Pentacyclic triterpenoids (PCTs) are a widely distributed class of plant secondary metabolites. These compounds have high bioactive properties, primarily antitumor and antioxidant activity. In this study, a method was developed for the quantitative analysis of pentacyclic triterpenoids in plants using supercritical fluid chromatography–tandem mass spectrometry (SFC-MS/MS). Separation of ten major PCTs (friedelin, lupeol, -amyrin, -amyrin, betulin, erythrodiol, uvaol, betulinic, oleanolic and ursolic acids) was studied on six silica-based reversed stationary phases. The best results (7 min analysis time in isocratic elution mode) were achieved on an HSS C18 SB stationary phase using carbon dioxide—isopropanol (8%) mobile phase providing decisive contribution of polar interactions to the retention of analytes. It was shown that the use of atmospheric pressure chemical ionization (APCI) is preferred over atmospheric pressure photoionization (APPI). The combination of SFC with APCI-MS/MS mass spectrometry made it possible to achieve the limits of quantification in plant extracts in the range of 2.3–20 gL1. The developed method was validated and tested in the analyses of birch outer layer (Betula pendula) bark, and licorice (Glycyrrhiza glabra) root, as well as lingonberry (Vaccinium vitis-idaea), cranberry (Vaccinium oxycoccos), apple (Malus domestica “Golden Delicious” and Malus domestica “Red Delicious”) peels.
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