NON-TARGETED ANALYSIS OF MUSHROOM-CONTAINING COFFEE PRODUCTS USING ION MOBILITY-HIGH RESOLUTION MASS SPECTROMETRY

Non-Targeted Analysis of Mushroom-Containing Coffee Products Using Ion Mobility-High Resolution Mass Spectrometry

Importance of the Topic

Over the past decade, functional mushrooms have gained prominence due to their reported health benefits, including anti-inflammatory properties and cognitive enhancement. This trend has driven the development of coffee products blended with mushroom extracts, creating complex matrices that require advanced analytical approaches for proper characterization and quality control.

Objectives and Study Overview

This study aimed to generate comprehensive chemical fingerprints of commercial mushroom-containing coffee products and compare them with traditional coffee and pure mushroom extracts. Key goals included:

• Characterizing up to three mushroom species—chaga, cordyceps, and lion’s mane—within coffee formulations.
• Employing non-targeted ion mobility-high resolution mass spectrometry (IMS-HRMS) coupled with multivariate statistics.
• Identifying distinguishing markers that differentiate mushroom coffees from conventional instant coffees and mushroom capsules.

Methodology

Sample Preparation:

• Retail samples included four blended mushroom coffees (M-A to M-D), two traditional instant coffees (T-A, T-B), and individual mushroom capsules.
• Products were reconstituted in hot water, centrifuged, and diluted 10-fold with water.
• All samples were analyzed in triplicate in randomized order, with pooled quality control injections.

Data Acquisition and Processing:

• IMS-HRMS data were acquired in negative ion mode using HDMSE acquisition.
• Liquid chromatography employed an ACQUITY UPLC I-Class PLUS system with an Atlantis Premier BEH C18AX column.
• Gradient elution from aqueous formic acid to acetonitrile over 17 minutes at 0.3 mL/min.
• Data processed via Driftscope and the UNIFI software; multivariate analysis performed with EZinfo (PCA and OPLS-DA).

Instrumentation Used

• SYNAPT XS IMS-QTOF mass spectrometer (Waters Corporation)
• ACQUITY UPLC I-Class PLUS system with Atlantis Premier BEH C18AX column (1.7 µm, 2.1 × 150 mm)
• Software: Driftscope, UNIFI 3.0.1, waters_connect, and EZinfo

Results and Discussion

Multivariate Analysis:

• PCA showed clear clustering, separating mushroom coffees from traditional coffees and pure capsules, with high reproducibility across replicates.
• S-plots (OPLS-DA) identified late-eluting oligosaccharides (e.g., hexaose, m/z 989.321) as key distinguishing markers of mushroom coffees.

Oligosaccharide Profiling:

• Oligosaccharides were enriched in cordyceps capsules and mushroom coffee products but absent in chaga or lion’s mane capsules and traditional coffee.
• Trehalose disaccharide adducts showed variable responses across mushroom coffee products, without correlation to specific mushroom species, suggesting extraction processes drive content.

Benefits and Practical Applications

The non-targeted IMS-HRMS workflow enables detailed characterization of polar constituents in complex beverage matrices, supporting:

• Quality control and batch consistency assessment of functional coffee products.
• Authentication of mushroom species contributions through unique oligosaccharide markers.
• Development of targeted assays for routine monitoring.

Future Trends and Applications

Potential directions include:

1. Extending non-targeted IMS-HRMS screening to other functional beverage and food matrices.
2. Integrating machine learning for automated marker discovery and classification.
3. Implementing targeted quantification methods for routine quality assurance and regulatory compliance.
4. Exploring health-related bioactivity correlations with specific oligosaccharide profiles.

Conclusion

This study demonstrated that ion mobility-high resolution mass spectrometry, combined with multivariate statistics, effectively differentiates mushroom-containing coffee products from traditional coffees and pure mushroom extracts. Oligosaccharides, notably hexaose and trehalose adducts, emerged as robust markers of mushroom additions, with extraction procedures influencing marker abundance more than species identity. These findings lay the groundwork for advanced quality control strategies in the functional beverage industry.

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