2.4. Quantification of triterpenoids and sterols by GC-MS and GC-FID

Sterol extraction of cell culture volumes corresponding to 10 OD₆₀₀ units was performed essentially as described by Hirz et al. (2013). In brief, cells were resuspended in 0.6 mL of methanol, 0.4 mL of 0.5% pyrogallol in methanol, and 0.4 mL of 60% KOH. Five µL of a 2 mg mL⁻¹ cholesterol solution in ethanol were added as internal standard. After heating the samples for 2 h at 90 °C, saponified lipids were extracted two times with 1 mL of n-heptane. Dried extracts were dissolved in 10 µL of pyridine and were derivatized with 50 µL of N,O-bis(trimethylsilyl)trifluoroacetamide. Samples were diluted with 200 µL of ethyl acetate and analyzed by gas chromatography–mass spectrometry (GC-MS) or gas chromatography - flame ionization detector (GC-FID). Quantification of analytes was performed by correlating the peak area of the internal standard cholesterol to the peak area of the respective compound.

A 7.5 m OPTIMA® delta-6 column (Macherey-Nagel; polysiloxane phase with autoselectivity 0.10 mm × 0.10 µm) was used on a Shimadzu QP2010 plus GCMS system equipped with a single quadrupole mass filter with electron impact ionization (EI 70 eV). Sample aliquots of 1 µL were injected in split mode (split ratio 15:1) at 270 °C injector and 300 °C detector temperatures with hydrogen as carrier gas at constant flow rate of 60 cm s⁻¹. The oven temperature program was as follows: 70 °C for 1 min, 30 °C min⁻¹ ramp to 320 °C (3 min). MSD was operated in a mass range of 50–550 amu with 6.6 scans/s and at electron multiplier voltage of 1.10 kV.

For routine analysis, a GC-FID method was developed. Therefore, a OPTIMA® delta-6 column (Macherey-Nagel; polysiloxane phase with autoselectivity; 7.5 m × 0.10 mm×0.10 µm) on a Hewlett-Packard 6890 GC equipped with a flame ionization detector (FID) was used. Sample aliquots of 1 µL were injected in split mode (split ratio 30:1) at 250 °C injector temperature and 320 °C detector temperature with hydrogen as carrier gas and a flow rate set to 0.4 mL min⁻¹ in constant flow mode (58 cm s⁻¹ linear velocity). The oven temperature program was as follows: 70 °C for 1 min, 30 °C min⁻¹ ramp to 310 °C (1 min).