Screening of Phosphodiesterase-5 Inhibitors and Their Analogs in Dietary Supplements by Liquid Chromatography-Hybrid Ion Trap-Time of Flight Mass Spectrometry.

An accurate and reliable method based on ion trap-time of flight mass spectrometry (IT-TOF MS) was developed for screening phosphodiesterase-5 inhibitors, including sildenafil, vardenafil, and tadalafil, and their analogs in dietary supplements. Various parameters affecting liquid chromatographic separation and IT-TOF detection were investigated, and the optimal conditions were determined. The separation was achieved on a reversed-phase column under gradient elution using acetonitrile and water containing 0.2% acetic acid at a flow rate of 0.2 mL/min. The chromatographic eluents were directly ionized in the IT-TOF system equipped with an electrospray ion source operating in the positive ion mode. The proposed screening method was validated by assessing its linearity, precision, and accuracy. Sequential tandem MS was conducted to obtain structural information of the references, and the fragmentation mechanism of each reference was proposed for providing spectral insight for newly synthesized analogs. Structural information, including accurate masses of both parent and fragment ions, was incorporated into the MSn spectral library. The developed method was successfully applied for screening adulterated dietary supplement samples.

Development of a specific fragment pattern-based quadrupole-Orbitrap mass spectrometry method to screen adulterated products of phosphodiesterase-5 inhibitors and their analogues.

Recently, adulterated supplements with phosphodiesterase-5 inhibitors (PDE-5i) have frequently observed. New synthetic analogues obtained from the chemical modification of parent compounds are frequently found in illicit products despite continuous efforts to inspect for these adulterants. A rapid and accurate method based on quadrupole-Orbitrap mass spectrometry was developed for simultaneously confirming and quantifying 85 PDE-5i and derived analogues present in illicit products for erectile dysfunction (ED). Common ions of PDE-5i according to their similar structures were proposed based on MS/MS fragmentations. These common ions could be an important diagnosis of their presence targets or new emerging analogues in supplements. Several validation parameters were employed, resulting in a limit of detection and quantification of 0.09-8.55 ng/mL and 0.24-17.10 ng/mL, respectively. The linear correlation coefficient (r2) was higher than 0.995, and mean recoveries of target compounds were in the range of 82-118%. A total of 187 illicit products, obtained from on/offline markets over a period of 3 years (2015-2017), were screened by the established method. Approximately 53% of them were adulterated with PDE-5i or derived analogues at concentrations of 0.1-726.0 mg/g in the illicit products. In the interests of public health, this study describes a rapid and accurate method to determine PDE-5i and new emerging analogues in adulterated products.

Simultaneous Detection of Three Phosphodiesterase Type 5 Inhibitors and Eight of Their Analogs in Lifestyle Products and Screening for Adulterants by High-Performance Thin-Layer Chromatography.

An HPTLC method is proposed to permit effective screening for the presence of three phosphodiesterase type 5 inhibitors (PDE5-Is; sildenafil, vardenafil, and tadalafil) and eight of their analogs (hydroxyacetildenafil, homosildenafil, thiohomosildenafil, acetildenafil, acetaminotadalafil, propoxyphenyl hydroxyhomosildenafil, hydroxyhomosildenafil, and hydroxythiohomosildenafil) in finished products, including tablets, capsules, chocolate, instant coffee, syrup, and chewing gum. For all the finished products, the same simple sample preparation may be applied: ultrasound-assisted extraction in 10 mL methanol for 30 min followed by centrifugation. The Rf values of individual HPTLC bands afford preliminary identification of potential PDE5-Is. Scanning densitometry capabilities enable comparison of the unknown UV spectra with those of known standard compounds and allow further structural insight. Mass spectrometric analysis of the material derived from individual zones supplies an additional degree of confidence. Significantly, the proposed screening technique allows focus on the already known PDE5 Is and provides a platform for isolation and chemical categorization of the newly-synthesized analogs. Furthermore, the scope could be expanded to other therapeutic categories (e.g., analgesics, antidiabetics, and anorexiants) that are occasionally coadulterated along with the PDE5-Is. The method was successfully applied to screening of 45 commercial lifestyle products. Of those, 31 products tested positive for at least one illegal component (sildenafil, tadalafil, propoxyphenyl hydroxyhomosildenafil, or dimethylsildenafil).