Zb 5msi capillary column

Gas chromatography–mass spectrometry (GC–MS) combined with solid phase microextraction in the gas phase (SPME Arrow, Arrow fiber, Shimadzu, Kyoto, Japan) was used for the separation, identification, and quantification of volatile organic compounds (VOC). VOCs were performed with Shimadzu GCMS QP 2020 Plus (Shimadzu, Kyoto, Japan) equipped with a ZB-5Msi capillary column (30 m × 0.25 mm × 0.25 µm; Phenomenex Ltd., Torrance, CA, USA).
The operational conditions of the GC were as follows: injection port 50 °C; temperature program started at 50 °C and held for 2 min, then at the of rate 3 °C·min⁻¹ to 180 °C, then at the rate of 20 °C·min⁻¹ to 270 °C, held for 5 min, 10 s; helium as carrier gas with flow 1 mL·min⁻¹; split 100 (SPME Arrow analysis).
The extraction of VOCs was performed with 1.10 mm DVB/C-WR/PDMS SPME Arrow fibre (Supelco, Bellefonte, PA, USA). Extraction was carried out in 20 mL headspace vials for 30 min at 45 °C. The extraction proceeded with incubation for 10 min at the same temperature. The analytes were desorbed under the conditions of the GC injection port for 3 min. MS operational conditions were as follows: interface temperature 250 °C; ion source temperature 250 °C; scan 40–400 m/z.

Sample analysis was performed through a gas chromatography (GC) system (Shimadzu, Nakagyo-ku, Kyoto, Japan) with a flame ionization detector (FID) using a ZB‐5MSi capillary column (30 m × 0.25 mm, 0.25-µm film thickness; Phenomenex, Torrance, CA, USA) as the stationary phase and H₂ as the carrier gas. The linear velocity of the mobile phase was set to 42.2 cm/s. A 1 µL sample was injected into the GC inlet in split mode with a ratio of 10, and the injection port temperature was set at 300 °C. The temperature program of the oven started from 80 °C for 5 min and then increased to 240 °C with a rate of 8 °C/min, and this was held for 8 min. For identification and quantification of fatty acids and fatty aldehydes, the corresponding standards at different concentrations were prepared, and calibration curves were constructed.

All solvents and chemicals were purchased from Sigma-Aldrich/Merck (Steinheim/Darmstadt, Germany), VWR International (Fontenay-sous-Bois, France), Carl Roth GmbH (Karlsruhe, Germany) or Fisher Scientific (Loughborough, UK) in best available purity and were used as received without further purification. HPLC tubes were bought from Macherey-Nagel (Düren, Germany) and the corresponding caps and inserts from Bruckner Analysentechnik (Linz, Austria). An Agilent Technologies 1100 Series executed the HPLC analysis, and a Shimadzu GCMS-QP2010 SE instrument equipped with an AOC-20i/s autosampler and injector unit together with a Zebron ZB-5MSi capillary column (30 m × 0.25 mm × 0.25 μm, Phenomenex) performed the GC-MS measurements. OD values were determined with an Eppendorf BioPhotometer plus. The CYP5035 coding regions, identified from the publicly available databases, were ordered as double-stranded DNA fragments from TWIST Bioscience. Cells of P. pastoris with expressed and versatile P450 3A4 were obtained from bisy GmbH (Hofstaetten, Austria) and used as a positive control for biotransformations. These cells had been cultivated, then stored as frozen pellets at −80°C. Figures were generated in the programmes GraphPad Prism 8 and CS ChemDraw Ultra.