of the injector was maintained at 230 °C. The temperature gradient was programmed as follows: the initial temperature was 40 °C (3 min) and then increased at 2 °C∙min⁻¹ to 250 °C (5.5 min) with a total analysis time of 60 min.
FID conditions: The detector temperature was set to 270 °C.
MS conditions: The interface temperature and the ion source temperature were maintained both at 200 °C. The mass spectrometer was operated in electron ionization mode (70 eV), and the detection of ions was performed in full scan mode at a range of 33–450 m/z.