Simultaneous quantitation and identification of 6 mushroom toxins in poisonous mushrooms using the SCIEX 5500+ system

Cheng Long¹, Lin Ji², Zhao Xianglong¹, Liu Bingjie¹, Guo Lihai¹ and Charlie Liu³

¹SCIEX China; ²Yunnan Provincial Center for Disease Control and Prevention; ³SCIEX Australia
Published date: May 29, 2024

Abstract

This technical note describes the simultaneous quantitation and identification of 6 mushroom toxins in a single injection using the SCIEX 5500+ system. The use of multiple reaction monitoring (MRM) to trigger the acquisition of enhanced product ion (EPI) scans produced both MRM data for quantitation and MS/MS spectra for qualitative identification (Figure 1). Here, the sensitivity of the SCIEX 5500+ system enabled quantitation of 6 mushroom toxins down to 0.1 ng/mL in-solvent, while MS/MS library matching provided orthogonal confirmation, in addition to ion ratio and retention times (RTs), for increased confidence during mushroom poisoning investigations.

Key benefits of the analysis of mushroom toxins using the SCIEX 5500+ system
 

• Fast 7-min method for both qualitative and quantitative analysis: Data-dependent acquisition (DDA) on the QTRAP system provided both quantitative MRM data and high-quality EPI-triggered MS/MS spectra in a single injection for the analysis of 6 common mushroom toxins
  
  
• Excellent quantitative performance: Good accuracies (85– 110%) and linearity (r² >0.999) were demonstrated for the calibration of the 6 target toxins. Excellent reproducibility (RSD <5%, n = 6) was also achieved for matrix spikes.
  
  
• Positive detection in a real-world sample: The detection of phallacidin at 0.64 µg/g in a wild mushroom sample was also supported by a high purity score of >99% from MS/MS library matching.

Introduction

China is home to over 100 different types of wild poisonous mushrooms. The predominant toxins in these mushrooms belong to a class of cyclic peptides. The structural composition of the amino acids classifies these cyclic peptides into 3 groups of toxins: amatoxins, phallotoxins and virotoxins.¹ Amatoxins include α-amanitin, β-amanitin and γ-amanitin, all of which are cyclic octapeptides. Phallotoxins consist of cyclic heptapeptides such as phallacidin, phalloidin and phallisacin. Virotoxins are single-ringed heptapeptides with relatively lower lethality and have been less studied.

Poisoning caused by amatoxins, specifically α-amanitin, has a high fatality rate and accounts for over 90% of mushroom poisoning-related deaths. Poisoning symptoms only become apparent after significant cellular damage upon which most of the toxin has entered the target organs. Detecting these toxins in biological fluids is challenging due to their low concentrations in urine and plasma.^2-4 Establishing a rapid and accurate detection method is crucial for early diagnosis and reducing mortality.

In this technical note, a quick 7-minute LC-MS/MS method was developed for the rapid and accurate analysis of 6 common mushroom toxins in wild mushrooms. The SCIEX 5500+ system enables sensitive quantitation as a triple quadrupole mass spectrometer, while the linear ion trap (LIT) technology offers unique scanning functionalities for qualitative analysis in the same injection. Here, the MS/MS spectra produced from the DDA MRM-triggered EPI method were used for the toxicological screening of wild mushrooms. Compilation of these spectra into a database for routine screening can be used for confirmatory analysis during mushroom poisoning investigations.

Methods

Sample preparation: 0.4 g of a mushroom sample was weighed into a 15 mL plastic centrifuge tube, followed by the addition of 6 mL of 80:20 (v/v) methanol/water. After vortexing for 1 min, the mixture was sonicated for 20 min, cooled to room temperature and centrifuged at 8000 rpm for 5 min. After transferring 4 mL of the supernatant to a new centrifuge tube, the sample was evaporated to near dryness under nitrogen at 45°C, then reconstituted with 4 mL of water and 1 mL of n-hexane. After vortexing for 1 min, the sample was centrifuged at 8000 rpm for 3 min, followed by discarding the upper hexane layer. The remaining water fraction was retained for clean-up.

Sample clean-up: Approximately 3 mL of the aqueous phase was loaded onto a solid phase extraction (SPE) cartridge (Waters Oasis HLB, 60 mg/3 mL). The analytes were first eluted with 3 mL of 5:95 (v/v) methanol/water, followed by another 2 mL of 30:70 (v/v) acetonitrile/methanol solution. After evaporation to dryness under nitrogen at 45℃, the sample was reconstituted with 1 mL of 95:5 (v/v) mobile phase A/B, vortexed for 1 min and filtered through a 0.22 µm membrane nylon filter prior to LC-MS/MS analysis.

Chromatography: Chromatographic separation was performed using a Luna Omega Polar C18 column (100 x 2.1 mm, 1.6 µm, Phenomenex, P/N: 00D-4748-AN). A flow rate of 0.4 mL/min, an injection volume of 1 µL and a column temperature of 40^oC were used. The LC gradient used is shown in Table 1.

Mass spectrometry: Analysis was performed in DDA mode in which scheduled MRM (sMRM) survey scans were used to trigger EPI scans in the same injection on the SCIEX 5500+ system in negative electrospray ionization mode. The EPI scan mode on the QTRAP system uses the LIT to collect the fragment ions produced from a range of collision energies in the collision cell. The fragment ions are then scanned out at one of three scan speeds, depending on the required fragment ion resolution. This provides highly sensitive and high-quality MS/MS data, because the fragment ions are accumulated in the LIT prior to detection. MS/MS spectra were acquired for a mass range of 50–1000 at a scan speed of 10,000 Da/sec and with a collision energy (CE) of 50 V and collision energy spread (CES) of ±20 V.

Table 2 lists the source and gas conditions used, while Table 3 lists the MRM transitions and optimized compound-dependent parameters.

Data processing: The data were processed using SCIEX OS software, version 2.0. MS/MS spectral matching was compared against a custom library containing reference spectra that were previously acquired using a SCIEX 7500 system. 

Chromatographic separation

Good chromatographic separation was achieved for the 6 target mushroom toxins, as demonstrated by Figure 2.

Calibration curve performance

Quantitation of the 6 target mushroom toxins was performed using a calibration curve that spanned at least 3 orders of magnitude with overall r² >0.999 (Table 4 and Figure 3). Acceptable accuracies of 85–110% were achieved across all levels of the calibration curve (0.1 – 200 ng/mL). The in-vial limit of quantitation (LOQ) based on the lowest calibration level meeting accuracy and precision acceptance criteria was 0.1 ng/mL for all 6 toxins.

Matrix spike reproducibility

A blank mushroom sample was spiked at 0.5 ng/mL, 5 ng/mL and 50 ng/mL to investigate instrument reproducibility in a complex food matrix. Excellent reproducibility (RSD <5%) was demonstrated for 6 replicate injections at all 3 spiking levels (Table 5).

Qualitative MS/MS library matching workflow

The combined triple quadrupole and QTRAP functionalities of the SCIEX 5500+ system simultaneously produced chromatograms of the 6 mushroom toxins for quantitation and EPI-triggered MS/MS spectra for qualitative analysis. Using the EPI scan on the QTRAP system yielded high-quality MS/MS spectra with a broader mass range coverage from fragmentation at different collision energies. The SCIEX OS software automatically screened the target analytes based on the specified RTs and compared their experimental MS/MS spectra against the custom-built library. For example, Figure 4 demonstrates the positive detection of α-amanitin (α-AMA) in a wild mushroom sample based on the retention time and ion ratio meeting the tolerance criteria against an authentic standard. Identification was further supported by the MS/MS match against the reference spectral library, with a purity score of 99. These full-scan MS/MS spectra provided orthogonal information for minimizing the risk of false positives and false negatives, which greatly increased the compound identification confidence.

Method applicability to quantify and identify mushroom toxins in a real-world sample

The method was applied to quantify and identify the 6 mushroom toxins in a batch of wild mushrooms collected from the Yunnan province in China. Phallacidin was positively detected at a concentration of 0.64 µg/g in one sample (Figure 5). Comparison of the experimental MS/MS spectrum against the reference library resulted in a library match with a purity score of 99.5, which further confirmed the identification. These results demonstrate that even low-concentration compounds in complex matrices can yield high-quality MSMS spectra.

Conclusion
 

• The SCIEX 5500+ system was used to develop a DDA-based method that enabled simultaneous MRM quantitation and EPI-triggered MS/MS library matching of 6 mushroom toxins in a single injection of wild mushroom extracts.
  
  
• Rapid and sensitive quantitation was performed with a 7-minute gradient with a dynamic range spanning >3 orders of magnitude and r² >0.999 and calibration accuracies in the range between 85% and 110%.
  
  
• The SCIEX Turbo V ion source design provides high ionization efficiency and outstanding contamination resistance, ensuring high sensitivity, stability and reproducibility during routine analysis of large batches of samples.
  
  
• The QTRAP-based EPI scanning mode allows for obtaining MS/MS fragment spectra across the entire mass range. Based on the comprehensive information from these MS/MS spectra, a dedicated standard library for mushroom toxins is established, enabling rapid qualitative screening and confirmation in routine operations.

References
 

1. State Administration for Market Regulation. Determination of six mushroom toxins including α-amanitin in mushrooms. BJS 202008. November 19, 2020.
   
   
2. Xiao, S. et al. Determination of amatoxins and phallotoxins in plasma and urine by ultra-performance liquid chromatography-tandem mass spectrometry. Food Sci. 2018, 39, 312-318.
   
   
3. Zhou, Y. et al. Determination of mushroom toxins in human blood plasma by UHPLC in hyphenation with MS/MS in tandem. Physical Testing and Chemical Analysis Part B: Chemical Analysis. 2019, 55, 1406-1411.
   
   
4. Chen, Z. et al. Historical development and present situation of detection methods for Amanita peptide toxins. Food Sci. 2014.