Hierarchical clustering of liquid chromatography-tandem mass spectrometry data for screening of phosphodiesterase type 5 inhibitors and their analogues in adulterated dietary supplements.

Sexual enhancement dietary supplements have often been adulterated with phosphodiesterase type 5 (PDE-5) inhibitors used for treatment of erectile dysfunction, and widely distributed through online markets. As the illegal adulterants, the original PDE-5 inhibitor drugs and a numerous number of synthetized analogues, more than 80, have already been found. Therefore, analytical methods that detect various PDE-5 inhibitors and uncover newly synthesized analogues are needed. In this study, we have developed a rapid and reliable screening method for PDE-5 inhibitors and their structural analogues by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by hierarchical clustering based on similarity of MS/MS spectra. Forty reference standards of PDE-5 inhibitors/analogues were measured using a quadrupole-orbitrap mass spectrometer in data-dependent mode. The 60 most intense fragment ions were extracted from each MS/MS spectra, and the ions observed within 1.5 mDa mass tolerance were considered to be the same ion. Based on fragment ion tables representing detected ions for each compound, hierarchical clustering was performed. The resulting dendrogram showed that the reference standards were separated into seven clusters according to their characteristic structures. Subsequently, two additional standards spiked into a herbal sample were analyzed. While herbal components were clearly separated from the clusters of the reference standards, the spiked standards were clustered closely with the structurally similar standards. Furthermore, application of our method to dietary supplements allowed for detection of sildenafil and tadalafil as adulterants. These results suggest that our screening method facilitates discovery of adulterant PDE-5 inhibitors/analogues by illustrating their structural similarity.