Isomeric Differentiation of Four Triterpene Acids Using Full Collision Energy Ramp-MS² Spectrum

Triterpene acids, a subclass of acidic triterpenoids existing in their free state, are widely distributed throughout traditional Chinese medicine (TCM). These compounds, characterized by their ring system structures, can be further categorized into several subtypes, primarily tetracyclic and pentacyclic triterpenoid acids. The structural variability is attributed to varying conjugation types and sites, resulting in numerous isomeric forms, thereby posing significant challenges to the precise identification of triterpene acids. Various methods have been employed to differentiate triterpene acid isomers, including liquid chromatography, mass spectrometry, and chemical derivatization coupled with mass spectrometry. However, these techniques often rely on reference standards or necessitate intricate sample preparation procedures, rendering them less suitable for the analysis of TCMs. Therefore, there is an urgent need to find a new method to distinguish triterpene acid isomers. Herein, four triterpene acid isomers were successfully differentiated using full collision energy ramp-MS² (FCER-MS²). Ultra-high performance liquid chromatography-quadrupole exactive-Orbitrap mass spectrometry (UPLC-QE-Qrbitrap-MS) was deployed to acquire high-resolution m/z values for four triterpene acid isomers, namely ursolic acid, oleanolic acid, α-boswellic acid and β-boswellic acid. Their mass fragmentation pathways were proposed by carefully assigning ion signals. The abundances of precursor and product ions at step-wise collision energy values were recorded using online energy-resolved mass spectrometry (online ER-MS). FCER-MS² spectra were configured by assembling sigmoid- and Gaussian-shaped breakdown graphs of those primary MS² spectral signals after appropriate data normalization. The parameters, such as collision energy at the half survival rate (CE₅₀) of precursor ions, the optimal collision energy (OCE) of primary fragment ion species and the maximal relative ion intensity (RII_max) were calculated from those regressive equations, and subsequently were applied for isomers differentiation. The four isomers were firstly divided into two groups, desinated as group I (ursolic acid and oleanolic acid) and group II (α-boswellic acid and β-boswellic acid) through the existences of the so-called diagnostic fragment ions. Moreover, the two isomers belonging to group I show different OCE and RII_max values for m/z 411 and 439, respectively. When concerning the two isomers in group II, significant differences occur on OCE values for m/z 163 and 121, and RII_max values for m/z 439, 219 and 205, particularly the RII_max ranking positions of m/z 439 and 219. FCER-MS² spectra also include m/z, CE₅₀, OCE and RII_max features, which can comprehensively cover the mass fragmentation behaviors of precursor ions. Therefore, it is an eligible tool to advance structural identification confidence of triterpene acids.