Chee-Leong Kee

Isolation and characterization of propoxyphenyl linked sildenafil and thiosildenafil analogues in health supplements

Two new phosphodiesterase-5 inhibitors (PDE-5) which consist of one sildenafil analogue and one thiosildenafil analogue have been found in heath supplements. The structural properties of these analogues have been elucidated by NMR, high resolution MS, MS(2), UV and IR spectroscopy. The sildenafil analogue is very similar to aildenafil and the thiosildenafil analogue is similar to thioaildenafil, except the ethoxy group bonded to phenyl ring is replaced by a propoxy group. Hence, the sildenafil analogue is named as propoxyphenyl aildenafil or propoxyphenyl methisosildenafil and the thiosildenafil analogue as propoxyphenyl thioaildenafil or propoxyphenyl thiomethisosildenafil.

Structural elucidation of a new sildenafil analogue using high-resolution Orbitrap mass spectrometry

RATIONALE One new phosphodiesterase type 5 (PDE-V) inhibitor, propoxyphenyl homohydroxysildenafil (PP-HHS), has been isolated from one health supplement, and analyzed using high-resolution Orbitrap mass spectrometry. High-resolution mass spectrometry (HRMS) is useful to elucidate unknown substances at low concentrations. METHODS Two isolated compounds, propoxyphenyl thiohomohydroxysildenafil (PP-THHS) and propoxyphenyl homohydroxysildenafil (PP-HHS), were infused into the Thermo Fischer Scientific LTQ Orbitrap XL™ hybrid FTMS system at a flow rate of 3 μL per min. The high-resolution MS(2) spectra were acquired using different high-energy collision dissolution (HCD) mode; 40 V for PPT-HHS and 45 V for PP-HHS. The accurate mass measurement was assisted with the aid of Mass Frontier software, version 5.0. RESULTS The fragmentation pattern of PP-HHS in the MS(2) spectrum is very similar to that of PP-THHS except the product ions at m/z 519, 501, 325, 299 and 283 are less than PP-THHS by 16 m/z units. This is a result of the replacement of sulfur atom by oxygen at the thiolactam moiety. All the mass errors are below 5.0 ppm. CONCLUSIONS High-resolution Orbitrap mass spectrometry is an alternative method to determine unknown compounds like PDE-V inhibitor analogues unambiguously by analyzing the product ions at high mass accuracy. PP-HHS is an unapproved drug and no pharmacological study has been reported. Hence, it could be harmful to unknowing consumers with undesirable side effects.

Structural elucidation of propoxyphenyl isobutyl aildenafil, adulterant in a health supplement using high-resolution Orbitrap mass spectrometry

A new sildenafil analogue, propoxyphenyl isobutyl aildenafil has been found in trace quantity from one health supplement. It has been purified by preparative high performance liquid chromatography (HPLC). The structural elucidation of this compound has been carried out using high-resolution Orbitrap mass spectrometry under two fragmentation modes, namely High energy Collision Dissolution (HCD) and Collision Induced Dissolution (CID). Under MS(3) experiments and CID mode, the isobutyl-bonded fragments of propoxyphenyl isobutyl aildenafil at m/z 313 and 297 have been compared with the reference ions derived from isobutyl sildenafil. The accurate mass measurement of each product ions has been carried out with the aid of Mass Frontier software (version 5.0). The mass error of all product ions is not more than 5.0ppm.

Analysis of 40 weight loss compounds adulterated in health supplements by liquid chromatography quadrupole linear ion trap mass spectrometry

In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) with scheduled multiple reaction monitoring (MRM) enhanced product ion (EPI) method was developed for simultaneous determination of 40 compounds with weight loss effect, including bisacodyl, phenolphthalein, and sibutramine and its metabolites, etc. They might be adulterated in health supplements to get prominent weight loss effect. The samples were analyzed using a Q-Trap 5500 coupled with high performance liquid chromatography (HPLC) and a CORTECS ultra performance liquid chromatography (UPLC) C18 column (100 mm x 2.1 mm x1.6 µm). Scheduled MRM was used as survey scan, MS2 spectra acquired in the EPI mode were used to perform library searching to increase the confidence of detection. Limits of detection were less than 10 ng/g for the majority of the analytes. A total of 447 weight loss products were tested in our laboratory in the past three years. Among these samples, 119 samples were found to be adulterated with one or more weight loss compounds, including sibutramine, its metabolites benzyl sibutramine and desmethyl sibutramine; phenolphthalein; bisacodyl; furosemide; liothyronine (T3); and thyroxine (T4). Copyright

Rapid determination of sildenafil and its analogues in dietary supplements using gas chromatography-triple quadrupole mass spectrometry.

Application of gas chromatography-triple quadrupole mass spectrometry for identification, confirmation and quantification of 6 phosphodiesterase-5 (PDE-5) inhibitors (sildenafil, dimethylsildenafil, homosildenafil, thiosildenafil, thiodimethylsildenafil and thiohomosildenafil) in dietary supplements was investigated. The MS was operated in multiple reaction monitoring mode, for better sensitivity and selectivity. In this manner, the method is adequate to reduce background noise with less interference from co-eluting compounds in the samples. Two different ionisation techniques, electron ionisation (EI) and chemical ionisation (CI), were studied and compared. The chromatographic separation was performed on a short 10 m non-polar capillary column without any derivatisation step. This permitted fast analysis for all analogues with retention time less than 11 min, for both techniques. Use of backflushing can aid method retention time reduction and improves column maintenance. Evaluation of method validation included limit of detection (LOD), lower limit of quantitation (LLOQ), linearity, precision and recovery were performed for both EI and CI techniques. The LOD obtained varied from 0.03 to 1.50 μg/g and the LLOQ ranged from 0.10 to 5.00 μg/g. Good calibration linearity was obtained for all analogues for both techniques, with correlation coefficients (r(2)) higher than 0.99. Mean recoveries of all analogues using CI show higher values (83.4-108.8%) than that of EI (61.9-91.1%). The intra- and inter-assay precisions were evaluated for all analogues at spiked concentration of 10 μg/g and the relative standard deviation was less than 15% for both methods. These methods were then successfully applied to dietary supplement samples without prior derivatisation, confirming that the samples were adulterated with sildenafil and/or its analogues.

Isolation and characterization of a tadalafil analogue, N-cyclopentyl nortadalafil in health supplement.

A tadalafil analogue was detected for the first time during the screening of a health supplement for undeclared sexual enhancement drugs. The compound had been isolated and purified by preparative high-pressure liquid chromatography (HPLC). Its chemical structure was elucidated using high-resolution mass spectrometry (HRMS), electrospray ionization tandem mass spectrometry (ESI-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The compound had a protonated molecular ion at m/z 444 with a chemical formula of C26H25N3O4. The data obtained from the MS analysis of the compound suggested that the N-methyl group on the piperazinedione moiety of tadalafil was substituted with a -C5H9 group. Analysis using NMR was performed and the -C5H9 group was characterized as a cyclopentyl moiety. The analogue was named N-cyclopentyl nortadalafil.

A review of synthetic phosphodiesterase type 5 inhibitors (PDE-5i) found as adulterants in dietary supplements

Graphical abstract Figure. No Caption available. Highlights80 PDE‐5i have been found as adulterants in dietary supplements from 2003 to Mar 2017.Sildenafil analogues (62%) are the top list and followed by tadalafil (26%) and vardenafil analogues (9%).Asia reported the highest number of PDE‐5i, (67%) and followed by Europe (22%) and North America (11%).Increased complexity of novel PDE‐5i which needs complementary analytical techniques. ABSTRACT To date, there are 80 synthetic PDE‐5i found as adulterants in dietary supplements. Analogues of sildenafil remain as the top list with 50 (62%) and are followed by analogues of tadalafil, 21 (26%), analogues of vardenafil, 7 (9%) and others, 2 (3%). The sildenafil group can be sub‐categorized into sulphonamide‐bonded (24, 48%), acetyl‐bonded (11, 22%), carbonyl or thiocarbonyl‐bonded (8, 16%) and other types (7, 14%) based on the functional group linked to pyrazolopyrimidine‐one moieties. Meanwhile, analogues of tadalafil have become popularly found as adulterants in dietary supplements like beverages and herbal extracts from 2015 to 2016. The uptrend has been observed with the increase in number and complexity with more trans‐oriented and dimerized tadalafil analogues being reported. Interestingly, there is no much increase for analogues of vardenafil. About two thirds of analogues have been reported from the Asian countries (67%), followed by Europe (22%) and North America (11%). South Korea and Singapore have reported the most number of analogues with a total number of 40 (50%). One plausible contributing factor to this trend is the convenient purchase of sexual enhancement dietary supplements, especially the on‐line purchase. In terms of analytical methodologies, high performance liquid chromatography (HPLC) hyphenated to ultra‐violet (UV) and/or mass spectrometry (MS) detection have been preferred in the screening analysis, i.e. 70 out of 77 compounds have been analysed by HPLC‐UV. In addition, the electrospray ionization multistage fragmentation experiments (ESI‐MSn) for acquiring low‐ and high‐resolution mass spectra have been successfully applied to detect and quantify PDE‐5i in adulterated products simultaneously. Nuclear magnetic resonance (NMR) is another important technique in the structural elucidation of novel analogues.

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.

Isolation and structural elucidation of flibanserin as an adulterant in a health supplement used for female sexual performance enhancement

A health supplement used for female sexual performance enhancement was sent to Health Sciences Authority of Singapore for testing. An unknown compound was detected and isolated from the health supplement and its structure was elucidated using LC-DAD, LC-FTMS, NMR and IR. The detected compound was identified to be flibanserin, a non-hormonal treatment developed for pre-menopausal woman with hypoactive sexual desire disorder (HSDD).

Isolation and characterization of a novel dithio-carbodenafil analogue from a health supplement

&NA; A novel dithio‐carbodenafil compound was isolated and identified from a health supplement. The structure of the unknown compound has been characterized using LC‐UV, Fourier Transform Infrared (FTIR), high‐resolution mass spectrometry, 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. It has been revealed as 3,5‐dimethylpiperazinyl dithio‐desmethylcarbodenafil as a result of two additional methyl groups on the piperazine ring. Graphical abstract Figure. No caption available. HighlightsIdentification of 3,5‐dimethylpiperazinyl dithio‐desmethylcarbodenafil by HRMS and NMR.It has two additional methyl groups on the piperazine ring as compared with dithio‐desmethylcarbodenafil.2D NMR (HMBC, HMQC) distinguish the position of carbons and protons on the piperazine.