Multicomponent Analysis of Metabolites in Chinese caterpillar fungus using gas chromatography-triple quadrupole mass spectrometry

Importance of the topic

Chinese caterpillar fungus (Cordyceps sinensis) is a prized traditional medicine and functional food, renowned for a diverse array of bioactive metabolites. Detailed profiling of these small molecules enhances understanding of its therapeutic effects, fosters quality assurance and streamlines modern research on this valuable natural product.

Objectives and study overview

The study aimed to develop a high-throughput method for simultaneous detection of up to 475 metabolites in C. sinensis using gas chromatography coupled with triple quadrupole mass spectrometry (GC-MS/MS). An MRM-based approach from the Smart Metabolites Database was applied to achieve broad coverage without requiring individual standards for each analyte.

Methodology and instrumentation

Sample Preparation:

• 30 mg dried fungus extracted in methanol/water/chloroform (2.5:1:1, v/v/v).
• Ribitol added as a substitute internal reference; 2-isopropylmalic acid introduced post-extraction for semi-quantitation.
• Extract dried under vacuum, followed by methyloximation and trimethylsilylation.

MRM Method Development:

• Retention times predicted using a C9–C33 n-alkane standard run under database parameters.
• Smart Metabolites Database provided MRM transitions for 475 targets, refined with experimental retention data.

Used instrumentation

GC-MS/MS System:

• Instrument: Shimadzu GCMS-TQ8050 triple quadrupole mass spectrometer
• Injection: Split mode (10:1), DB-5 column (30 m×0.25 mm×1.0 µm)
• Oven: 100 °C (4 min) → 4 °C/min → 320 °C (8 min)
• Carrier gas: Helium; Collision gas: Argon; Ionization: EI; Interface 280 °C; Ion source 200 °C

Main results and discussion

From the optimized MRM method, 141 metabolites spanning amino acids, organic acids, sugars, nucleosides and related compounds were detected in C. sinensis. Semi-quantitative data were obtained by comparing peak areas to the 2-isopropylmalic acid internal standard. Typical MRM traces illustrated clear separation of key analytes such as glycine-TMS, valine-TMS, adenosine-TMS and dopamine-TMS. The absence of individual standards did not compromise identification, owing to the comprehensive transition library.

Benefits and practical applications

The workflow offers:

• Rapid screening of hundreds of metabolites in a single run.
• Robust semi-quantitation using a single internal standard.
• Enhanced quality control for herbal products and nutraceutical research.

Future trends and potential applications

Further developments may include expansion of the metabolite library, integration of absolute quantitation using isotope-labeled standards, and application of this platform to other botanicals and complex biological matrices. Automation of data processing and incorporation of chemometric tools will advance comprehensive metabolomics studies.

Conclusion

An MRM-based GC-MS/MS approach leveraging the Smart Metabolites Database enables efficient semi-quantitative profiling of over 140 metabolites in Cordyceps sinensis without individual standards, offering a valuable tool for research and quality assessment of this important medicinal fungus.