METHODOLOGY FOR DETECTION AND STRUCTURAL CHARACTERIZATION OF PHOSPHODIESTERASE-5 (PDE-5) INHIBITOR ADULTERANTS IN AN HERBAL COFFEE PRODUCT

Significance of the Topic

Adulteration of herbal coffee products with undeclared phosphodiesterase-5 (PDE-5) inhibitors poses serious health risks due to potential interactions with prescription medications and unpredictable dosages.
Consumers seeking natural functional foods may unknowingly ingest potent pharmaceuticals, underlining the need for reliable analytical screening methods.

Objectives and Overview of the Study

The main goal was to establish a rapid, robust workflow for detecting and structurally characterizing PDE-5 inhibitor adulterants in two commercial herbal coffee samples.
The study combined ambient mass spectrometry screening with confirmatory high-resolution liquid chromatography and spectroscopic techniques.

Methodology and Instrumentation

Two coffee samples were analyzed without extensive preparation using direct analysis in real time (DART) coupled to a single-quadrupole mass detector (QDa).
Positive-ion mode screening identified protonated molecular ions characteristic of caffeine (m/z 195), sildenafil (m/z 475) and tadalafil (m/z 390), as well as an unknown ion at m/z 505.
Confirmation employed ultra-performance liquid chromatography (UPLC) with photodiode array detection (PDA) and high-resolution tandem mass spectrometry (HRMS) in data-independent acquisition mode (MSE).
Mass-directed preparative supercritical fluid chromatography (SFC) isolated the unknown compound for nuclear magnetic resonance (1H, 13C NMR) and UV spectroscopic characterization.

Used Instrumentation

• DART source with ACQUITY QDa single-quadrupole mass detector
• ACQUITY UPLC H-Class system with Xevo G2-XS QTof
• UNIFI Scientific Information System and ChemSpider structural database
• Preparative SFC for compound isolation
• 1H/13C NMR spectrometer and UV-Vis PDA detector

Results and Discussion

Rapid DART-QDa screening confirmed caffeine and known PDE-5 drugs in Sample 1.
Sample 2 exhibited a prominent ion at m/z 505, consistent with a sildenafil analogue.
ChemSpider and UNIFI database searching proposed thiohomosildenafil (C23H32N6O3S2). Fragment-match scoring (34 matches) and matching UV absorbance confirmed the identification.
NMR and UV spectra of the isolated material aligned with published data, despite minor signal broadening attributed to salt formation.
Quantitative UV analysis revealed approximately 60 mg of thiohomosildenafil per sachet, equivalent to a therapeutic dose range.

Benefits and Practical Applications

• Enables rapid, minimal-preparation screening of functional foods for illicit drug adulterants.
• Combines ambient MS with high-resolution LC-MS for confident identification and quantification.
• Supports regulatory and quality-control laboratories in consumer safety monitoring.

Future Trends and Opportunities

• Integration of portable ambient MS instruments for on-site screening.
• Expansion of spectral libraries and machine-learning algorithms for automated identification of novel analogues.
• Development of real-time monitoring protocols in manufacturing and distribution chains.
• Advances in preparative isolation methods to facilitate structural elucidation of unknown adulterants.

Conclusion

The presented workflow demonstrates a streamlined approach for detecting and characterizing PDE-5 inhibitor adulterants in herbal coffee.
Ambient DART-QDa screening coupled with UPLC-HRMS, SFC isolation and NMR/UV analysis provides high confidence in identification and quantification.
This strategy enhances consumer protection by enabling rapid, reliable testing of functional food products.

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