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Hydroxycarbodenafil, an analogue of carbodenafil, was detected in a dietary supplement in China in 2020. However, previous reports have not identified some carbon signals from the piperazine ring in nuclear magnetic resonance (NMR) experiments. Because the compound contains an amide bond, the reaction was suggested to be characteristic of compounds with rotational isomers. Variable-temperature NMR is used to determine the rotational barrier between different conformations by changing the measurement temperature. Using this technique, we succeeded in obtaining the first distinct data, including the carbon signals of the piperazine ring in the NMR spectrum of hydroxycarbodenafil. We also confirmed that this technique could be applied to other carbodenafil analogues. Multi-stage mass spectrometry (MS n ) measurements with a high-resolution mass spectrometer specific to the substructures were performed to develop a protocol for the structural determination of the carbodenafil analogues. In addition, hydroxycarbodenafil was analysed using X-ray crystallography, and its inhibitory activity against phosphodiesterase type 5 (PDE5) was measured. The IC 50 value of the inhibitory activity of hydroxycarbodenafil for PDE5A1, a PDE5 isoform, of 2.9 nM was lower than the 4.5 nM for sildenafil, a positive control., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The rapid and accurate detection of illegal adulteration of chemical drugs into dietary supplements is a big challenge in the food chemistry field. Detection of compounds without a standard reference is even more difficult; however, this is a common situation. Here in this study, a novel "standard-free detection of adulteration" (SFDA) method was proposed and phosphodiesterase-5 inhibitor derivatives were used as an example to figure out the possibility and reliability of this SFDA method. After analysis by quadrupole coupled time of flight-tandem mass spectrometry detection and multivariable statistics, six common fragment ions were chosen to indicate whether adulteration was present or not, while 20 characteristic fragment ions indicated whether adulteration was by nitrogen-containing heterocycles or by anilines. Furthermore, the quantitative methods were conducted by high-performance liquid chromatography-tandem mass spectrometry. In a word, this strategy allows for a quick determination of dietary supplement adulteration without any need for standard materials, improving the efficacy of food safety testing., (© 2024 John Wiley & Sons Ltd.)

The aim of individuals consuming health supplements is to attain a robust state through nutritional regulation. However, some unscrupulous manufacturers, motivated by profit, fraudulently incorporate drugs or unauthorized components with therapeutic effects into the product for instant product performance enhancement. The long-term use of these products may inadvertently inflict harm on human health and fail to promote nutritive healthcare. The illegal inclusion of these substances is prevalent in kidney-tonifying and sexuality-enhancing products. Developing effective analytical methods to identify these products and screen for illegal added ingredients can effectively prevent such products from reaching and remaining on the market. A target screening method for the detection and quantification of 90 phosphodiesterase type 5 inhibitors (PDE-5is) in 5 kinds of health products was developed and validated. The type of dietary supplements varied from tablets, capsules, and protein powder to wine and beverages. Sample preparation was completed with a one-step liquid phase extraction. The screening process of 90 PDE-5is was done efficiently within 25 min by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) using the dynamic multiple reaction monitoring (dMRM) technique. The LODs of 90 PDE-5is were detected at levels ranging from 25 to 85 ng/g or ng/mL. This novel targeting methodology was effective and can be applied to routine market supervision. Among 286 batches of samples, 8 batches were found to be positive. Three kinds of PDE-5is were first detected in healthy products. The screening method demonstrated herein will be a promising and powerful tool for rapid screening of PDE-5is.

The last few decades have witnessed the increasing consumption of functional foods, leading to the expansion of the worldwide market. However, the illegal addition drugs in functional foods remains incessant despite repeated prohibition, making it a key focus of strict crackdowns by regulatory authorities. Effective analytical tools and procedures are desperately needed to rapidly screen and identify illegally added drugs in a large number of samples, given the growing amount and diversity of these substances in functional foods. The MRSIT-HRMS (Multiple Sample Rapid Introduction combined with High Resolution Mass Spectrometry) without chromatographic separation, after direct sampling, utilizes NIST software (National Institute of Standards and Technology) matching with a home-built library to target identification and non-targeted screen of illegal additives. When applied to 50 batches of suspicious samples, the targeted method detected illegal added drugs in 41 batches of samples, while the non-targeted method screened a new phosphodiesterase-5 (PDE-5) inhibitor type structural derivative. The positive results obtained by the targeted method were consistent with LC-MS/MS (QQQ). The novel MRSIT-HRMS with a limit of quantification (LOD) of 1 μg/mL achieved 100 % correct identification for all 50 batches of actual samples, demonstrating its potential as a highly promising and powerful tool for fast screening of illegally added drugs in functional food, especially when compared to traditional LC-MS/MS methods. This is essential for ensuring drug safety and public health., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ziyan Hu reports financial support was provided by State Administration for Market Regulation of China. Ziyan Hu reports financial support was provided by Jiangsu market supervision and Administration Burea. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Erectile dysfunction (ED) is one of the most common chronic diseases affecting men and its incidence increases with aging. Due to its substantial influence on the quality of life, phosphodiesterase type-5 (PDE5) inhibitors have been implemented to treat ED by increasing the penile blood flow that results in improving erection. PDE5 inhibitors is a class of drugs that affects many pharmacological sectors, and it is essential to review the different analytical methods described for their determination. Few reviews were published concerning this group of drugs. For this reason, this review article gathers the different analytical methods used to determine PDE5 inhibitors in pharmaceutical and biological samples over the past 20 years. Different analytical techniques were used to analyze these compounds in different matrices such as separation methods (capillary electrophoresis, LC-MS, UPLC-MS/MS, and GC-MS), spectroscopic methods (UV-visible methods, FT-IR spectroscopy and spectrofluorometry) and electrochemical methods (polarography, voltammetry and potentiometry). This review focuses on the different electrochemical methods and their use in analytical determination of PDE5 inhibitors in pharmaceutical dosage forms and biological samples. Moreover, it discusses the different modified electrodes used for their electroanalytical determination and the behavior of the studied drugs at different modified electrodes. Additionally, this review discusses the pharmacokinetics of the studied compounds and their interactions with other co-administered drugs especially the metabolic interactions between the studied compounds and other co-administered drugs in different matrices. This literature survey would provide a beneficial guide for future analytical investigation of PDE5 inhibitors.

This article is an up-to-date review of 112 unapproved phosphodiesterase type 5 inhibitors (PDE-5i) found as adulterants in sexual enhancement dietary supplements and other products from 2003 to July 2023. Seventy-five of these unapproved PDE-5i are analogues of sildenafil (67%), followed by 26 analogues of tadalafil (23%), 9 analogues of vardenafil (8%) and 2 other type of compounds (2%). The products have been formulated in various packaging, primarily in capsule, tablet, and powder forms. Common screening techniques allowing detection of such analogues include high performance or ultra-high performance liquid chromatography in tandem with ultra-violet detector (HPLC-UV or UPLC-UV) (50%) and thin-layer chromatography in tandem with ultra-violet detection (TLC-UV) (7%). Screening by mass spectrometry (MS) is relatively less common with the use of single-, triple-quadrupole or time-of-flight (TOF) mass spectrometers (9%). Meanwhile, the combined detection by UV-MS has been recorded at 10% usage. Screening by proton nuclear magnetic resonance spectroscopy (NMR) (11%) has also been applied. For compound characterization, i.e. structural elucidation, NMR spectroscopy has been preferred (100 out of 112 compounds), followed by high-resolution mass spectrometry (HRMS) (74 out of 112 compounds) and Fourier-transform infrared spectroscopy (FTIR) (44 out of 112 compounds). Over the past two decades, analytical technology has been evolving with enhanced sensitivity and resolution. Despite this, structural elucidation of the new emerging analogues in adulterated dietary supplements remains a challenge, especially when the analogues involve complex structural modification. Therefore, the above-mentioned techniques may not be adequate to characterize the analogues. Additional work involving chiroptical methods, two-dimensional (2D) NMR experiments and X-ray crystallography are likely to be required in the future.

Sexual enhancement products adulterated with phosphodiesterase 5 inhibitors (PDE-5i) pose a serious public health concern. Tadalafil and its analogues (Tds) are PDE-5i frequently detected as adulterants. In this study, a Td detector tube for the rapid detection of Tds was developed based on the color change reaction between sulfuric acid and Tds. The specificity of this test method was evaluated using 13 Tds, all of which elicited positive results. Additionally, 30 commonly found adulterants in dietary supplements, 11 active pharmaceutical ingredients of psychotropic drugs and 18 food ingredients were tested and obtained no false-positive results, except levomepromazine. The test tube accurately detected the presence or absence of Tds in 54 commercially available products. The visual detection limit was 2-50 and 5-20 μg/ml for Tds and tadalafil-spiked samples with matrix, respectively. The applicability of the developed detector tube to a semiquantitative test using digital image analyses were investigated using red, green, and blue color values. The results of the recovery test suggested that the tube test was affected by the dark-colored matrix. The results of semiquantitative analyses of tadalafil for five marketed products were consistent with the liquid chromatographic quantification results, except for the blue value. The detector tube developed in this study can facilitate with the rapid screening of Tds in adulterated sexual enhancement products., (© 2022 John Wiley & Sons Ltd.)

The detection and identification of phosphodiesterase type 5 enzyme (PDE-5) inhibitors in dietary supplements poses an analytical challenge due to the large number of analogs and isomers currently available and the continued introduction of novel analogs. The use of trapped ion mobility spectrometry (TIMS) in conjunction with liquid chromatography (LC) and electrospray ionization tandem mass spectrometry (MS/MS) was explored for the analysis of two groups of isomeric PDE-5 inhibitor analogs using a 5-minute method. Of the eight compounds studied, six were resolved by a combination of LC and TIMS; the two remaining isomers were distinguished by one or more unique product ions in the MS/MS spectrum. The results revealed that separation by LC corresponded to differences in substitution on the piperazine moiety of the PDE-5 inhibitors, while separation by TIMS corresponded to the position of a nitrogen atom in the fused ring region of the molecules. Samples prepared by spiking mixtures of varying amounts of the Group 2 isomers into a representative dietary supplement matrix were analyzed and concentrations determined from the mobility-adjusted extracted ion chromatograms exhibited relative standard deviations of 6.0 % or less for 17 of 20 measurements and recoveries between 80 % and 120 % for all measurements. Quantitative measurements from a short LC gradient were possible due to the reduced chemical background associated with the TIMS separation of co-eluting matrix compounds, which enabled acquisition of rapid and qualitatively relevant broadband collision induced dissociation spectra that didn't require precursor ion isolation; the reduced chemical background permits non-targeted detection of novel analogs and eliminates the need for a separate method for quantitative measurement., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Consumption of foods and dietary supplements (DS) adulterated with unprescribed or non-permitted phosphodiesterase-5 inhibitors (PDE-5i) and their analogs can cause serious risk to human health. This study aims to analyze 93 PDE-5i and their analogs present in adulterated foods and DS using an established and validated method involving high-performance liquid chromatography (HPLC). The method was validated in solid and liquid samples, resulting in a limit of detection and quantitation of 0.03-0.5 and 0.08-1.6 μg/mL, respectively. Using the validated method, a total of 404 samples were screened. It was found that 32% of 404 samples were illegally adulterated with PDE-5i and their analogs; moreover, 16.9% of the adulterated samples were found to contain more than three compounds. HPLC-quadrupole-time-of-flight (TOF)/mass spectrometry (MS) analysis was conducted on all the samples to confirm the detected compounds accurately based on fragmentation ion patterns. In addition, sildenafil and tadalafil were detected from the capsule shells of DS unusually. Subsequently, the detected compounds were identified and quantified using HPLC at concentrations ranging from 0.007 to 370.0 mg/g. NMR analysis was carried out to confirm the accurate chemical structure of a compound found during the TOF/MS analysis, which did not match with the 93 reference standards.; it was identified to be N-desmethylthiosildenafil. In this study, various PDE-5i compounds and their analogs were detected from low to high concentrations in a sample. Therefore, the study sheds light on the misuse of PDE-5i and their analogs in consumable products, which pose a severe threat to public health., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Sexual enhancement dietary supplements have often been adulterated with phosphodiesterase type 5 (PDE-5) inhibitors used for treatment of erectile dysfunction, and widely distributed through online markets. As the illegal adulterants, the original PDE-5 inhibitor drugs and a numerous number of synthetized analogues, more than 80, have already been found. Therefore, analytical methods that detect various PDE-5 inhibitors and uncover newly synthesized analogues are needed. In this study, we have developed a rapid and reliable screening method for PDE-5 inhibitors and their structural analogues by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by hierarchical clustering based on similarity of MS/MS spectra. Forty reference standards of PDE-5 inhibitors/analogues were measured using a quadrupole-orbitrap mass spectrometer in data-dependent mode. The 60 most intense fragment ions were extracted from each MS/MS spectra, and the ions observed within 1.5 mDa mass tolerance were considered to be the same ion. Based on fragment ion tables representing detected ions for each compound, hierarchical clustering was performed. The resulting dendrogram showed that the reference standards were separated into seven clusters according to their characteristic structures. Subsequently, two additional standards spiked into a herbal sample were analyzed. While herbal components were clearly separated from the clusters of the reference standards, the spiked standards were clustered closely with the structurally similar standards. Furthermore, application of our method to dietary supplements allowed for detection of sildenafil and tadalafil as adulterants. These results suggest that our screening method facilitates discovery of adulterant PDE-5 inhibitors/analogues by illustrating their structural similarity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

The use of herbal supplements for improved sexual performance is a common practice amongst the youth and some senior citizens in Ghana. These products are considered 'natural' and greatly preferred over synthetic alternatives due to the assurance of little to no adverse effects by producers. However, the high rate of adulteration often compromises their safety. Forty herbal supplements, of which 25 were previously shown to result in medium to high intake of phosphodiesterase type-5 (PDE-5) inhibitors using a PDE-Glo bioassay, were further investigated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to examine the reliability of the bioassay and whether the observed higher responses could be ascribed to inherent plant constituents or adulterants. Results showed significant amounts of vardenafil, tadalafil and especially sildenafil, in 2, 1 and 10 samples, respectively, with total concentration levels resulting in estimated daily intakes (EDIs) above 25 mg sildenafil equivalents with six supplements even having EDIs above 100 mg sildenafil equivalents. Only one sample contained a natural ingredient (icariin), but its concentration (0.013 mg g -1 ) was too low to explain the observed potency in the bioassay. The estimated concentrations of PDE-5 inhibitors in 35 supplements, according to the bioassay, were in line with those of the LC-MS/MS analysis. However, discrepancies were observed for five supplements. Further examination of one of the latter supplements using the PDE-Glo bioassay to select the positive fraction and further examination with LC-MS/MS and 1 H-NMR revealed the presence of hydroxythiohomosildenafil, a sildenafil analogue not yet included in the liquid chromatography-mass spectrometry reference library. This study demonstrates the significance of applying a tiered approach, where the use of a bioassay is followed by chemical analysis of bioactive samples in order to identify unknown bioactive compounds.

Herbal medicines are commonly used in many countries all around the world. In Western countries they are now gaining more and more popularity, whereas in countries like China and India they have been entrenched for millenniums. Some of these perceived herbal medicines claim to help when suffering from erectile dysfunction. Nevertheless, many of these products are adulterated with PDE5 inhibitors like sildenafil or α-blockers. Patients who suffer from high blood pressure sometimes resort to herbal products, as they are not allowed to take sildenafil because of negative drug-drug interactions with nitrates (often utilized as treatment for coronary diseases). Products which are then adulterated with PDE5 inhibitors, can seriously harm patients. Therefore, this study reports the instant screening of alleged herbal products by employing atmospheric pressure solids analysis probe and high-resolution mass spectrometry to determine adulterants. Three out of 12 investigated products contained sildenafil in ranges from 0.5% to 18%. Multivariate analysis of ambient mass spectrometry measurements revealed encouraging outcomes for distinguishing non-sildenafil and sildenafil adulterated samples. Atmospheric pressure solids analysis probe is therefore a promising method for the rapid determination of sildenafil in herbal products with possible downstream semiquantitative analysis., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The surge in the consumption of food products containing herbal aphrodisiacs has driven their widespread adulteration. A rapid screening strategy is, therefore, warranted to curb this problem. This study established an enzyme inhibition assay to screen phosphodiesterase 5 (PDE5) inhibitors as adulterants in selected food products. Fluorescein-labelled cyclic-3',5'-guanosine monophosphate was utilised as substrates for the PDE5A1 enzyme, aided by the presence of nanoparticle phosphate-binding beads on their fluorescence polarisation. The sample preparation was optimised to improve the enzyme inhibition efficiency and applied to calculate the threshold values of six blank food matrices. The assay was validated using sildenafil, producing an IC 50 of 4.2 nM. The applicability of the assay procedure was demonstrated by screening 55 distinct food samples. The results were subsequently verified using confirmatory liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis. Altogether, 49 samples inhibited the PDE5 enzyme above the threshold values (75.7%-105.5%) and were registered as potentially adulterated samples. The remaining six samples were marked as nonadulterated with percentage inhibition below the threshold values (-3.3%-18.2%). The LC-HRMS analysis agreed with the assay results for all food products except for the instant coffee premix (ICP) samples. False-positive results were obtained for the ICP samples at 32% (8/25), due to possible PDE5 inhibition by caffeine. Contrarily, all other food samples were found to produce 0% (0/30) false-positive or false-negative results. The broad-based assay, established via a simple mix-incubate-read format, exhibited promising potential for high-throughput screening of PDE5 inhibitors in various food products, except those with naturally occurring phosphodiesterase inhibitors such as caffeine., (© 2020 John Wiley & Sons, Ltd.)

The presence of erectile dysfunction (ED) drugs in adulterated dietary supplements, mainly in pharmaceutical dosage forms, is frequently addressed in the literature. Little attention is given to food products despite their increasing adulteration trend. To address this knowledge gap targeted, suspected-target, and non-targeted strategies were utilised to analyse ED drugs and their analogues in powdered drink mix (PDM), honey, jelly, hard candy, and sugar-coated chewing gum using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). The method was optimised and validated using 23 target analytes, representing different ED drugs with structural similarities. The modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction exhibited insignificant matrix effect (ME) within - 9.2-8.8% and provided complete coverage of target analytes with acceptable extraction recovery (RE) within 75.5-123.9%, except for carbodenafil in the PDM matrix. Based on the ME and RE performance, the analytical method was validated to analyse 25 food samples that claimed to enhance male sexual performance. The method exhibited good specificity and linearity with a limit of detection within 10-70 ng/mL and limit of quantification of 80 ng/mL. Similarly, the accuracy and precision were satisfactory within 77.4-122.0% and< 16.7%RSD, respectively. The LC-HRMS targeted analysis, together with suspected-target and non-targeted screenings, identified and detected ten ED drugs from 24 food samples. The modified QuEChERS extraction with LC-HRMS-based method was demonstrated to be universally applicable to various food products, covering an extensive range of known and potentially novel ED drugs, which is valuable for routine casework., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The lucrative market of herbal remedies spurs rampant adulteration, particularly with pharmaceutical drugs and their unapproved analogues. A comprehensive screening strategy is, therefore, warranted to detect these adulterants and, accordingly, to safeguard public health. This study uses the data-dependent acquisition of liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS) to screen phosphodiesterase 5 (PDE5) inhibitors in herbal remedies using suspected-target and non-targeted strategies. For the suspected-target screening, we used a library comprising 95 PDE5 inhibitors. For the non-targeted screening, we adopted top-down and bottom-up approaches to flag novel PDE5 inhibitor analogues based on common fragmentation patterns. LC-QTOF-MS was optimised and validated for capsule and tablet dosage forms using 23 target analytes, selected to represent different groups of PDE5 inhibitors. The method exhibited excellent specificity and linearity with limit of detection and limit of quantification of <40 and 80 ng/mL, respectively. The accuracy ranged from 79.0% to 124.7% with a precision of <14.9% relative standard deviation. The modified, quick, easy, cheap, effective, rugged, and safe extraction provided insignificant matrix effect within -9.1%-8.0% and satisfactory extraction recovery of 71.5%-105.8%. These strategies were used to screen 52 herbal remedy samples that claimed to enhance male sexual performance. The suspected-target screening resulted in 33 positive samples, revealing 10 target analytes and 2 suspected analytes. Systematic MS and tandem MS interrogations using the non-targeted screening returned insignificant signals, indicating the absence of potentially novel analogues. The target analytes were quantified from 0.03 to 121.31 mg per dose of each sample. The proposed strategies ensure that all PDE5 inhibitors are comprehensively screened, providing a useful tool to curb the widespread adulteration of herbal remedies., (©2020 John Wiley & Sons, Ltd.)

Objectives: The present work aims to develop and validate a simple, rapid, cost-effective, sensitive and extractive spectrophotometric methods for the determination of phosphodiesterase type 5-inhibitor; vardenafil HCl (VARD) in pure and in dosage forms., Methods: The developed methods are based on the formation of ion-pair complexes between vardenafil HCl and dyes, namely, bromocresol green (BCG), bromocresol purple (BCP), bromophenol blue (BPB), bromothymol blue (BTB) and eriochrom black T (EBT) in acidic buffer solutions. Different factors affecting the reactions between VARD and the dyes were studied and optimized., Results: The formed complexes were extracted with methylene chloride and measured at 418, 410, 415, 417 and 520nm using BCG, BCP, BPB, BTB and EBT, respectively. The beer's law was obeyed in the ranges 1.0-10, 1.0-16, 0.5-8.0, 2.0-20 and 1.0-14μgmL -1 for BCG, BCP, BPB, BTB and EBT, respectively under the optimum conditions. The composition of the ion-pairs was found 1:1. The molar absorptivity's, Sandell's sensitivity, limits of detection and the limits of quantification were calculated. Other method validation parameters, such as accuracy, intra-day and inter-day precision, robustness, ruggedness and selectivity, have been evaluated., Conclusion: The proposed methods have been applied successfully for the analysis of vardenafil HCl in pure and dosage forms. The reliability of the methods was further ascertained by performing recovery studies using the standard addition method. Statistical comparison of the results with the reported method was performed by applying student's t- and F-tests and no significant statistical differences were obtained., (Copyright © 2020 Académie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.)

With the rise in consumption of dietary supplements for various ailments such as erectile dysfunction (ED), there is concern that these supplements may contain illegally added phosphodiesterase type 5 (PDE-5) inhibitor and its analogs. HPLC or LC is a general separation method, and MS is a detection technique, together LC/MS/MS technology provides the mass spectral confirmation in identifying sildenafil, vardenafil, tadalafil and their analogs. In our present study, a sample extraction technique with 1:1 acetonitrile: water solvents and sonication was used for screening, then identification was performed using an LC coupled with Velos Pro linear ion trap mass spectrometry. This was a simple and reliable method for a variety of matrices of dietary supplements and pharmaceutical formulations in tablet, capsule or liquid form. The run time is only 6.5   min, allowing for a quick screening and identification of all of analytes of ED drugs using full scan and data-dependent scan MS/MS, except for tadalafil and aminotadalafil (MS/MS/MS). To conclude this study, Sildenafil, tadalafil, vardenafil, and other 16 analogs in dietary supplements could be quickly screened and identified by HPLC coupled with ion trap MS using data dependent scanning function. The main method using the short column is very rapid, and saves a lot of running time and solvents, and the identification is further confirmed by MS/MS information. The current study develops and validates a quick and reliable method to screen for ED drugs.

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(3 H )-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 IC 50 value of the inhibitory activity for PDE5A1 (one of the PDE5 isoforms) was 2.0 nM (sildenafil IC 50 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).

Illegal dietary supplements adulterated with phosphodiesterase type 5 inhibitors (PDE-5i) are increasingly widely distributed through internet markets and underground routes. For this reason, it demands development of reliable screening methods to determine a wide range of PDE-5i drugs in various types of dietary supplements. Herein, we developed a screening method using gas chromatography-mass spectrometry (GC-MS) for simultaneous detection of 53 PDE-5i drugs in supplements. Common formulations (such as capsule, powder, pill, and tablet) of supplements with complicated matrices were treated by simple liquid-liquid extraction and trimethylsilyl (TMS) derivatization. With the aid of TMS derivatization, 53 PDE-5i drugs could be successfully separated and detected within 15 min, using a short microbore GC column (15 m). Moreover, owing to enhanced detection sensitivity and selectivity of PDE-5i TMS derivatives, 0.5 mg of sample was sufficient to screen and confirm targeted PDE-5i drugs. In this study, specific common ions according to structural characteristics of PDE-5i drugs were found under the electron ionization (EI) of their TMS derivatives. These specific common fragments could reflect the common pharmacophores for 4 classes of PDE-5i drugs (sildenafil, other sildenafil, vardenafil, and tadalafil analogues). Based on characteristic EI fragment ions, extracted common ion chromatograms (ECICs) and discriminant analysis (DA) were effectively used for reliable screening and classification of various types of PDE-5i drugs. Specific ECICs and DA using characteristic EI fragments here will aid in identification of newly emerging PDE-5i counterfeits in supplements. This study will be helpful to supervise illegal adulteration of PDE-5i drugs in dietary supplements to protect public health and consumer safety., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 MS n spectral library. The developed method was successfully applied for screening adulterated dietary supplement samples.

Phosphodiesterase-5 (PDE-5) inhibitors and endothelin receptor antagonists (ERAs) are standard therapies for pulmonary arterial hypertension (PAH). The inter-individual variability of these pharmacokinetics is reported remarkably large, and therapeutic drug monitoring (TDM) can be useful to improve the likelihood of the desired therapeutic and safety outcomes. This study aimed to develop a LC-MS method to determine the concentrations of five PAH drugs (PDE-5 inhibitors: sildenafil and tadalafil, ERAs: bosentan, macitentan, and ambrisentan) from plasma samples using a simple process followed by a single mass spectrometric run, and to validate this approach through pharmacokinetic analyses in patients. A solid extraction method was used for sample preparation of the drugs from human plasma. The total run time for a single injection was within 10 min. The calibration curves for all drugs were linear, and the lower limits of quantitation were 1 (sildenafil), 2 (tadalafil), 5 (ambrisentan), and 10 ng/mL (bosentan, macitentan). The accuracy and precision values suggested that the assay had high accuracy and reliability. To prove the utility of this method, the plasma concentrations of the five PAH drugs were determined after their oral administration to nine PAH patients.

A novel combination of tamsulosin hydrochloride and tadalafil is recently available for treatment of benign prostatic hyperplasia and erectile dysfunction. For the first time, four simple, accurate, smart and robust spectrophotometric methods have been suggested for their simultaneous quantification. The methods, namely; first derivative, ratio difference, derivative ratio and mean centering of ratio spectra, successfully resolved the spectral overlap of their challenging binary mixture. Calibration curves were linear at 2.0-40.0 and 2.0-55.0 μg/mL for tamsulosin hydrochloride and tadalafil, respectively. The methods were validated according to ICH guidelines and statistically compared with the official ones, revealing no considerable difference with respect to accuracy and precision. Specificity of the developed methods was assessed by evaluating various laboratory prepared mixtures. Furthermore, the methods were successfully applied for the quantification of the two drugs in their combined dosage form., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 IC 50 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.

As a widely consumed beverage, coffee tends to be a target for intentional adulteration. This study describes the application of modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) coupled to liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for simultaneous screening, identification, and quantification of undeclared phosphodiesterase 5 (PDE5) inhibitors in instant coffee premixes (ICPs). The mass spectrometer was operated in auto MS/MS acquisition for simultaneous MS and MS/MS experiments. Qualitative establishments from the suspected-target screening and targeted identification processes led to an unambiguous analyte assignment from the protonated molecule ([M+H] + ) precursor ion which is subsequently used for quantification of 23 targeted PDE5 inhibitors. The analytical method validation covered specificity, linearity, range, accuracy, limit of detection (LOD), limit of quantification (LOQ), precisions, matrix effect (ME), and extraction recovery (RE). The specificity was established using the optimised chromatographic separation as well as the distinguishable [M+H] + precursor ion. The linearity of each target analyte was demonstrated with a coefficient of determination (r 2 ) of >0.9960 over the expected range of sample concentrations. The accuracy ranged from 88.1%-119.3% with LOD and LOQ of <70 ng/mL and 80 ng/mL, respectively. Excellent precisions were established within 0.4%-9.1% of the relative standard deviation. An insignificant ME within -5.2% to +8.7% was achieved using three different strategies of chromatography, sample extraction, and sample dilution. The RE was good for all target analytes within 84.7%-123.5% except for N-desethylacetildenafil at low (53.8%) and medium (65.1%) quality control levels. The method was successfully applied to 25 samples of ICPs where 17 of them were found to be adulterated with PDE5 inhibitors and their analogues. Further quantification revealed the total amount of these adulterants ranged from 2.77 to 121.64 mg per sachet., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In this paper, we propose a novel framework to select the most relevant X-Ray Fluorescence (XRF) energy values (i.e., features) to enhance the clustering (grouping) of counterfeit and illicit medical tablets. The framework is based on the integration of multidimensional scaling (MDS) and Procrustes analysis (PA) multivariate techniques. MDS provides a projection of the original data into a lower dimension, while PA finds a projection matrix from the original data. Such outputs give rise to a feature importance index that guides an iterative feature selection process; after each feature is inserted in the subset, an optimization procedure based on a greedy search method is carried out to maximize the clustering quality assessed through the Silhouette Index (SI). The inorganic chemical fingerprinting of 41 commercial samples (Viagra®, Cialis®, Lazar®, Libiden®, Maxfil®, Plenovit®, Potent 75®, Rigix®, V-50®, Vimax® and Pramil®) and 56 seized counterfeit samples (Viagra and Cialis) was used to validate the proposed framework. From the original 2048 data points in the full spectra, we identified a subset comprised of 41 energy values that substantially improved clustering quality; the obtained groups were assessed by visual inspection of the PCA plots., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The introduction of sildenafil (SDF) to treat erectile dysfunction has solved a widespread condition with negative on the quality of life. Recently, the co-administration of tramadol (TMD) with SDF to manage premature ejaculation has illegally increased and thus drug-drug interaction studies of these drugs became of great importance. Although certain biological functions have been altered upon co-administration of the two drugs, methods for their determination in vivo to understand their interactions have yet to be published. Herein, therefore, an HPLC method with photometric detection was developed for the determination of a binary mixture of TMD and SDF in rabbit plasma after oral administration. In this study, a reversed-phase chromatography was performed at room temperature on a C 18 column with a mobile phase composed of 10 mM Na 2 HPO 4 solution (pH 7.5): acetonitrile (45:55, v/v) at a flow rate of 0.8 mL min -1 using caffeine (CAF) as an internal standard. The detector was set at 220 nm. The total analysis time was 6 min. Calibration graphs were linear in the concentration ranges of 0.1-10 and 0.05-10 μg mL -1 with a detection limit of 0.05 and 0.02 μg mL -1 for TMD and SDF, respectively. The method was validated in terms of accuracy, precision, limit of detection and quantitation, recovery, and stability as per US FDA bioanalytical guidelines. In addition, the metabolites N-desmethylsildenafil (UK-103,320) and O-desmethyltramadol were quantified in rabbit plasma after 2 h of oral administration using LC-MS/MS. The simultaneous administration of TMD with SDF has affected peak plasma concentration (C max ), T max , area under the concentration-time curve (AUC), and the elimination rate constant (K el ) of SDF. The present study is the first to give valuable insights into the drug-drug interaction and the pharmacokinetic implications associated with the co-administration of SDF and TMD., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The worldwide spread of illegal sexual enhancement products is posing a threat to public health. The aim of this study was to investigate illegal products claiming to be effective in improving sexual performance through the online or offline markets between 2014 and 2017; these products include foods, dietary supplements, counterfeit drugs, and herbal medicines. These samples were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the presence of 80 PDE-5 inhibitors (PDE-5i) and analogues. The developed method was validated as follows: LODs and LOQs spiked in solid- and liquid-type negative samples (0.03-3.33 ng/mL and 0.08-10.00 ng/mL), linearities (R 2  > 0.997), recoveries spiked negative samples (82.2-109.3 %), accuracies (81.6-118.9 %), precisions (≥ 6.5%, RSD) of intra-day and inter-day, and stability (≥10.0%, RSD). Out of 362 measured samples, 145 were adulterated samples mostly detected in food (51%). Sildenafil group (50%) was frequently observed, followed by tadalafil group (41%). Although sildenafil and tadalafil were mainly detected in adulterated samples, their analogues were also found. In particular, new analogues have appeared steadily on illicit erectile dysfunction (ED) products even after they were first discovered. The concentration of detected samples ranged from 0.1 to 826.0 mg/g, and sildenafil of them contained a considerable amount in illicit ED products in 2014, posing a potential toxicology risk of public health. The testing method is fast and reliable making it suitable for both routine screening and up-to-date quantitative analysis of PDE-5i and their analogues in suspicious foods, dietary supplements, and counterfeit drugs., (Copyright © 2019. Published by Elsevier B.V.)

A novel method for hierarchical screening of illegal adulterants in Fur seal ginseng pills products was developed by multi-dimensional fingerprint profiling analysis. Fingerprint feature of the samples was acquired by high-performance liquid chromatography analysis of 11 batches of samples with diode array detector and fluorescence detector, and then potential illegal adulterants including phosphodiesterase type-5 inhibitors, androgens, α receptor antagonists and yohimbine, were further separated at multiple wavelengths to reduce or remove interferences from sample matrix for highlight their chromatographic characteristics. Accordingly, a hierarchical screening strategy was designed by first-order and second-order fingerprints combined with spectral examination to achieve high accuracy and reliability. The method was successfully applied to screening of illegal adulterants in real samples, and it also exhibited good quantification performance through validation tests. From 16 batches of samples, three suspected samples were confirmed to be positive, containing 9.37μg/g of testosterone, 18.8 μg/g of tadalafil, and 48.5 μg/g of sildenafil, respectively. The recoveries and relative standard deviations were in the range of 83.6-103.1% and 4.2-6.8%, respectively. The proposed method provided a simple, efficient and promising alternative to monitoring functional foods., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The practice of drug counterfeiting is an important challenge due to the extremely rapid growth rate of this disturbing trend and its immense potential harmfulness. According to WHO even 10% of counterfeit medicines can be exact copies of genuine medicines: they have exactly the same quantitative and qualitative composition in terms of both API and excipients. Thus, the identification of such drugs using chemical analysis methods can be very difficult or even impossible. The aim of this study was to verify the effectiveness of using computed microtomography in the identification of counterfeit medicines. The General Electric v|tome|x s microtomography system was used in the study. The recorded microtomographic scans were subjected to analysis and image processing. The following parameters of image analysis and processing were identified: mean brightness, homogeneity, contrast, quadratic tree decomposition. The original and falsified 100-mg Viagra® tablets (Pfizer) were compared. 8 original Viagra® tablets (hereinafter referred to as T) and 8 falsified tablets (hereinafter referred to as F1-F8) were tested. The range of variation for the genuine medicines against fake products was: brightness: 90.9-117.1 vs 33.8, 50.1, homogeneity: 0.84-0.92 vs 0.94-1.01 and quadratic tree decomposition for the 1 × 1 mask: 55768-58792 vs 0-439. The proposed method of microtomographic image analysis and processing enables to identify solid dosage forms, including those that are an accurate chemical copy, with high sensitivity and specificity, 94.5% and 97%, respectively. The advantage of the μCT method is its high efficiency and speed, whereas the disadvantages include the possibility of using only solid dosage forms and high equipment costs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Erectile dysfunction medicines such as Cialis and Viagra are very popular worldwide and are between the most prevalent counterfeit medicines in Brazil. A range of analytical methods has been used to analyze Cialis and Viagra, such as ATR-FTIR, GCMS and UPLC-MS. Until now, there are no data available of DSC methods for analysis of counterfeit medicines of Cialis and Viagra. DSC is a thermal analysis that provides useful information of physico-chemical events, and however is almost not used for forensic purposes. In this study, thermal analysis of 25 counterfeit Viagra and Cialis seized by Brazilian Federal Police were performed by DSC and compared to their authentic medicines and analytical standards, along with chemometric tools. Authentic samples of Viagra displayed a similar thermal profile with the API, while Cialis were different with additional endothermic peaks, that could be related to excipients interference. Thermograms of Viagra counterfeit samples were similar to authentic samples, while Cialis showed an enlargement and displacement of endothermic peaks. Also, some Cialis counterfeit samples showed melting peaks attributed to sildenafil, the API of Viagra, instead tadalafil, confirming previous results obtained by UPLC-MS. Multivariate analysis with application of Hierarchical Cluster Analysis classified different groups of samples, including a cluster with counterfeit Cialis and Viagra, indicating the use of same API for both counterfeit medicines and possibly the same illicit production; and a cluster with authentic Viagra and counterfeit Cialis, confirming the addition of sildenafil instead tadalafil to Cialis counterfeit samples. Here for the first time we described the use of DSC for chemical profiling of Cialis and Viagra and showed that even when applied to a small group of samples, DSC along with chemometric tools can be considered as a good auxiliary method in forensic casework samples. DSC provided useful data to perform the identification of counterfeit and authentic medicines, with low cost and a simple method., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Popularity of natural-based preparations supporting the sexual potency significantly increased in recent years, which also led to the increase of illegal use of phosphodiesterase type 5 inhibitor (PDE-5) in sexual performance enhancement products. In this study, a rapid U-HPLC‒HRMS/MS method has been developed to simultaneously determine 59 PDE-5 inhibitors and their analogues. Within the development of sensitive method for analysis of 59 PDE-5 inhibitors and their analogues, both sample preparation procedure, as well as separation / detection conditions have been optimized. Extraction efficiency of particular extraction solvents, influence of different mobile phase additives on target analytes separation, as well as impact of various settings of mass analyzer on sensitivity of detection were examined. Data were collected in the 'full MS/data dependent MS/MS' acquisition mode (full MS-dd-MS/MS). Before the U-HPLC‒HRMS/MS method was used for analysis of real samples, proper validation had been conducted. The precision of the method expressed as the relative standard deviation (RSD) was ≤4.2% and ≤5.2% at spiking concentrations 5 μg/g and 0.25 μg/g, respectively. The limits of quantification were in the range 0.25 - 0.05 μg/g and the recovery ranged between 71 and 90%. The optimized method was successfully applied for analysis of 64 real samples, and 10 of them were proved to contain both registered or unregistered synthetic PDE-5 inhibitors. Additionally, the acquired U-HPLC‒HRMS/MS fingerprints were demonstrated to serve as an efficient tool for revealing of other type of possible fraud in products labeling. Retrospective mining of markers of herbs declared on dietary supplements packaging allowed to assess the trueness / untruth in the declaration of medical herbs composition., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

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 MS 3 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.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The presence of illegally adulterated sildenafil and related compounds in dietary supplements was qualitatively analyzed by employing ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Samples were extracted by ultrasonic method using methanol. The analysis was performed on an Agilent Poroshell 120 SB-C18 column (75 mm×3.0 mm, 2.7 μm) using gradient elution with a mixture of aqueous formic acid solution (0.1%, v/v) and acetonitrile as the mobile phase at a flow rate of 0.4 mL/min. Q-TOF-MS equipped with ESI ion source was operated in a positive ionization mode. Qualitative analysis was based on the retention time and the accurate relative molecular mass of elemental compositions of the precursor ion and product ions. The limits of detection (LODs) of sildenafil and other 99 compounds were found to be 0.1-50 mg/kg or 0.1~50 mg/L in 10 different matrices. In this manuscript, the fragmentation behavior of 98 phosphodiesterase-5 inhibitors are categorically summarized, which can provide information for referencing and identification of suspicious compounds. The method was successfully applied to the analysis of actual samples, of which 19 compounds combated the illegal adulteration behavior effectively.

A novel "Prediction and Confirmation" (PC) strategy was proposed for characterizing phosphodiesterase-5 inhibitor (PDE-5) derivatives in botanical dietary supplements (BDSs) for on-site detection. Discovery Studio (DS) and density functional theory (DFT) calculations were used for the "Prediction" step in order to estimate PDE-5 derivative structures and theoretical Raman shifts without synthesizing the derivatives. After 11 potentially bioactive sildenafil derivatives were acquired through DS, 32 common calculated Raman shifts were obtained through DFT. The mean absolute wavenumber deviation (δ, peak range) of the major bands and the minimum number (τ) of Raman spectral peaks matching the calculated common shifts were optimized, so that a positive result of an unknown sample could be reasonably produced. In this study, δ was set at ±10 cm -1 and the corresponding τ was set at 4-5 after optimization. Surface plasmon resonance (SPR) biosensor and surface-enhanced Raman scattering (SERS) detection were the "Confirmation" step to validate the reliability and accuracy of DS and DFT in the "Prediction" step, respectively. The optimized δ and τ criteria were used as indexes for on-site SERS detection after thin-layer chromatographic (TLC) separation of six real-world samples, one of which was preliminarily identified as "suspected positive samples." This strategy allows for a quick determination of the BDSs adulterated with sildenafil or its derivatives, independent of any standard materials.

The commerce of falsified drugs has substantially grown in recent years due to facilitated access to technologies needed for copying authentic pharmaceutical products. Attenuated Total Reflectance coupled with Fourier Transform Infrared (ATR-FTIR) spectroscopy has been successfully employed as an analytical tool to identify falsified products and support legal agents in interrupting illegal operations. ATR-FTIR spectroscopy typically yields datasets comprised of hundreds of highly correlated wavenumbers, which may compromise the performance of classical multivariate techniques used for sample classification. In this paper we propose a new wavenumber interval selection method aimed at selecting regions of spectra that best discriminate samples of seized drugs into two classes, authentic or falsified. The discriminative power of spectra regions is represented by an Interval Importance Index (III) based on the Two-Sample Kolmogorov-Smirnov test statistic, which is a novel proposition of this paper. The III guides an iterative forward approach for wavenumber selection; different data mining techniques are used for sample classification. In 100 replications using the best combination of classification technique and wavenumber intervals, we obtained average 99.87% accurate classifications on a Cialis ® dataset, while retaining 12.5% of the authentic wavenumbers, and average 99.43% accurate classifications on a Viagra ® dataset, while retaining 23.75% of the authentic wavenumbers. Our proposition was compared with alternative approaches for individual and interval wavenumber selection available in the literature, always leading to more consistent and easier to interpret results., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Chinese tonic liquor is an important dietary supplement in daily use, but it often happens that illicitly adulterated drugs in Chinese tonic liquor could lead to food safety issues. In this survey, an analytical method consisting of a liquid chromatography-quadrupole-time of flight mass spectrometer (LC-Q-TOF-MS), coupled with quick easy cheap effective rugged safe (QuEChERS) pretreatment, was established for identification of phosphodiesterase type 5 enzyme (PDE-5) inhibitors in Chinese tonic liquors. 86 PDE-5 inhibitors were qualitatively analysed by employing information dependent acquisition mass spectrometry (IDA-MS) in comparison with the accurate mass of the protonated molecular ion, isotopic pattern, library and chemical formula. This method was used to test 28 Chinese tonic liquor samples. The results revealed that the IDA-MS screening method is suitable for qualitative analysis of 86 PDE-5 inhibitors. Four samples were found to be adulterated with sildenafil, tadalafil, vardenafil, isopiperazinoafil, nortadalafil and desmethylsidenafil, which was 14.3% of the tested samples.

In this work a step forward in investigating the use of prescription drugs, namely erectile dysfunction products, at European level was taken by applying the wastewater-based epidemiology approach. 24-h composite samples of untreated wastewater were collected at the entrance of eight wastewater treatment plants serving the catchment within the cities of Bristol, Brussels, Castellón, Copenhagen, Milan, Oslo, Utrecht and Zurich. A validated analytical procedure with direct injection of filtered aliquots by liquid chromatography-tandem mass spectrometry was applied. The target list included the three active pharmaceutical ingredients (sildenafil, tadalafil and vardenafil) together with (bio)transformation products and other analogues. Only sildenafil and its two human urinary metabolites desmethyl- and desethylsildenafil were detected in the samples with concentrations reaching 60 ng L -1 . The concentrations were transformed into normalized measured loads and the estimated actual consumption of sildenafil was back-calculated from these loads. In addition, national prescription data from five countries was gathered in the form of the number of prescribed daily doses and transformed into predicted loads for comparison. This comparison resulted in the evidence of a different spatial trend across Europe. In Utrecht and Brussels, prescription data could only partly explain the total amount found in wastewater; whereas in Bristol, the comparison was in agreement; and in Milan and Oslo a lower amount was found in wastewater than expected from the prescription data. This study illustrates the potential of wastewater-based epidemiology to investigate the use of counterfeit medication and rogue online pharmacy sales., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Recently, illegal sildenafil analogues have emerged, causing serious social issues. In spite of the importance of sildenafil analogues, their metabolic profiles or clinical effects have not been reported yet. In this study, new metabolites of illegal sildenafil analogues such as hongdenafil, homosildenafil, and hydroxyhomosildenafil were determined using liquid chromatography quadrupole-time of flight mass spectrometry (LC-Q-TOF-MS) and tandem mass spectrometry (LC-Q-TOF-MS/MS). To prepare metabolic samples, in vitro and in vivo studies were performed. For in vivo metabolites analysis, urine and feces samples of rats treated with sildenafil analogues were analyzed. For in vitro metabolites analysis, human liver microsomes incubated with sildenafil analogues were extracted and analyzed. All metabolites were characterized by LC-Q-TOF-MS and LC-Q-TOF-MS/MS. As a result, five, six, and seven metabolites were determined in hongdenafil, homosildenafil, and hydroxyhomosildenafil treated samples, respectively. These results could be applied to forensic science and other analytical fields. Moreover, these newly identified metabolites could be used as fundamental data to determine the side effect and toxicity of illegal sildenafil analogues., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: Due to the chaos in the legislation in the Middle East, male enhancement nutraceuticals may be sold without any registration or evaluation. These products need to be evaluated with respect to safety and efficacy. Furthermore, cultural and social considerations in the Middle East prevent the use of international evaluations schemes for erectile dysfunction., Aim: Evaluating the safety and efficacy parameters of generic and nutraceutical products for erectile dysfunction in the Middle East through a custom-designed, representable and simple system tailored to the regional culture., Methods: 74 healthy male volunteers were enrolled into a comparative, simple randomized, single dose, double blind, and crossover clinical study incorporated with a tailored-designed questionnaire. Safety assessment included laboratory analysis for liver functions and measuring blood pressure., Main Outcome Measures: Subjective data regarding safety and efficacy were assessed from the validated questionnaire. Blood pressure was measured. Blood samples were collected to assess the drug/adulterants concentration and liver and kidney functions., Results: All tested nutraceuticals showed undeclared Sildenafil citrate in patients. Questionnaire results showed high inter-patient variability with respect to efficacy and comparable safety profile compared to Viagra®., Conclusion: The validated tailored-designed questionnaire effectively assessed the efficacy and safety of male enhancement products. The male enhancement nutraceuticals, sold in Egypt, claimed to be 100% natural are adulterated and of questionable safety profile., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

High-performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS 2 ) with a triple quadrupole is proposed for determining the synthetic phosphodiesterase-5 inhibitors, sildenafil, tadalafil and vardenafil, and the active metabolite N-desmethyl sildenafil. The method was successfully applied to the analysis of waters of different origins and human urine samples. Dispersive liquid-liquid microextraction (DLLME) was applied in the classical way for water analysis, whereas a previous extraction into an organic solvent was necessary for urine samples, the acetonitrile extract being used as dispersant solvent in the DLLME step. The analytes were determined by LC-ESI-MS 2 in the multiple reaction monitoring mode. Detection limits were in the 5-50 and 15-250ngL -1 ranges for water and urine samples, respectively. The repeatability was calculated using the relative standard deviation, obtaining values of between 3.6% and 10.1%. The enrichment factors were between 75 and 81. Accuracy of the procedure was calculated through recovery assays and average recoveries ± SD (n = 48) of 93.6 ± 3.5 and 91.1 ± 3.5 were obtained for water and urine samples, respectively. None of the samples analyzed contained the target compounds, at least above the corresponding detection limits., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Adulteration of herbal health supplements with phosphodiesterase-5 (PDE-5) inhibitors and their analogues is becoming a worldwide problem. The aim of this study was to investigate herbal and food products sold in the Malaysian market for the presence of these adulterants. Sixty-two products that claim to enhance men's sexual health were sampled between April 2014 and April 2016. These products included unregistered products seized by the Pharmacy Enforcement Division of the Ministry of Health (n = 39), products sent to the National Pharmaceutical Regulatory Agency for pre-registration testing (n = 9) and products investigated under the post-registration market surveillance programme (n = 14). The products were tested against an in-house spectral library consisting of 61 PDE-5 inhibitors and analogues using a validated liquid chromatography-mass spectrometry ion-trap-time-of-flight (LC-MS IT-TOF) method. Thirty-two (82%) of the unregistered products and two (14%) of the registered products were found to be adulterated with at least one PDE-5 inhibitor or analogue, while none of the pre-registration products contained adulterants. A total of 16 different adulterants were detected and 36% of the adulterated products contained a mixture of two or more adulterants. This study has demonstrated that the adulteration of unregistered herbal products in the Malaysian market is an alarming issue that needs to be urgently addressed by the relevant authorities.

A facile method based on electrospray mass spectrometry was established and validated for the differentiation of enantiomeric tadalafil isomers without using chiral chromatographic separation. The enantiomers were coupled with a chiral selector to form diastereomeric complex ions. Nickel-tadalafil complexes, [Ni II (tadalafil)(l-Trp)-H] + , produced a characteristic fragment ion at m/z 524 by loss of 1-methyl-1,6-dihydropyrazine-2,5-dione via collision-induced dissociation. The relative abundance of this fragment ion to the precursor contributed to differentiate tadalafil enantiomers, and energy-resolved product-ion spectra were applied to determine the molar composition of tadalafil in the mixture (R,R and S,S) as well. In addition, the other two forms of stereomeric isomers of tadalafil (R,S and S,R) could be also distinguished and analyzed by this method. The method was validated in different types of mass spectrometers (AB quadrupole time-of-flight and Bruker ion trap) and also verified by a chiral high-performance liquid chromatography coupled with quadrupole time-of-flight. The chiral determination of tadalafil using MS method proved to be rapid (1-min run time for each sample) and to have the same accuracy and precision comparable to chiral liquid chromatography mass spectrometry methods. This method provides an alternative to commonly used chromatographic technique for chiral determination and is particularly useful in rapid screening in enantioselective synthesis and enantiomeric impurity detection in pharmaceutical industry. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

A novel facile method for the detection of the phosphodiesterase type 5 enzyme inhibitors added illegally into health products was established using thin-layer chromatography and surface enhanced Raman spectroscopy combined with BP neural network. When the detection conditions were optimized in detail, a repetitive adding procedure of silver colloids with the total amount keeping constant was used to improve the enhancement effect of surface enhanced Raman spectroscopy. According to the main Raman peaks and the retention factor of analyte, the data predictive model was established. Under the optimized experimental conditions, this method was successful to apply to detect the artificially produced model samples, and the limit of detection less than 5 mg/kg was obtained. Based on the excellent sensitivity of this method, the real samples have been detected accurately and the detection results were confirmed by high-performance liquid chromatography. In addition, the developed method was suitable for the detection of other adulterants, especially those that have similar chromatographic or spectroscopic behaviors., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The sildenafil analogue adulterant previously identified as a nitroso derivative (nitrosoprodenafil) in a dietary supplement (DS) marketed to increase sexual performance and sold in Europe in the early 2010s is the same as that found in the same type of DS available in Japan whose structure was established as a nitro derivative (mutaprodenafil or nitroprodenafil). Indeed, the compound isolated from the Man Power DS has identical UV, IR, NMR and MS spectroscopic characteristics and hydrolysis behavior than nitrosoprode-nafil. By revisiting its NMR assignments and MS and MS/MS data interpretation, it is demonstrated that the compound is actually a nitrothioimidazole-methisosildenafil hybrid, i.e. nitroprodenafil, whose structure is unequivocally confirmed by X-ray crystallography and synthesis experiments. Because the product is converted to methisosildenafil by hydrolysis, it is named nitropromethisosildenafil., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Erectile dysfunction prevalence globally is noticeably high. This is accompanied by an increase in the use of nutraceuticals for male enhancement. However, the global market is invaded by counterfeit and adulterated nutraceuticals claimed to be of natural origin sold with a therapeutic claim. The objective of this article is to review male enhancement nutraceuticals worldwide with respect to claim, adulterants, and safety. The definition of such products is variable across countries. Thus, the registration procedures differ as well. This facilitates the manipulation of the process, which leads to widespread adulterated and counterfeit products without control. The tele-advertisement and Internet pharmacies aided the widespread sale of male enhancement nutraceuticals, unfortunately, the spurious ones. Finally, based on literature, most of these products were found to be adulterated with active pharmaceutical ingredients (API) and mislabeled as being natural. These products represent a major health hazard for consumers due to lack of clear regulations.

A sensitive analytical method was developed for the simultaneous determination of sildenafil and tadalafil in legal drugs, illicit/counterfeit drugs, and wastewater samples. Chromatographic separation of two analytes was achieved on a C18 column with a mobile phase including 50 mM phosphate buffer at pH 6.0 and acetonitrile (35 + 65, v/v) at the flow rate of 1.0 mL/min. Analytes were separated from each other in 6 min with high resolution. LOD/LOQ values were calculated as 28/92 ng/mL for sildenafil citrate and 39/129 ng/mL for tadalafil. Calibration plots for both analytes were linear with correlation coefficients >0.9993. A validated method was successfully applied to legal and illicit erectile-dysfunction drug samples consumed in Istanbul, Turkey, and to wastewater samples. Nine different samples were analyzed for qualitative and quantitative measurement of their ingredients, and the results were compared with the values written on the labels of the drugs. The wastewater sample was also analyzed for its sildenafil and tadalafil content. To calculate the recoveries, a spiking experiment was performed and recovery rates for sildenafil and tadalafil were calculated as 101.30 ± 3.43 and 102.68 ± 1.59, respectively.

This work presents a simple, sensitive and generic high-performance liquid chromatography with diode array detection method for the simultaneous determination of seven drugs prescribed for the treatment of erectile dysfunction and premature ejaculation. Investigated drugs include the phosphodiesterase-5 inhibitors: sildenafil, tadalafil, and vardenafil, in addition to the selective serotonin reuptake inhibitors: dapoxetine, duloxetine, fluoxetine, and paroxetine. The drugs were separated using a Waters C8 column (4.6 × 250 mm, 5 μm) with the mobile phase consisting of phosphate buffer pH 3, acetonitrile and methanol in the ratio 60:33:7. The flow rate was 1.2 mL/min, and quantification was based on measuring peak areas at 225 nm. Peaks were perfectly resolved with retention times 3.3, 3.9, 6.4, 7.5, 9.5, 10.7, and 13.4 min for vardenafil, sildenafil, paroxetine, duloxetine, dapoxetine, fluoxetine, and tadalafil, respectively. The developed method was validated with respect to system suitability, linearity, ranges, accuracy, precision, robustness, and limits of detection and quantification. The proposed method showed good linearity in the ranges 5-500, 2-200, 2-200, 3-300, 1.5-150, 2-200, and 2-200 μg/mL for sildenafil, tadalafil, vardenafil, dapoxetine, duloxetine fluoxetine, and paroxetine, respectively. The limits of detection were 0.18-0.38 μg/mL for the analyzed compounds. The applicability of the proposed method to real life situations was assessed through the analysis of commercial tablets, and satisfactory results were obtained., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To evaluate the distribution of a daily phosphodiesterase type 5 inhibitor dose (mirodenafil) in rat plasma and bladder and prostate tissue in a model of atherosclerosis-induced chronic pelvic ischemia., Methods: Thirty-two 18-week-old male Sprague Dawley rats were divided into two groups. Group I (n = 16) comprised a chronic pelvic ischemia model treated with mirodenafil and group II (n = 16) comprised a sham-operated model also treated with mirodenafil. The mirodenafil concentrations in each organ were measured at specific time points after 14 days of daily mirodenafil administration. The drug distribution ratio of group I to group II of each organ was measured, and the bladder tissue-to-plasma and prostate tissue-to-plasma ratios were calculated., Results: The mean drug concentration in the bladder of the rats in group I did not differ significantly from that of group II after mirodenafil administration. In the prostate, the mean drug concentration of group I was significantly higher than that of group II at 1 and 4 hours after drug administration. The drug concentration was higher in the bladder tissue than in the prostate tissue and the bladder tissue-to-plasma ratio was significantly higher than the prostate tissue-to-plasma ratio., Conclusion: Our results suggest that mirodenafil levels might be sufficient in the target tissue after daily treatment in an ischemia-induced aging model. Considering the difficulties of tissue distribution study in human subjects, the results of this investigation provided meaningful evidence of the application of daily doses of mirodenafil for treating lower urinary tract symptoms in an aging population., (Copyright © 2016. Published by Elsevier Inc.)

Three approaches for the development of a screening method to detect adulterated dietary supplements, based on Stationary Phase Optimised Selectivity Liquid Chromatography were compared for their easiness/speed of development and the performance of the optimal method obtained. This comparison was performed for a heterogeneous group of molecules, i.e. slimming agents (Part I) and a group of structural analogues, i.e. PDE-5 inhibitors (Part II). The first approach makes use of primary runs at one isocratic level, the second of primary runs in gradient mode and the third of primary runs at three isocratic levels to calculate the optimal combination of segments of stationary phases. In each approach the selection of the stationary phase was followed by a gradient optimisation. For the PDE-5 inhibitors, the group of structural analogues, only the method obtained with the third approach was able to differentiate between all the molecules in the development set. Although not all molecules are baseline separated, the method allows the identification of the selected adulterants in dietary supplements using only diode array detection. Though, due to the mobile phases used, the method could also be coupled to mass spectrometry. The method was validated for its selectivity following the guidelines as described for the screening of pesticide residues and residues of veterinary medicines in food., (Copyright © 2015 Elsevier B.V. All rights reserved.)