UPLC-ESI-MRM/MS for Absolute Quantification and MS/MS Structural Elucidation of Six Specialized Pyranonaphthoquinone Metabolites From Ventilago harmandiana

Abstract

Pyranonaphthoquinones (PNQs) are important structural scaffolds found in numerous natural products. Research interest in these specialized metabolites lies in their natural occurrence and therapeutic activities. Nonetheless, research progress has thus far been hindered by the lack of analytical standards and analytical methods for both qualitative and quantitative analysis. We report here that various parts of Ventilago harmandiana are rich sources of PNQs. We developed an ultraperformance liquid chromatography–electrospray ionization multiple reaction monitoring/mass spectrometry method to quantitatively determine six PNQs from leaves, root, bark, wood, and heartwood. The addition of standards in combination with a stable isotope of salicylic acid-D6 was used to overcome the matrix effect with average recovery of 82% ± 1% (n = 15). The highest concentration of the total PNQs was found in the root (11,902 μg/g dry weight), whereas the lowest concentration was found in the leaves (28 μg/g dry weight). Except for the root, PNQ-332 was found to be the major compound in all parts of V. harmandiana, accounting for ∼48% of the total PNQs quantified in this study. However, PNQ-318A was the most abundant PNQ in the root sample, accounting for 27% of the total PNQs. Finally, we provide novel MS/MS spectra of the PNQs at different collision induction energies: 10, 20, and 40 eV (POS and NEG). For structural elucidation purposes, we propose complete MS/MS fragmentation pathways of PNQs using MS/MS spectra at collision energies of 20 and 40 eV. The MS/MS spectra along with our discussion on structural elucidation of these PNQs should be very useful to the natural products community to further exploring PNQs in V. harmandiana and various other sources.

High-Resolution QTOF-MRM for Highly Accurate Identification and Quantification of Trace Levels of Triterpenoids in Ganoderma lucidum Mycelium

“…Our previous study reported that the use of MS/MS spectra derived from a single collision energy may be insufficient to determine the complete fragmentation pathway of a specialized metabolite. 33 This is due to the complex structures and the stability of the fragment ions. 40 MRM Ion Selection, Specificity, Sensitivity, and Matrix Effect.…”

“…For all of the triterpenoids, three calibration curves derived from the three sets of calibration standards resulted in different slopes, demonstrating that there was a matrix effect (Figure S5). In this regard, standard addition 33 was applied for the quantitation of triterpenoids in the mycelium and fruiting body samples.…”

“…Since MS/MS analysis of a number of triterpenoids analysis have already been explored, − we propose here only the MS/MS fragmentation pathways of Lu-B and Lu-C which have never been discussed. Our previous study reported that the use of MS/MS spectra derived from a single collision energy may be insufficient to determine the complete fragmentation pathway of a specialized metabolite . This is due to the complex structures and the stability of the fragment ions .…”

“…The extraction of triterpenoids in mycelium and fruiting body samples of G. lucidum was performed using our previously published protocol. 33 In brief, dried mushroom sample (∼30 mg) was spiked with 0.02 μmol of Sal-d 6 . The sample was extracted with 1 mL of MeOH using an ultrasonic bath (Bandelin, Berlin, Germany) at 50 °C for 30 min.…”

“…The extraction of triterpenoids in mycelium and fruiting body samples of G. lucidum was performed using our previously published protocol . In brief, dried mushroom sample (∼30 mg) was spiked with 0.02 μmol of Sal- d 6 .…”