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.

Evaluation of carboxamide-type synthetic cannabinoids as CB1/CB2 receptor agonists: difference between the enantiomers.

Recently, carboxamide-type synthetic cannabinoids have been distributed globally as new psychoactive substances (NPS). Some of these compounds possess asymmetric carbon, which is derived from an amide moiety composed of amino acid derivatives (i.e., amides or esters of amino acids). In this study, we synthesized both enantiomers of synthetic cannabinoids, N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(2-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA 2-fluorobenzyl isomer), N-(1-amino-1-oxo-3-phenylpropan-2-yl)-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (APP-CHMINACA), ethyl [1-(5-fluoropentyl)-1H-indazole-3-carbonyl]valinate (5F-EMB-PINACA), ethyl [1-(4-fluorobenzyl)-1H-indazole-3-carbonyl]valinate (EMB-FUBINACA), and methyl 2-[1-(4-fluorobenzyl)-1H-indole-3-carboxamido]-3,3-dimethylbutanoate (MDMB-FUBICA), which were reported as NPS found in Europe from 2014 to 2015, to evaluate their activities as CB1/CB2 receptor agonists. With the exception of (R) MDMB-FUBICA, all of the tested enantiomers were assumed to be agonists of both CB1 and CB2 receptors, and the EC50 values of the (S)-enantiomers for the CB1 receptors were about five times lower than those of (R)-enantiomers. (R) MDMB-FUBICA was shown to function as an agonist of the CB2 receptor, but lacks CB1 receptor activity. To the best of our knowledge, this is the first report to show that the (R)-enantiomers of the carboxamide-type synthetic cannabinoids have the potency to activate CB1 and CB2 receptors. The findings presented here shed light on the pharmacological properties of these carboxamide-type synthetic cannabinoids in forensic cases.

Isomeric discrimination of synthetic cannabinoids by GC-EI-MS: 1-adamantyl and 2-adamantyl isomers of N-adamantyl carboxamides.

N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (APINACA) and N-(1-adamantyl)-1-pentyl-1H-indole-3-carboxamide (APICA) are carboxamide-type synthetic cannabinoids comprising indazole/indole-3-carboxylic acid and adamantan-1-amine moieties. However, in the case of compounds like APINACA or APICA, adamantyl positional isomers exist, wherein either adamantan-1-amine or adamantan-2-amine is present. These adamantyl positional isomers have not been reported in previous studies, and no analytical data are available. To avoid misidentification of adamantyl carboxamide-type synthetic cannabinoids, it is important to develop methods to discriminate these adamantyl positional isomers. In this study, we report the analytical characterization by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS). For providing analytical standards, we synthesized eight carboxamide-type synthetic cannabinoids (APINACA 2-adamantyl isomer, APICA 2-adamantyl isomer, 5 F-APINACA 2-adamantyl isomer, 5 F-APICA 2-adamantyl isomer, 5Cl-APINACA, 5Cl-APINACA 2-adamantyl isomer, adamantyl-THPINACA, 2-adamantyl-THPINACA) and purchased four 1-adamantyl derivatives (APINACA, APICA, 5 F-APINACA, 5 F-APICA). Although the retention times of the isomers are similar, 1-adamantyl carboxamides can be clearly discriminated from their 2-adamantyl isomers based on their different fragmentation patterns in the EI-MS spectra. Specifically, EI-MS spectra for adamantylindazole carboxamides showed remarkable differences between the 1-adamantyl and 2-adamantyl isomers. On the other hand, EI-MS spectra for adamantylindole carboxamides were similar, but the diagnostic ions of the 2-adamantyl isomers were observed. The method described herein was applicable to all compounds tested in this study and is expected to be of use for isomeric differentiation between other untested adamantyl carboxamide-type synthetic cannabinoids. Copyright © 2016 John Wiley & Sons, Ltd.

Enantioseparation of the carboxamide-type synthetic cannabinoids N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-1H-indazole-3-carboxamide and methyl [1-(5-fluoropentyl)-1H-indazole-3-carbonyl]-valinate in illicit herbal products.

Synthetic cannabinoids, recently used as alternatives to Cannabis sativa, are among the most frequently abused drugs. Identified in 2014, the synthetic cannabinoids N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-1H-indazole-3-carboxamide (5F-AB-PINACA) and methyl [1-(5-fluoropentyl)-1H-indazole-3-carbonyl]-valinate (5F-AMB) are carboxamides composed of 1-(5-fluoropentyl)-1H-indazole-3-carboxylic acid and valine amide/methyl ester. Because of their composition, these molecules have pairs of enantiomers derived from the chiral center of their amino acid structures. Previous studies on the identification of 5F-AB-PINACA and 5F-AMB did not consider the existence of enantiomers, and there have been no reports on the enantiopurities of synthetic cannabinoids. We synthesized both enantiomers of these compounds and then separated the enantiomers by liquid chromatography-high-resolution mass spectrometry using a column with a chiral stationary phase consisted with amylose tris (3-chloro-4-methylphenylcarbamate). Under the optimized conditions, the enantiomer resolutions were 2.2 and 2.3 for 5F-AB-PINACA and 5F-AMB, respectively. Analysis of 10 herbal samples containing 5F-AB-PINACA and one herbal sample containing 5F-AMB showed that they all contained the (S)-enantiomer, but the (R)-enantiomer was only detected in two samples and at a ratio of less than 20%.

Simultaneous identification of 18 illegal adulterants in dietary supplements by using high-performance liquid chromatography-mass spectrometry.

We developed a method for the identification of 18 illegal adulterants in dietary supplements for erectile dysfunction by using high-performance liquid chromatography-mass spectrometry. The separation was achieved on a Cosmosil 3C18-EB column. The mobile phase consisted of 0.1% formic acid solution and 0.1% formic acid in acetonitrile, with gradient elution at a flow rate of 0.15 mL/min. The proposed method may be useful for the identification of illegal adulterants and for quality control of dietary supplements.

Simultaneous identification of hydroxythiohomosildenafil, aminotadalafil, thiosildenafil, dimethylsildenafil, and thiodimethylsildenafil in dietary supplements using high-performance liquid chromatography-mass spectrometry.

We developed a method for the separation and identification of illegal adulterants (hydroxythiohomosildenafil, aminotadalafil, thiosildenafil, dimethylsildenafil, and thiodimethylsildenafil) from dietary supplements using high-performance liquid chromatography-mass spectrometry. The separation was achieved on a C18 column: the mobile phase consisted of 5 mM ammonium formate (pH 6.3)-acetonitrile (75 : 25, v/v) and acetonitrile, with gradient elution at a flow rate of 0.2 mL/min. The proposed method could also be used to separate vardenafil, homosildenafil, and dimethylsildenafil, all of which have the same molecular weight. Furthermore, the proposed method could simultaneously separate hydroxythiohomosildenafil, aminotadalafil, thiosildenafil, dimethylsildenafil, thiodimethylsildenafil, vardenafil, and homosildenafil. Thus, this method may be useful to identify medicinal ingredients for erectile dysfunction and their analogs and to control the quality of dietary supplements.

A simple and selective detection method for aristolochic acid in crude drugs using solid-phase extraction.

The official Japanese method for analyzing aristolochic acid I (AA-I) in Asiasarum root using conventional high-performance liquid chromatography (HPLC) is described in the Japanese Pharmacopoeia, Sixteenth Edition. Interfering peaks of AA-I sometimes appear after HPLC analysis of crude drugs. A selective analytical method is needed to determine definitively whether AA-I is present in crude drugs. In this study, we developed a selective method that combined solid-phase extraction and liquid chromatography/mass spectrometry (LC/MS) which may be useful for identifying AA-I in crude drugs and for quality control.

Rapid determination of atropine and scopolamine content in scopolia extract powder by HPLC.

A rapid method that does not require a complicated preparation was developed for determining by HPLC the content of atropine (At) and scopolamine (Sc) in a sample of scopolia extract powder. The sample solution for HPLC was extracted using 0.1 mol/L HCl/methanol (8:2). At and Sc were separated using a pentafluorophenylpropyl column and detected at a wavelength of 210 nm. Acetonitrile-10 mmol/L ammonium acetate adjusted to pH 5.0 (8:2, v/v) was used as the mobile phase. The linearity was good in the 5.0-500 µg/mL range for At and 0.5-500 µg/mL range for Sc. The specificity for both At and Sc was satisfactory. The quantitation limits were 5.0 µg/mL for At and 0.5 µg/mL for Sc. The quantitative values of total alkaloid calculated using this method were higher (1.3-3.7%) than those obtained using the Japanese Pharmacopoeia Fifteenth Edition (JP15) method. The precision of this method, measured as the standard deviation, was found to be satisfactory and comparable to that of the JP15 method, determined by an analysis of 3 commercial scopolia extract powder samples.

Simple and rapid analysis of sennoside A and sennoside B contained in crude drugs and crude drug products by solid-phase extraction and high-performance liquid chromatography.

The sennoside A (SA) and sennoside B (SB) contents of various samples of crude drugs were determined using solid-phase extraction (SPE) and HPLC. The samples examined were crude drugs (senna leaf, senna pods, and rhubarb), conventional crude drug products, and Kampo formulations. The sample solution was purified using an Oasis MAX cartridge, which has strong anion-exchange and reversed-phase properties. The samples containing SA and SB were dissolved in a solution of methanol-0.2% sodium bicarbonate (7:3, v/v) and applied to the Oasis MAX cartridge. The cartridge was washed with a solution of methanol containing 1% acetic acid. SA and SB were eluted with methanol-water-formic acid (70:30:2, v/v), and the eluate was used as the sample solution for HPLC analysis. SA and SB were analyzed using a conventional octadecylsilyl (ODS) column at a detection wavelength of 380 nm; water-acetonitrile-phosphoric acid (800:200:1, v/v) was used as the mobile phase. The SA and SB components in most samples were completely separated from other interfering constituents within 10 min. In particular, several interfering peaks adjacent to the SB peak were eliminated by SPE using the Oasis MAX cartridge. On subjecting the Kampo extracts to an additional recovery experiment, high recovery rates of SA and SB were obtained. The method employed in this study proved to be a simple and rapid method for the quantification of SA and SB.