Neutral fragment filtering for rapid identification of new diester-diterpenoid alkaloids in roots of Aconitum carmichaeli by ultra-high-pressure liquid chromatography coupled with linear ion trap-orbitrap mass spectrometry

Abstract

A rapid and effective method was developed for separation and identification of diester-diterpenoid alkaloids (DDA) in the roots of Aconitum carmichaeli by ultra-high-pressure liquid chromatography coupled with high resolution LTQ-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MS(n)). According to accurate mass measurement and the characteristic neutral loss filtering strategy, a total of 42 diester-diterpenoid alkaloids (DDA) were rapidly detected and characterized or tentatively identified. Meanwhile, the proposed fragmentation pathways and the major diagnostic fragment ions of aconitine, mesaconitine and hypaconitine were investigated to trace DDA derivatives in crude plant extracts. 23 potential new compounds were successfully screened and characterized in Aconitum carmichaeli, including 16 short chain fatty acyls DDA, 4 N-dealkyl DDA and several isomers of aconitine, mesaconitine and hypaconitine.

Figure 1. LC-MS spectra of the extract of daughter roots of A. carmichaeli.

(a). Total ion chromatogram (TIC); (b). Neutral fragment chromatogram (NFC) for 60 Da.

Figure 2. The structures of typical DDA, MDA and DEDA in A. carmichael.

Figure 3. The key fragmentation patterns of aconitine, mesaconitine and hypaconitine.

Figure 4. Schematic workflow of the automated data processing and rapid identification of DDA in A. carmichaeli.

Figure 5. The key fragmentation pathway of two isomers of hypaconitine.

Figure 6. Mass spectra of peak 23 and 25. (

a). Extracted ion chromatogram (EIC) of m/z 646 (amplified); (b). MS² of peak 23 (t _R: 11.20 min); (c). MS³ of peak 23; (d). MS² of peak 25.

Figure 7. Proposed fragmentation pattern of 2,3-didehydro-hypaconitine.

Figure 8. Proposed fragmentation pattern of N-deethyl-aconitine.

Figure 9. The mass spectra of peak 15 (8-scn-benzoylaconine). (

a). Extracted ion chromatogram (EIC) of m/z 704; (b). Full scan mass spectrum of peak 15; (c). MS² spectrum of peak 15; (d). MS³ spectrum of peak 15.

Figure 10. The mass spectra of peak 2, 5 and 8.

(a). EIC of m/z 690; (b₁). MS² of peak 2; (b₂). MS³ of peak 2; (c₁). MS² of peak 5; (c₂). MS³ of peak 5; (d₁). MS² of peak 8; (d₂). MS³ of peak 8.