A novel approach to simultaneous screening and confirmation of regulated pharmaceutical compounds in dietary supplements by LC/MS/MS with an information-dependent acquisition method

Abstract

The commercial success of synthetic phosphodiesterase-5 (PDE-5) inhibitors (viz. sildenafil, vardenafil and tadalafil) for erectile dysfunction (ED) has led to their widespread use as adulterants in dietary supplements (DSs). Reports on adulteration by ED drugs or their analogues in DSs suggest they may cause a serious threat to human health. The problem is becoming more complex as hidden and structurally modified analogues are continuously being reported. To analyse known drugs and their analogues, three commonly used PDE-5 inhibitors, naturally existing icariin and yohimbin, and their 19 analogues were analyzed in this study. They were identified using ion-spray liquid chromatography/tandem mass spectrometry (LC/MS/MS) using multiple reaction monitoring (MRM). This MRM procedure gave a limit of detection of less than 0.02 ng ml(-1) for the 24 compounds, selectivity of fragmentation using MRM for 2.5 - 8.5 min in a single run and peak height repeatability of coefficient of variation of 3.9 - 31.8%. An IDA method using the MRM scans to detect the presence of known analytes was set up and added to a built-in library for screening for PDE-5 inhibitors. These MRM experiments were used to trigger product ion scans using a hybrid quadrupole-linear ion trap instrument. The product ion scan was compared and confirmed by a library search of MS/MS spectra acquired from a reference standard. To search for new analogues of PDE-5 inhibitors, a precursor ion scan of an expected ion m/z 283, which was one of the mass fragments from the analogues of sildenafil or vardenafil, was performed and fragmentation of the precursor ion, by combining a precursor ion scan with automatic confirmation using EPI spectra, was acquired. Of the 37 DSs tested, two were eventually found to be adulterated with yohimbin and vardenafil, respectively. The approach proposed in this study would be valuable in characterizing chemical constituents of drug residues and their analogues with identical chemical substructures from complex natural and synthetic sources in DSs using an information-dependent acquisition-enhanced product ion (IDA-EPI) scan.

Simultaneous determination of 38 phosphodiestrase-5 inhibitors in illicit erectile dysfunction products by liquid chromatography–electrospray ionization-tandem mass spectrometry

“…Generally, in case of the LC-MS/MS experiment, baseline resolution is not too critical for the multiple compound analysis because MS data provides more certain information to confirm the target compounds [3,23]. However, because some compounds (dimethylsildenafil/homosildenafil and dimethylthiosildenafil/thiohomosildenafil) that have very similar structure and fragmentation pattern with the same precursor molecular weight, each compound must be separated to avoid interference …”

“…Additionally, ammonium formate and acetic acid (with formic acid or not) were selected as a solvent modifier [3,14,15,21,22]. According to previous research, MeOH was not good for separation between sildenafil and vardenafil [14,20], and formic acid was not suitable for analysis of the compounds having amino groups such as sildenafil, homosildenafil, and hydroxyhomosildenafil [18].…”

“…ACN and MeOH have been widely used as organic solvent in PDE-5 inhibitors analysis using LCMS and LC-MS/MS [3,[13][14][15]21]. Additionally, ammonium formate and acetic acid (with formic acid or not) were selected as a solvent modifier [3,14,15,21,22].…”

“…A gas chromatography-mass spectrometry (GC-MS) method was used to analyze thioketone analogues of sildenafil [16]. Although 24 PDE-5 inhibitors and their analogues were analyzed using LC-MS/MS [3], almost all previous studies focused on specific compounds such as sildenafil, tadalafil, and vardenafil.…”

“…Due to increase of dietary supplement consumption for treatment of ED in males, the adulteration of unapproved phosphodiestrase type 5 (PDE-5) inhibitors (sildenafil, tadalafil and vardenafil) and analogues in dietary supplements has been widely reported [1][2][3][4][5]. Dishonest supplement manufacturers add phosphodiesterase type 5 (PDE-5) inhibitors and their analogues into dietary supplement products to improve the effect of products.…”