Identification of Psychotropic Substances in Mushrooms and Chocolate by UHPLC/MS

Significance of the Topic

Forensic laboratories increasingly encounter cases involving psychotropic compounds in complex food matrices such as mushrooms and chocolates. Traditional techniques often lack the sensitivity, selectivity, or resolution to identify thermally labile substances like psilocybin and psilocin without extensive sample preparation or coelution problems. A rapid, robust liquid chromatography–mass spectrometry approach can overcome these limitations and streamline forensic workflows.

Objectives and Study Overview

This application note describes the development and validation of an ultra high performance liquid chromatography–single quadrupole mass spectrometry (UHPLC/MS) method to detect, separate, and confirm psilocybin and psilocin in mushroom and chocolate samples. Key goals included minimizing analysis time, reducing sample preparation complexity, and achieving clear chromatographic resolution for forensic confirmation.

Methodology

Sample Preparation:

• Weighed 10 mg of dried mushroom or chocolate material.
• Extracted with 2 mL of methanol by vortexing and centrifugation.
• Filtered the supernatant and performed a 50-fold dilution before injection.

Chromatographic Conditions:

• Column: Hypersil GOLD PFP, 1.9 µm, 100 × 2.1 mm.
• Mobile phase: water, acetonitrile, and methanol each with 0.06 % acetic acid in a rapid gradient.
• Flow rate: 1 mL/min; column temperature: 45 °C; injection volume: 2 µL partial loop.

Instrumentation Used

The system comprised a Thermo Scientific Accela UHPLC coupled to an MSQ Plus single quadrupole mass spectrometer. Electrospray ionization in positive mode scanned from m/z 100–400 with a 0.2 s scan time, cone voltage of 90 V, and probe temperature of 500 °C.

Main Results and Discussion

Psilocybin and psilocin standards eluted at 0.65 min and 2.25 min, respectively, with baseline resolution. Full-scan MS detection confirmed molecular ions at m/z 285 (psilocybin) and m/z 205 (psilocin). Authentic mushroom extracts and chocolate samples exhibited peaks matching the standard retention times and mass spectra. Consecutive blank injections demonstrated negligible carryover.

Practical Benefits and Applications

This UHPLC/MS approach offers several advantages for forensic and quality-control laboratories:

• Rapid turnaround: total run time under 5 minutes per sample.
• Minimal sample preparation reduces solvent use and sample consumption.
• High sensitivity and selectivity for low-level psychotropic compounds.
• Robust method with low carryover and minimal matrix interference.

Future Trends and Opportunities

Advancements may include coupling UHPLC with high-resolution mass spectrometry for enhanced structural confirmation, expanding spectral libraries for novel analogues, and integrating automated sample preparation. Portable UHPLC/MS platforms could enable on-site forensic screening, while method miniaturization may further reduce solvent consumption and analysis cost.

Conclusion

The described UHPLC/MS workflow provides a fast, reliable, and sensitive means to identify psilocybin and psilocin in complex matrices. Its streamlined sample preparation, rapid chromatographic separation, and clear mass spectral confirmation make it well suited for forensic and analytical laboratories.