Development of a gas chromatography-mass spectrometry (GC-MS) method for the characterization and quantification of triterpenic acids.

Triterpenic acids (TA) are widely distributed secondary metabolites/specialized metabolites in plants. Here a sensitive and comprehensive method for the characterization and quantitative analysis of 20 TA derivatives by means of GC-MS has been developed. The TA were efficiently derivatized using TMSCHN and BSTFA before analysis. Chromatographic separation using a DB-5 column (40 m, 0.18 mm ID, 0.25 μm film) enabled the selective detection of naturally occurring and modified TA derivatives. Sample preparation involved the removal of water-soluble matrix compounds and the extraction with ethyl acetate. The electron ionization (EI) led to distinct fragments allowing a structural characterization and specific detection. The method was applied on the analysis of apple peels of different varieties. TA concentrations of up to 3.5 g/100 g dry weight (DW) were found. Ursolic acid (up to 2.9 g/100 g DW) was the main TA, followed by oleanolic acid (up to 500 mg/100 g DW) and pomolic acid (up to 130 mg/100 g DW). Recovery of added TA was good (70-140%) and determined concentrations of naturally occurring TA were in high agreement (75-125%) with those obtained by GC-FID analysis, demonstrating the accuracy of the developed method. The method showed high precision with low intra-day and inter-day variability (≤ 22%). The described method allows for the first time the qualitative and quantitative analysis of a wide range of TA derivatives. This will help to gain more insights into the occurrence of abundant triterpenic acids such as ursolic acid but also low concentrated derivatives such as pomolic acid in plants.