UPLC-MS/MS-Based Target Screening of 90 Phosphodiesterase Type 5 Inhibitors in 5 Dietary Supplements.

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.

A novel standard-free detection of adulteration method for sildenafil derivatives in dietary supplements.

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.

Variable temperature-nuclear magnetic resonance experiment and high-resolution MS/MSn measurement of hydroxycarbodenafil, and its PDE5 inhibitory activity.

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 (MSn) 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 IC50 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.

Rapid screening and identification of targeted and non-targeted illegal added drugs in functional foods by MRSIT-HRMS based on NIST screening database.

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.

Analytical methods for the detection and characterization of unapproved phosphodiesterase type 5 inhibitors (PDE-5i) used in adulteration of dietary supplements- a review.

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.

Combining trapped ion mobility spectrometry with liquid chromatography and tandem mass spectrometry for analysis of isomeric PDE-5 inhibitor analogs.

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.

Development of a detector tube for screening tadalafil and its analogues in adulterated sexual enhancement products.

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.

Hierarchical clustering of liquid chromatography-tandem mass spectrometry data for screening of phosphodiesterase type 5 inhibitors and their analogues in adulterated dietary supplements.

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.

Detection of 94 PDE-5is and Their Analogs Including N-Desmethylthiosildenafil in Various Formulations of Dietary Supplements and Food Samples Using HPLC and LC-Q-TOF/MS.

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.

Identification of phosphodiesterase type-5 (PDE-5) inhibitors in herbal supplements using a tiered approach and associated consumer risk.

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 1H-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.

Fast and semiquantitative screening for sildenafil in herbal over-the-counter formulations with atmospheric pressure solid analysis probe (ASAP) to prevent medicinal adulteration.

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.

Liquid chromatography-high-resolution mass spectrometry analysis of erectile dysfunction drugs and their analogues in food products.

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.

Fluorescence polarisation for high-throughput screening of adulterated food products via phosphodiesterase 5 inhibition assay.

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 IC50 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.

Sensitive spectrophotometric determination of vardenafil HCl in pure and dosage forms.

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.

Suspected-target and non-targeted screenings of phosphodiesterase 5 inhibitors in herbal remedies using liquid chromatography-quadrupole time-of-flight-mass spectrometry.

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.

Identification of Erectile Dysfunction Drugs in Dietary Supplements by Liquid Chromatography Ion Trap Mass Spectrometry.

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.

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

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

Reliable screening and classification of phosphodiesterase type 5 inhibitors in dietary supplements using gas chromatography / mass spectrometry combined with specific common ions.

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.

Screening of Phosphodiesterase-5 Inhibitors and Their Analogs in Dietary Supplements by Liquid Chromatography-Hybrid Ion Trap-Time of Flight Mass Spectrometry.

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

Simultaneous LC-MS analysis of plasma concentrations of sildenafil, tadalafil, bosentan, ambrisentan, and macitentan in patients with pulmonary arterial hypertension.

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.