[Identification of Descarbonsildenafil in an Adulterated Dietary Supplement and Evaluation of Its Inhibitory Activity for Phosphodiesterase Type 5 (PDE5)].

Some illegal dietary supplements contain phosphodiesterase type 5 (PDE5) inhibitors, such as sildenafil, for exerting "therapeutic" effects in erectile dysfunction. This is apparently dangerous, and thus, should be appropriately regulated. Identification of descarbonsildenafil was first reported in Singapore in a coffee sample labeled to exert male sexual performance enhancement effects. However, it is unclear whether the compound possesses PDE5 inhibitory activity. We encountered during our survey of dietary supplements, a sexual enhancement product commercially available in Tokyo, in which a peak presumed to be of descarbonsildenafil was detected by LC-UV and electrospray ionization-tandem MS (ESI-MS/MS). The compound was isolated and identified as descarbonsildenafil with liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS), NMR, and X-ray crystal structural analysis. In addition, descarbonsildenafil showed PDE5 inhibitory activity in PDE5 inhibition assay, and its IC50 value for PDE5A1 was found to be 30 nmol/L. The results of INADEQUATE NMR and X-ray crystal structural analysis in this study provide information for the identification of descarbonsildenafil. Since this study indicates that this compound is a PDE5 inhibitor having adequate activity, it is regulated as a drug component in Japan.

[LC/Tribrid Orbitrap Analysis of Phosphodiesterase-5 Inhibitors and Their Analogs as Adulterants in Dietary Supplements].

LC/Tribrid Orbitrap was developed to determine phosphodiesterase-5 (PDE-5) inhibitors and their analogs as adulterants in dietary supplements. High-resolution MS/MS and MS3 spectra of PDE-5 inhibitors and their analogs were obtained by LC/Tribrid Orbitrap using both higher-energy collisional dissociation and collision-induced dissociation. We investigated dietary supplements that claim to enhance men's sexual performance, and detected PDE-5 inhibitors and their analogs. We also estimated the structures of the PDE-5 inhibitor analogs and the impurities of PDE-5 inhibitors and their analogs in the dietary supplements.

Identification of N-octylnortadalafil and its Stereoisomers in Dietary Supplements with Chiral Liquid Chromatography-Circular Dichroism.

A direct chiral liquid chromatography-circular dichroism (LC-CD) method was developed for the simple and rapid identification of N-octylnortadalafil [(6R, 12aR)-6-(1,3-benzodioxol-5-yl)-2-octyl-2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione; RR-OTDF] and its stereoisomers in dietary supplements. Samples were extracted with methanol. Compounds were then separated by chiral LC-CD using Chiralcel OD-RH (4.6 × 150 mm, 5 µm) with 5 mM ammonium formate (pH 3)/0.1% formic acid in acetonitrile (95:5, v/v) mixture solution (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The isocratic elution used was mobile phase A / mobile phase B (3:7, v/v) at a flow rate of 1 ml/min. The column temperature was held at 30°C. RR-OTDF and its stereoisomers were separated within 20 min with the resolution factors being over 2.0. Using this method, RR-OTDF and (6R, 12aS)-6-(1,3-benzodioxol-5-yl)-2-octyl-2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione were detected in a dietary supplement.

Stability assessment and content determination test of oral liquid forms of beclomethasone dipropionate prepared in hospital pharmacy.

OBJECTIVES: Oral beclomethasone dipropionate (BDP) is known for its use as a therapeutic medicine for gastrointestinal graft-versus-host disease (GI-GVHD). Despite growing demand for oral BDP formulation, no commercial forms have yet been marketed. Therefore, at the Tokyo Metropolitan Cancer and Infectious Disease Centre Komagome Hospital, pharmacists prepare oral liquid forms of BDP for patients with upper GI-GVHD. This study aims to develop a new high performance liquid chromatography (HPLC) method for measuring BDP in the prepared formulations and assessing its quality. METHODS: We developed a new HPLC method for measuring BDP in prepared formulations validated according to international guidelines. Three types of formulations were prepared and analysed using the validated HPLC method. One contains 1 mg of BDP per 30 mL aqueous solution, and the others using ethanol for preparation contain 1 mg of BDP per 15 mL aqueous solution. For stability assessment, the BDP contents were assayed while formulations were stored in plastic bottles for 8 weeks under two different conditions of 25°C in bright light and 4°C in darkness. A content determination test was also conducted to assess the individual contents of BDP and lot-to-lot variation in dosage units. RESULTS: A stability test demonstrated that the remaining BDP content after the storage period was greater than 90% of the initial content in almost all samples regardless of storage conditions. A content determination test showed thattwo new ethanol-containing formulations contained about 0.1 mg more BDP than the original ethanol-free formulation and it was close to the target BDP content of 1 mg. Furthermore, new formulations had less lot-to-lot BDP variation in dosage units than the original formulation. CONCLUSIONS: A new HPLC method for measuring BDP in prepared formulations was developed and validated. The results of the stability test and content determination test indicated that the newly designed formulations were superior to the conventional formulation.

Analytical characterization and differentiation between threo- and erythro-4-fluoroethylphenidate.

PURPOSE: Methylphenidate analogs appeared on the drug market during the last years. Its analogs contain two chiral centers and, thus, have potential varying configurations (i.e., threo and erythro forms). This study presents the analytical characterization of 4-fluoroethylphenidate (4-FEP) and its differentiation between threo- and erythro-4-FEP. METHODS: Analysis of the samples included high-performance liquid chromatography (HPLC), gas chromatography-electron ionization-mass spectrometry (GC-EI-MS), high-resolution mass spectrometry (HRMS) analyses, nuclear magnetic resonance (NMR) spectroscopy and X-ray crystal structure analysis. RESULTS: NMR spectroscopic investigations confirmed the differences between threo- and erythro-4-FEP, and demonstrated that both isomers could be separated using HPLC and GC methods. Two samples obtained from one vendor in 2019 consisted of threo-4-FEP, whereas the other two samples obtained from a different vendor in 2020 consisted of a mixture of threo- and erythro-4-FEP. CONCLUSIONS: Several analytical approaches including HPLC, GC-EI-MS, HRMS analyses, NMR spectroscopy and X-ray crystal structure analysis enabled the unambiguous identification of threo- and erythro-4-FEP. The analytical data presented in this article will be useful for identifying threo- and erythro-4-FEP included in illicit products.

Structure elucidation of a novel analog of sildenafil detected as an adulterant in a dietary supplement using LC-UV and LC/MS.

A sildenafil-related compound was detected in a dietary supplement marketed as an aphrodisiac. The compound was detected during analysis of the dietary supplement using LC-UV and LC/electrospray ionization-MS. The structure of the compound was established using high resolution MS, NMR spectrometry, and X-ray crystal structure analysis. The compound was identified as 5-(5-((3,5-dimethylpiperazin-1-yl)sulfonyl)-2-ethoxyphenyl)-l-methyl-7-((1-methyl-4-nitro-1H-imidazol-5-yl)thio)-3-propyl-1H-pyrazolo[4,3-d] pyrimidine. Based on this structure, the compound was named nitroprodenafil. The dietary supplement was found to contain 90 mg nitroprodenafil/capsule. This article describes the structural characterization of a new sildenafil-related compound. The compound was detected during analysis of a dietary supplement using LC-UV and LC/electrospray ionization (ESI)-MS. The structure was established using high resolution MS (HRMS), NMR spectrometry, and X-ray crystal structure analysis. The structures of methisosildenafil, thiomethisosildenafil, and this new analog, named nitroprodenafil (21), are shown in Figure 1. In the Demizu et al. report, the compound is named mutaprodenafil instead ofnitroprodenafil. Considering the naming right, the authors of this paper think the use of mutaprodenafil is appropriate as the compound name, although nitroprodenafil is used.

[Improvement Method of Confirmation Test of the In-pharmacy Formulation as Topical Medicine No. 37-①].

Topical Medicine No. 37-① is one of the in-pharmacy formulation that specifies a confirmation test (referred hereafter as the conventional method) to identify its ingredient diphenhydramine (DH) by colorimetric test. However, the conventional method is environmentally unfriendly due to the large amount of sample and organic solvent used, and the extraction process is complicated. We therefore developed three methods in view of improving the currently confirmation testing process: a simplified version of the conventional method, a TLC method, and an LC/photodiode array detector (PDA) method. The LC/PDA method was also examined for the quantitation of DH in order to determine its content in the medicine when needed. The LC/PDA method also demonstrated sufficient linearity in the calibration curve and recovery rate. We also evaluated whether the three methods developed in this study could be applied to Topical Medicine No. 37-①, which is made of absorptive cream containing different parabens.

Differentiation between sulfoaildenafil and its analogs.

An analog of aildenafil, which is a potent and highly selective inhibitor of phosphodiesterase 5, was found in a dietary supplement marketed for enhancement of sexual function. The compound was isolated by silica gel column chromatography, and its structure was identified by means of 13C-NMR spectrometry, 1H-NMR spectrometry, high-resolution MS, and X-ray structure determination. The compound was identified to be sulfoaildenafil (other names: thioaildenafil, dimethyl sildenafil thione, and thiomethisosildenafil). Sulfoaildenafil is very similar to the compound thiohomosildenafil. As it is difficult to distinguish between them by LC-photodiode array detector analysis, ultra-performance LC (UPLC)/MS, ion trap LC/MS/MS (LC/IT-MS/MS), and GC/MS were performed. The mass spectra of thiohomosildenafil by UPLC/MS and LC/IT-MS/MS showed mass fragments of m/z 58, 72, and 355, and the mass spectrum by GC/MS showed mass fragments of m/z 56, 72, and 420. Some of these fragments had low intensities, but they were useful for distinguishing between the two compounds. The relationship between aildenafil (other names: dimethylsildenafil and methisosildenafil) and homosildenafil is similar to that between sulfoaildenafil and thiohomosildenafil. Therefore, these compounds were also examined.

Structure elucidation of a PDE5 inhibitor detected as an illegal adulteration in a libido-boosting dietary supplement.

A compound with potent inhibitory activity for phosphodiesterase type 5 (PDE5) was identified as an illegal adulteration in a libido-boosting dietary supplement being sold at a store in Tokyo. This compound was identified as 5,6-diethyl-2-{5-[(4-methylpiperazin-1-yl)sulphonyl]-2-propoxyphenyl}pyrimidin-4(3H)-one using liquid chromatography-diode array detector (LC-DAD), liquid chromatography-tandem mass spectrometer (LC-MS), LC-HRMS, nuclear magnetic resonance (NMR), and X-ray crystallography. The IC50 value of the inhibitory activity for PDE5A1 (one of the PDE5 isoforms) was 2.0 nM (sildenafil IC50 value was 4.5 nM). This compound was previously synthesised as a PDE5 inhibitor by Shanghai Institute of Materia Medica. The dietary supplement contained 85 mg of this compound in a capsule, which was about 26% of the capsule content (320 mg).