Rapid simultaneous determination of pentacyclic triterpenoids by mixed-mode liquid chromatography-tandem mass spectrometry.

Pentacyclic triterpenoids (PCTs) possess high biological activity, including antitumor, anti-inflammatory, antiviral and hepatoprotective properties and are widespread in a plant biomass. Due to significant differences in polarity and other physicochemical properties, the simultaneous determination of different classes of PCTs by the methods of reversed phase liquid chromatography is difficult. In the present study, we proposed a new approach to chromatographic separation of such compounds based on the use of a stationary phase with a mixed retention mechanism combining hydrophobic, weak anion exchange and hydrophilic interactions. The use of the Acclaim Mixed-Mode WAX-1 column and tuning the selectivity by changing the contributions of different types of analyte-stationary phase interactions allowed the separation of 10 PCTs (betulin, erythrodiol, uvaol, friedelin, lupeol, β-amyrin, α-amyrin, betulinic, oleanolic and ursolic acids) belonging to four different classes (monools, diols, ketones and triterpenic acids) during 7.5 min in isocratic elution mode. The combination of this approach with atmospheric pressure chemical ionization tandem mass spectrometric detection and pressurized liquid extraction of analytes with methanol allowed to develop a rapid, accurate and highly sensitive method for analyzing PCTs in plant tissues with a total duration of the analytical cycle (including sample preparation steps) of not more than 40 min. It provides the detection limits in plant biomass extracts of 3-12 µg L (44 µg L for friedelin). The developed method was validated and successfully tested in the analyses of real birch bark and lingonberry peels.