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.

Isolation and identification of a new sildenafil analogue, hydroxycarbodenafil, found as an adulterant in a health supplement.

During routine screening of illegal adulterants in health supplements, a novel sildenafil analogue was discovered, and subsequently isolated by recrystallization. Its structure was elucidated by extensive analyses of high resolution mass spectrometry (HRMS), one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) data. The analogue was finally determined as hydroxycarbodenafil, featuring a hydroxyethyl group instead of an ethyl group on piperazine ring in comparison with carbodenafil.

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.

Isolation and characterisation of a novel sildenafil analogue adulterant, desmethylpiperazinyl propoxysildenafil, in a dietary supplement.

A new sildenafil analogue was detected during routine screening of dietary supplements suspected to be adulterated with an erectile dysfunction drug(s) using HPLC-DAD. The UV spectrum of this compound was highly similar to that of sildenafil and almost identical to that of desmethylpiperazinyl sildenafil. The analogue was purified by using semi-preparative HPLC and structurally elucidated by performing mass spectrometric and NMR spectroscopic experiments. The spectral data revealed that this sildenafil analogue bears an n-propoxy group instead of an ethoxy group and possesses no methylpiperazinyl moiety. The isolated compound, structure of which was further confirmed by spectral comparison with synthetic one, was thus named as desmethylpiperazinyl propoxysildenafil.

Characterization of a new illicit phosphodiesterase-type-5 inhibitor identified in the softgel shell of a dietary supplement.

A new sildenafil analog has been identified in the softgel shell of a dietary supplement. The compound was investigated by UV spectroscopy and high-resolution MS analysis, leading to the proposed structure 1-methyl-5-{5-[2-(4-methylpiperazin-1-yl)acetyl]-2-propoxyphenyl}-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one. A synthetic reference compound with the proposed structure was prepared, and the two sets of analytical data were compared, confirming the structure of the new compound. The compound was named propoxyphenyl noracetildenafil from its structure and similarity with the known compound.

Identification and structural elucidation of a new sildenafil analogue, dithiopropylcarbodenafil, from a premixed powder intended as a dietary supplement.

A new sildenafil analogue was detected during the monitoring of a premixed powder intended as a dietary supplement. The ultraviolet (UV) spectrum of the unknown compound was similar to that of dithiodesmethylcarbodenafil and dithiodesethylcarbodenafil, although their corresponding HPLC peaks were observed at different retention times. The chemical structure of the unknown compound was characterized by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-TOF/MS), followed by nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. The comparison of its structure with that of dithiodesmethylcarbodenafil, revealed that the N-methyl group on the piperazine ring is replaced by a propyl group. This new sildenafil analogue was identified as 5-(2-ethoxy-5-(4-propylpiperazine-1-carbonothioyl)phenyl)-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidine-7-thione and designated as a dithiopropylcarbodenafil. To the best of our knowledge, this is the first study reporting the identification and characterization of dithiopropylcarbodenafil.

Solid-phase extraction assisted dispersive liquid-liquid microextraction based on solidification of floating organic droplet to determine sildenafil and its analogues in dietary supplements.

A novel analytical method for the simultaneous determination of the concentration of sildenafil and its five analogues in dietary supplements using solid-phase extraction assisted reversed-phase dispersive liquid-liquid microextraction based on solidification of floating organic droplet combined with ion-pairing liquid chromatography with an ultraviolet detector was developed. Parameters that affect extraction efficiency were systematically investigated, including the type of solid-phase extraction cartridge, pH of the extraction environment, and the type and volume of extraction and dispersive solvent. The method linearity was in the range of 5.0-100 ng/mL for sildenafil, homosildenafil, udenafil, benzylsildenafil, and thiosildenafil and 10-100 ng/mL for acetildenafil. The coefficients of determination were ≥0.996 for all regression curves. The sensitivity values expressed as limit of detection were between 2.5 and 7.5 ng/mL. Furthermore, intraday and interday precisions expressed as relative standard deviations were less than 5.7 and 9.9%, respectively. The proposed method was successfully applied to the analysis of sildenafil and its five analogues in complex dietary supplements.

A Case Report of Fatal Desmethyl Carbodenafil Toxicity.

We present the case report of a 34-year-old Hispanic male who was found unresponsive in the carport of his residence. Surveillance video footage from a security camera showed that he collapsed as he was walking to his vehicle. The decedent had no medical history and no history of illicit drug use. Initial toxicology testing revealed no alcohol or illicit drugs. Autopsy findings indicated a need for additional toxicological analysis due to a lack of trauma and the paucity of pathophysiologically significant natural disease. Liquid chromatography time-of-flight mass spectrometry of postmortem blood revealed the presence of two large peaks corresponding to desmethyl carbodenafil, an unapproved sildenafil analogue and its hydroxy metabolite. Species that are probable desmethyl and hydroxydesmethyl metabolites of desmethyl carbodenafil were also found. The mass and retention time of the parent compound in the decedent's sample were matched to those of a commercial standard. Based on this preliminary match, a method was developed and validated to quantify desmethyl carbodenafil in human blood. This is the first known case of fatal intoxication by desmethyl carbodenafil, a phosphodiesterase-5 inhibitor that is not approved for use in the United States. Over the past several years, retailers have issued voluntary recalls for dietary supplements marketed as sexual performance enhancers on the basis that these supplements may contain undeclared desmethyl carbodenafil.

Isolation and identification of a novel sildenafil analogue adulterant in herbal products.

A novel sildenafil analogue found in herbal products by a routine drug-adulteration screening programme was isolated by column chromatography. On the basis of extensive 1D- and 2D-nuclear magnetic resonance (NMR) spectroscopy and mass spectral analysis, the structure of a new compound YJ-07 was established as 1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl) benzenesulfonamide. It common name is aminosildenafil.

Identification of a new sildenafil analogue adulterant, desethylcarbodenafil, in a herbal supplement.

In a maca-containing herbal supplement claimed to remedy erectile dysfunction, a new sildenafil analogue was found using adulterant screening with TLC, GC-MS and LC-MS/MS. This compound was isolated by column chromatography and HPLC, and identified by extensive 1D- and 2D-NMR and mass spectral analyses. The structure of this new compound was established as 5-[2-ethoxy-5-(piperazine-1-carbonyl) phenyl]-1-methyl-3-propyl-1,6-dihydro-pyrazolo[4,3-d]pyrimidin-7-one, and was named desethylcarbodenafil.

Development and evaluation of an immunochromatographic strip for rapid screening of sildenafil-type compounds as illegal additives in functional foods.

Sildenafil is a phosphodiesterase-5 inhibitor (PDE-5) for the treatment of erectile dysfunction. Undeclared sildenafil and related analogues adulterated in functional foods are a threat to public health. To screen these illegal drugs rapidly in herbal samples, an immunochromatographic (IC) assay was developed based on polyclonal antibodies specific to both sildenafil and its analogues. A group that is pharmacological necessary for sildenafil and its analogues was employed as a representative hapten for the generation antibodies against the target compounds. The desired antisera showed satisfactory specificities to sildenafil and major analogues with IC50 values ranging from 19.3 to 34.6 ng ml(-1) in a referring enzyme-linked immunosorbent assay (ELISA). The optimised IC assay showed detection thresholds in the range 5.0-20 µg g(-1) for sildenafil and major analogues in herbal samples. Sixty herbal food supplements were screened and six were found to be positive using the IC strip. It was confirmed by ELISA and UPLC-PDA-MS/MS that positive samples contain target illegal additives in levels of 10-40 mg g(-1) (1-4%). In this range, sensitivity of the IC strip is adequate to screen sildenafil-type compounds in herbal commodities under a dilution ratio of 1:10(3). Thus, the current IC assay is a suitable tool for screening sildenafil and its analogues as illegal additives in herbal food supplements.

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).

Application of Orbitrap-mass spectrometry to differentiate isomeric sildenafil- and thiosildenafil-like analogues used for the adulteration of dietary supplements.

Two groups of isomeric phosphodiestrase-type 5 inhibitors (PDE-5), consisting of four sildenafil- and three thiosildenafil-like analogues, have been successfully differentiated using high-resolution MS/MS. The optimised MS/MS data obtained from each compound were used to build a database with the aid of mass processing software. Isomeric compounds with very close chromatographic separation like dimethylsildenafil and homosildenafil could be distinguished by their unique fingerprint fragment ions in the MS/MS database. All fragment ions were within the mass tolerance of 5 ppm. One case study using an adulterated dietary supplement is included to provide more insights into this application.