Identification and quantification of triterpenoids by gas chromatography coupled to a flame ionization detector and mass spectrometer (GC-FID/MS)

An Agilent Technologies 7890 A gas chromatograph equipped with a 5975C mass spectrometric detector was used for qualitative and quantitative analyses. Samples dissolved in diethyl ether:methanol (5:1, v/v) were applied (in a volume of 1–4 μL) using a 1:10 split injection. The column used was a 30 m × 0.25 mm (L × I.D), 0.25 μm particle size (HP-5MS UI, Agilent Technologies, Santa Clara, CA, USA). Helium was used as the carrier gas at a flow rate of 1 mL/min. The separation was made either under isothermal conditions at 280 °C or at the programmed temperature; an initial temperature of 160 °C held for 2 min, then increased to 280 °C at 5 °C/1 min, and the final temperature of 280 °C held for a further 44 min. Additional parameters were employed as follows: inlet and FID temperature 290 °C; MS transfer line temperature 275 °C; quadrupole temperature 150 °C; ion source temperature 230 °C; EI 70 eV; m/z range 33–500; FID gas (H2) flow 30 mL/min (hydrogen generator); and airflow 400 mL/min. Individual compounds were identified by comparing their mass spectra with library data from Wiley 9th ED. and NIST 2008 Lib. SW Version 2010 or previously reported data, and by comparison of their retention times and corresponding mass spectra with those of authentic standards, when available. Quantitation was performed using an external standard method based on calibration curves determined for the compounds belonging to representative triterpenoid classes; α-amyrin for triterpene alcohols, oleanolic acid methyl ester for triterpene acid methyl esters, and sitosterol for steroids.