Monitoring Metabolite Production of Aflatoxin Biosynthesis by Orbitrap Fusion Mass Spectrometry and a D-Optimal Mixture Design Method

Abstract

Aflatoxins, highly toxic and carcinogenic to humans, are synthesized via multiple intermediates by a complex pathway in several Aspergilli, including Aspergillus f lavus. Few analytical methods are available for monitoring the changes in metabolite profiles of the aflatoxin biosynthesis pathway under different growth and environmental conditions. In the present study, we developed by a Doptimal mixture design a solvent system, methanol/dichloromethane/ethyl acetate/formic acid (0.36/0.31/0.32/0.01), that was suitable for extracting the pathway metabolites. The matrix effect from dilution of cell extracts was negligible. To facilitate the identification of these metabolites, we constructed a fragmentation ion library. We further employed liquid chromatography coupled with high-resolution mass spectroscopy (UHPLC-HRMS) for simultaneous quantification of the metabolites. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.002−0.016 and 0.008−0.05 μg/kg, respectively. The spiked recovery rates ranged from 81.3 to 100.3% with intraday and interday precision less than 7.6%. Using the method developed to investigate the time-course aflatoxin biosynthesis, we found that precursors, including several possible toxins (with a carcinogenic group similar to aflatoxin B 1 ), occurred together with aflatoxin, and that production increased rapidly at the early growth stage, peaked on day four, and then decreased substantially. The maximum production of aflatoxin B 1 and aflatoxin B 2 occurred 1 day later. Moreover, the dominant branch pathway was the one for aflatoxin B 1 formation. We revealed that the antiaflatoxigenicity mechanism of Leclercia adecarboxylata WT16 was associated with a factor upstream of the aflatoxin biosynthesis pathway. The design strategies can be applied to characterize or detect other secondary metabolites to provide a snapshot of the dynamic changes during their biosynthesis.

Current research and prevention of aflatoxins in China

“…A reverse-phase chromatography column is subsequently used to separate metabolites in the aflatoxin pathway. For the analysis of these metabolites both high-energy collision-induced dissociation (HCD) and electrospray ionization (ESI) should be carried out with an Orbitrap Fusion mass spectrometer (Xie et al, 2018). Another technique is the development of a specific nanobody-polyclonal antibody sandwich ELISA for detection of the aflatoxin-producing fungi A. flavus and A. parasiticus in agricultural products (Wang et al, 2017).…”

Fungi population metabolomics and molecular network study reveal novel biomarkers for early detection of aflatoxigenic Aspergillus species

“…We used a full-scan MS coupled with data-dependent fragmentation (DDA) mode to acquire the mass spectrometry data (Xie et al, 2018). The positive and negative ionization mode data were separately acquired.…”

Applications of mixture experiments for response surface methodology implementation in analytical methods development

“…Xie et al 47 proposed the use of a D ‐optimal design to monitor the production of 19 metabolites extracted from Aspergillus flavus 73 and Leclercia adecarboxylata WT16. The mixture design was used to evaluate the proportion of methanol, dichloromethane, acetonitrile, and ethyl acetate.…”

“…10,20,21,25,27,29,31,38 Most of the mixture experiments requires a single simplex design to optimize one or multiple responses of the studied system. 23,24,[39][40][41][42][43][44][45][46] However, several complex situations require innovative strategies to solve cases such as the application of constraints 18,19,29,32,35,37,47 or more than one mixture design (mixture-mixture design). 10,28,30,31,48 In addition, process (independent) and mixture (dependent) factors were also simultaneously optimized.…”