Development of Un-targeted Screening Method for Detection of Synthetic PDE-5 Inhibitors and Analogues Adulterated in Health Supplements on LCMS-IT-TOF

Importance of the Topic

Adulteration of health supplements with unapproved synthetic PDE-5 inhibitors and their analogues poses serious health risks and regulatory challenges. Conventional targeted methods may miss unknown adulterants, highlighting the need for high-resolution untargeted screening.

Objectives and Study Overview

The study aimed to establish an untargeted LCMS-IT-TOF screening workflow using a compound database and MetID software. Thirty-two known PDE-5 inhibitors and analogues were used to evaluate method performance in five locally sourced supplements.

Methodology and Instrumentation

Sample Preparation:

• Capsule contents extracted with methanol (1 g in 10 mL)
• Sonication for 20 min, filtration (0.2 µm PTFE)

Chromatography:

• Shim-pack C18 column (150×2.0 mm, 5 µm)
• Mobile phases A: water + 0.1% formic acid, B: acetonitrile + 0.1% formic acid
• Gradient: 10% B → 75% B (0–20 min) → 10% B (20–25 min)

Mass Spectrometry:

• LCMS-IT-TOF high-resolution mass spectrometer with ESI in positive mode
• Multi-event TIC acquisition divided into 50 Da windows covering m/z 200–600
• MetID Solution software for peak detection and database search (±5 ppm mass error)

Used Instrumentation

• Shimadzu UFLC system
• LCMS-IT-TOF mass spectrometer
• Shim-pack C18 analytical column
• Electrospray ionization (ESI) source

Results and Discussion

• Multi-event vs. single-event: multi-event TIC showed approximately 2× higher peak areas for 32 analytes.
• All 32 compounds were detected at 0.5 ppm in five supplement matrices using untargeted screening.
• Core-structure search enabled identification of tadalafil analogues not present in the original database.
• Matrix effects varied widely by sample and analyte, with some compounds showing strong suppression.
• A tentative detection limit of 0.5 ppm was established for reliable untargeted screening under current conditions.

Benefits and Practical Applications

This workflow enables comprehensive monitoring of both known and emerging PDE-5 analogues in dietary supplements, supporting regulatory compliance, consumer safety, and QA/QC in pharmaceutical and nutraceutical laboratories.

Future Trends and Potential Applications

• Expansion of compound libraries to include other adulterant classes and novel analogues
• Integration of machine-learning algorithms for automated candidate prioritization
• Extension to broader illicit compound screening (e.g., weight-loss drugs, stimulants)
• Coupling with metabolomic profiling for holistic safety assessment

Conclusion

An untargeted LCMS-IT-TOF method with multi-event acquisition and MetID database matching was developed and validated for detecting synthetic PDE-5 inhibitors in health supplements, offering improved sensitivity, broad coverage of analogues, and a practical screening limit of 0.5 ppm.

References

• B.J. Venhuis et al. J. Pharm. Biomed. Anal. 69 (2012) 196–208
• D.N. Patel et al. J. Pharm. Biomed. Anal. 87 (2014) 176–190
• J.H. Lee et al. Food Addit. Contam. A 30(11) (2013) 1849–1857