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Isolation and structural characterization of a new tadalafil analog (2-hydroxyethylnortadalafil) found in a dietary supplement

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Highlights

  • A new synthetic phosphodiesterase type-5 (PDE-5) inhibitor analog has been isolated and characterized.

  • The new analog was detected in an “all-natural” dietary supplement marketed for “enhanced sexual performance”.

  • The analog was characterized by HPLC-UV, LC–MSn, high-resolution accurate mass MS, and NMR.

  • The analog is structurally similar to tadalafil.

Abstract

A screen for known PDE-5 inhibitors in a dietary supplement product marketed for “enhanced sexual performance” detected a compound that structurally resembled tadalafil. The compound was isolated from the supplement matrix using high-performance liquid chromatography with ultraviolet detection (HPLC-UV) and a fraction collector, and was further characterized using nuclear magnetic resonance (NMR), liquid chromatography–mass spectrometry (LC–MS), as well as high-resolution accurate mass mass spectrometry (HRAM-MS). The analog had an accurate mass of m/z 420.15614 (error is 1.77235 ppm) for the protonated species [M + H]+, corresponding to a molecular formula of C23H22N3O5. Mass spectral fragmentation data suggested that the modification occurred in place of the single bondCH3 located on the pyrazinopyridoindole-1,4-dione of tadalafil. NMR was utilized to further elucidate the configuration of the substitution. The analysis indicated that the moiety is a single bondCH2CH2OH, hydroxyethyl group. The new analog has been named 2-hydroxyethylnortadalafil.

Introduction

It has become increasingly common to detect synthetic, phosphodiesterase type-5 (PDE-5) inhibitors including sildenafil, vardenafil, and tadalafil in products that are labeled as “all-natural” herbal supplements that offer “sexual performance enhancement” [1], [2], [3], [4], [5]. Additionally, many analogs of these PDE-5 inhibitors have been discovered and added to the established screening methods typically run in regulatory agencies [6], [7]. The analogs are structurally similar to their approved correlative drug compounds, displaying minor alterations while retaining the active moiety [8]. The analogs are not declared on the labeling, and little to no information is available regarding their toxicological or pharmacological effects. This presents a substantial risk to public health. As regulatory agencies become more adept at detecting and identifying PDE-5 inhibitor analogs in adulterated dietary supplements, the producers of these supplements are becoming more creative in their synthesis of different compounds to avoid detection. A rapid and quantitative LC–MS/MS screening method for 71 known erectile dysfunction drugs was recently published [9] and new analogs are continuously being discovered in adulterated dietary supplements [10], [11], [12], [13], [14], [15], [16], [17], [18], [19].

This article describes the structural characterization of 2-hydroxyethylnortadalafil (“Compound 1”), an analog of tadalafil (Fig. 1). Compound 1 was detected during a routine screening of a “sexual performance enhancing” dietary supplement using liquid chromatography–ion trap mass spectrometry (LC–MSn). The compound was isolated from the supplement matrix using HPLC-UV coupled to an analytical scale fraction collector. Accurate mass and NMR were performed to fully elucidate the structure of Compound 1.

Section snippets

Sample and chemicals

A dietary supplement product was obtained during an undercover purchase from an online company that listed their manufacturing site as Hong Kong and was submitted to the laboratory for analysis. The product was supplied in a paper board box containing two foil blister packages, each containing six tablets. The product was composited by grinding five tablets to a white powder with a mortar and pestle, placing the ground tablets into a scintillation vial, and using a vortex mixer to thoroughly

HPLC-UV analyses

The isolation of Compound 1 utilized retention time as the trigger for fraction collection, and the peaks were characterized by their UV spectra in comparison to the spectrum obtained from tadalafil. The UV spectrum, compared to that of tadalafil, displayed almost identical characteristics (see Fig. 2).

Based on comparisons of the peak areas to those obtained from tadalafil standard solutions, Compound 1 was present at approximately 34 mg per tablet.

Mass spectrometry

The protonated molecular formula of Compound 1

Conclusion

In the present study, a new tadalafil analog was isolated from a dietary supplement and its structure was elucidated using UV, NMR, and high-resolution MS. The data indicate that the difference in the structure is due to substitution of the N-methyl group on the piperazinedione ring with a single bondCH2CH2OH moiety. The compound was given the name 2-hydroxyethylnortadalafil. To our knowledge, this is the first identification of this analog.

References (19)

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    Various solvents were used for 1H NMR spectra recording but the nature of solvent does not in fact influence the chemical shift of H6. Indeed, the H6 δ of cis-tadalafil are 6.13, 6.15 and 6.17 ppm in DMSO-d6 [70], CDCl3 [74] and CD3OD [44] respectively. The only significant change of chemical shift is observed for homotadalafil in pyridine-d5 with a H6 at 6.54 ppm [69].

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