High Sensitive Detection and Quantification of Thirty-Two Synthetic PDE-5 Inhibitors and Analogues Adulterated in Health Supplements Using LC/MS/MS

Significance of the Topic

The unapproved addition of synthetic phosphodiesterase type 5 (PDE-5) inhibitors to herbal supplements poses health risks and undermines regulatory compliance. Reliable screening methods are essential to protect consumers and ensure product integrity.

Objectives and Study Overview

This study aimed to develop a highly sensitive LC-MS/MS method to detect and quantify thirty-two synthetic PDE-5 inhibitors and analogues in health supplements. The method targets both approved drugs and emerging structural analogues spiked into real supplement matrices.

Methodology

• Sample Preparation: Powdered supplement samples (0.2 g) were extracted with 2 mL methanol, sonicated for 20 minutes, and filtered through a 0.2 µm PTFE membrane.
• Calibration: Mixed standards of thirty-two compounds prepared at ten concentration levels (0.1–500 ppb) in methanol.
• MRM Optimization: Transitions optimized via auto-optimization; one quantifier and one or two qualifier transitions selected per analyte.
• Chromatography: Nexera UHPLC with Shim-pack XR-ODS-III column (150×2.0 mm, 2 µm), gradient elution (water/0.1% formic acid and acetonitrile/0.1% formic acid), flow rate 0.3 mL/min, injection volume 2 µL.
• Mass Spectrometry: Shimadzu LCMS-8040 triple quadrupole; positive ESI, MRM mode, block 400 °C, DL 250 °C, CID gas Ar 230 kPa, nebulizing gas N₂ 3 L/min, drying gas N₂ 15 L/min.

Instrumentation

• Shimadzu Nexera UHPLC System
• Shimadzu LCMS-8040 Triple Quadrupole Mass Spectrometer
• Shim-pack XR-ODS-III UHPLC Column (150×2 mm, 2 µm)

Main Results and Discussion

• Sensitivity: Limits of detection ranged from 0.05 to 2 ppb and quantitation from 0.2 to 6.7 ppb in methanol standards (2 µL injection).
• Linearity: All analytes exhibited linear calibration curves (R² > 0.999) over LOQ to 500 ppb.
• Repeatability: Relative standard deviations of peak areas were 1.3–8.8% at 10 ppb and 0.4–7.8% at 100 ppb (n=6).
• Matrix Effects: Evaluated in five supplement extracts; most analytes showed matrix effects between 70% and 150%, with some suppression or enhancement. Estimated detection limits in extracts are 0.1–4 ppb (1–40 ng/g in powder, 10× dilution).
• Screening Reliability: MRM transitions with retention time windows of ±2.5 min provided robust detection across diverse matrices. Confirmation ions ensure specificity.

Benefits and Practical Applications

• Enables routine screening of adulterated supplements for a broad panel of synthetic PDE-5 inhibitors and analogues.
• Supports regulatory laboratories and quality control units in rapid, accurate identification and quantification.
• High sensitivity and specificity minimize false negatives and false positives in complex herbal matrices.

Future Trends and Potential Applications

Advances in high-resolution mass spectrometry and alternative ionization sources may further improve sensitivity and matrix tolerance. Expanding target lists to include new analogues and integrating automated data processing will enhance throughput. Ambient ionization techniques could enable rapid on-site screening.

Conclusion

A robust LC-MS/MS method was established for the sensitive detection and quantification of thirty-two synthetic PDE-5 inhibitors and analogues in health supplement matrices. The approach demonstrates excellent linearity, repeatability, and low detection limits, addressing critical needs for consumer safety and regulatory compliance.

References

• Patel D.N. et al. Journal of Pharmaceutical and Biomedical Analysis 87 (2014) 176–190
• Lee J.H. et al. Food Additives & Contaminants: Part A 30(11) (2013) 1849–1857
• Zou P. et al. Journal of Pharmaceutical and Biomedical Analysis 47 (2008) 279–284
• Xing J., Aravind D., Sun Z., Ang M.Y., Zhan Z. ASMS 2015 Poster No. 1685 (2015)