title: 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.
abstract: 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 triterpene diesters are present in the samples as well. However, the corresponding quasi-molecular ions could not be detected by APCI-Exactive Orbitrap HR-MS. Instability of triterpene diesters and loss of a fatty acid residue, respectively, in the ion-source made their MS detection challenging. Thus, a set of new APCI-QTOF-MS methods (using the TripleTOF 5600+ mass spectrometer) were developed which made 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 sensitivity scan mode with low collision energy and multi-channel averaging acquisition (MCA) allowed the detection of quasi-molecular ions of triterpene diesters for the first time and unequivocally confirmed the presence of faradiol 3,16-dimyristate and -dipalmitate, as well as the corresponding mixed diesters faradiol 3-myristate,16-palmitate and faradiol 3-palmitate,16-myristate. Preferential loss of the fatty acid in 16-position made it possible to distinguish the mixed diesters by MS/MS spectra. Their chromatographic separations 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-house synthesized 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 of various triterpenes (faradiol, maniladiol, arnidiol, arnitriol A and lupane-3β,16β,20-triol esters) in the n-hexane extract of C. officinalis with high mass resolution and good mass accuracy.
authors.affiliation: Institute of Pharmaceutical Sciences, Pharmaceutical Biology and Biotechnology, University of Freiburg, Stefan-Meier-Strasse 19, D-79104 Freiburg, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Nicolaus
authors.affiliation: Institute of Pharmaceutical Sciences, University of Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Sievers-Engler
authors.affiliation: Escuela de Quimica and CIPRONA, Universidad de Costa Rica, 2060 San Jose, Costa Rica.
authors.forename: Renato
authors.initials: R
authors.lastname: Murillo
authors.affiliation: Laboratorio di Chimica Bioorganica, Università degli Studi di Trento, Via Sommarive, 14, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: D'Ambrosio
authors.affiliation: Institute of Pharmaceutical Sciences, University of Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany. Electronic address: michael.laemmerhofe@uni-tuebingen.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Lämmerhofer
authors.affiliation: Institute of Pharmaceutical Sciences, Pharmaceutical Biology and Biotechnology, University of Freiburg, Stefan-Meier-Strasse 19, D-79104 Freiburg, Germany. Electronic address: irmgard.merfort@pharmazie.uni-freiburg.de.
authors.forename: Irmgard
authors.initials: I
authors.lastname: Merfort
mesh_terms: D020838:Calendula; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D035264:Flowers; D053978:Pentacyclic Triterpenes / Q000032:analysis* / Q000737:chemistry; D010936:Plant Extracts / Q000032:analysis* / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D053978:Pentacyclic Triterpenes; D010936:Plant Extracts
keywords: (U)HPLC-APCI-QTOF-MS; C30 stationary phase; Calendula officinalis; Triterpene mono- and novel diesters; non-aqueous RPLC