Microwave-Assisted Extraction of Multiple Trace Levels of Intermediate Metabolites for Camptothecin Biosynthesis in Camptotheca acuminata and Their Simultaneous Determination by HPLC-LTQ-Orbitrap-MS/MS and HPLC-TSQ-MS

Camptothecin (CPT) has strong antitumor activity and is used as an anticancer therapeutic agent. To better understand and decipher the pathway of CPT biosynthesis in , the main purpose here was focused on creating an effective extraction strategy for a rich intermediate metabolite profile. In the pr...

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Bibliographic Details
Published in:Molecules (Basel, Switzerland) Switzerland), 2019-02, Vol.24 (4), p.815
Main Authors: Jin, Zhaoxia, Wan, Ruyi, Yan, Ruxue, Su, Yingying, Huang, Honglan, Zi, Lihan, Yu, Fang
Format: Article
Language:English
Subjects:
Acids
Biosynthesis
Camptothecin
Design optimization
High performance liquid chromatography
HPLC-LTQ-MS/MS
Identification
Intermediates
Irradiation
Liquid chromatography
Mass spectrometry
Mass spectroscopy
Metabolic pathways
Metabolism
Metabolites
microwave assisted extraction
Orthogonal arrays
Quadrupoles
Qualitative analysis
Scientific imaging
Solvents
Taguchi experimental design
Trace levels
Variance analysis
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Summary:Camptothecin (CPT) has strong antitumor activity and is used as an anticancer therapeutic agent. To better understand and decipher the pathway of CPT biosynthesis in , the main purpose here was focused on creating an effective extraction strategy for a rich intermediate metabolite profile. In the present study, a 70% aqueous acetonitrile was verified as an optimal extraction solvent for microwave-assisted extraction (MAE) of metabolites by spiking experiments. Based on multi-objective optimization, the best extraction conditions of a solid-liquid ratio of 1:20, microwave power of 230 W, and a time of 4 min were achieved using a full factorial 3⁴ experimental design. Crude extracts obtained from the shoot apex of using MAE have been qualitatively profiled by high-performance liquid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry (HPLC-LTQ-Orbitrap-MS/MS) and a HPLC triple quadrupole-MS (HPLC-TSQ-MS) analysis was conducted for their metabolite content in different tissues. CPT, and ten related metabolites and their isomers, including tryptamine, loganic acid, secologanic acid, strictosidinic acid, strictosamide, strictosamide epoxide, strictosamide diol, strictosamide ketolactam, pumiloside, and deoxypumiloside, were detected and tentatively identified. Scanning electron microscopy (SEM) imaging of the shoot apex demonstrated that severe cell disruption was evident after intensified extraction processes. The study showed the difference of metabolite profiles and the enhancement of metabolite content after microwave-pretreated techniques, and the established MAE procedure is an effective methodology to preserve valuable metabolite compounds for analysis.
ISSN:1420-3049
1420-3049
DOI:10.3390/molecules24040815