[Attempt to Calculate the Efficiency of Methylation Using 1H-NMR for Analyzing Brominated Vegetable Oil].

We studied the efficiency of methylation for analyzing brominated vegetable oil (BVO). In this report, we investigated whether 1H-NMR is an applicable method for assessing the efficiency of methylation to analyze BVO. 1H-NMR sufficiently calculated the efficiency of methylation using each integral and the numbers of protons derived from the methyl group, which is characteristic in products, and the methine group, which is characteristic in unreacted substances. Additionally, the efficiency of methylation calculated via 1H-NMR was in good agreement with changes in the peak area of BVO fatty acid methyl esters (BVOFAMEs) after various heating times obtained from GC-FID analysis. Therefore, 1H-NMR is applicable for calculating the efficiency of methylation to analyze BVO.

[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.

Rapid identification of a narcotic plant Papaver bracteatum using flow cytometry.

In May 2011, numerous poppy plants closely resembling Papaver bracteatum Lindl., a type of narcotic plant that is illegal in Japan, were distributed directly from several large flower shops or through online shopping throughout Japan, including the Tokyo Metropolitan area. In order to better identify the narcotic plants, the relative nuclear DNA content at the vegetative stage was measured by flow cytometric (FCM) analysis in 3 closely-related species of the genus Papaver section Oxytona, namely P. orientale, P. pseudo-orientale, and P. bracteatum, based on the difference between the chromosome numbers of these species. The results showed that the nuclear DNA content differed between these 3 species, and that most of the commercially distributed plants examined in this study could be identified as P. bracteatum. The remaining plants were P. pseudo-orientale, a non-narcotic plant. In addition, the FCM results for the identification of P. bracteatum completely agreed with the results obtained by the morphological analysis, the inter-genic spacer sequence of rpl16-rpl14 (PS-ID sequence) of chloroplast DNA, and the presence of thebaine. These results clearly indicate the usefulness of FCM analysis for the identification of P. bracteatum plants, including when they are in their vegetative stage.

Peyote identification on the basis of differences in morphology, mescaline content, and trnL/trnF sequence between Lophophora williamsii and L. diffusa.

Genus Lophophora (Cactaceae) has two species: Lophophora williamsii Coulter, which is called peyote, and L. diffusa Bravo. Although it was reported that L. williamsii contained mescaline and L. diffusa did not, we found L. williamsii specimens that did not contain mescaline. This finding indicated that the two species could not be differentiated in terms of mescaline content. Moreover, the relationship between mescaline content and morphology of the two species is also unknown. In this study, we attempted to clarify the difference in morphology, mescaline content, and DNA alignment of the chloroplast trnL/trnF region between L. williamsii and L. diffusa. As a result, L. williamsii specimens were classified into two groups. Group 1 had small protuberances on the epidermis, contained mescaline, and the analyzed region on the trnL/trnF sequence was 881 base pairs (bp) long in all except one (877 bp). Group 2 had large protuberances on the epidermis, did not contain mescaline, and the analyzed region was 893 bp long. On the other hand, L. diffusa had medium-sized protuberances on the epidermis, did not contain mescaline, and the analyzed region was 903 bp long. Also investigated was the potential application of the PCR-restriction fragment length polymorphism (RFLP) method as a means of identification based on the trnL/trnF sequence. By applying the PCR-RFLP method, the two species could be distinguished and L. williamsii specimens could be differentiated into group 1 and group 2.

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.