Mastering analytical challenges for the characterization of pentacyclic triterpene mono- and diesters of Calendula officinalis flowers by non-aqueous C30 HPLC and hyphenation with APCI-QTOF-MS

Pentacyclic triterpene mono- and diesters have been isolated from Calendula officinalis flowers. GC–MS,APCI-Exactive Orbitrap HR-MS and NMR allowed to identify the triterpene skeleton in various samples(different triterpene mixtures from Calendula n-hexane extract). NMR provided evidence that triter-pene diesters are present in the samples as well. However, the corresponding quasi-molecular ionscould not be detected by APCI-Exactive Orbitrap HR-MS. Instability of triterpene diesters and loss ofa fatty acid residue, respectively, in the ion-source made their MS detection challenging. Thus, a set ofnew APCI-QTOF-MS methods (using the TripleTOF 5600+ mass spectrometer) were developed whichmade it eventually possible to solve this problem and confirm the diester structures by MS via quasi-molecular ion [M + H]+detection. Direct infusion APCI-QTOF MS experiments in MS/MS high sensitivityscan mode with low collision energy and multi-channel averaging acquisition (MCA) allowed the detec-tion of quasi-molecular ions of triterpene diesters for the first time and unequivocally confirmed thepresence of faradiol 3,16-dimyristate and -dipalmitate, as well as the corresponding mixed diesters fara-diol 3-myristate,16-palmitate and faradiol 3-palmitate,16-myristate. Preferential loss of the fatty acid in16-position made it possible to distinguish the mixed diesters by MS/MS spectra. Their chromatographicseparations turned out to be challenging due to their bulkiness and extended molecular dimensions.However, separation could be achieved by an uncommon non-aqueous RPLC mode with an in-housesynthesized C30 phase. Finally, two (U)HPLC-APCI-QTOF-MS methods with C18- and C30-based non-aqueous RPLC provided suitable, sensitive assays to monitor the presence of monoesters and diesters ofvarious triterpenes (faradiol, maniladiol, arnidiol, arnitriol A and lupane-3,16,20-triol esters) in then-hexane extract of C. officinalis with high mass resolution and good mass accuracy.