Benchtop NMR Coupled with Chemometrics: A Workflow for Unveiling Hidden Drug Ingredients in Honey-Based Supplements.

Recently, benchtop nuclear magnetic resonance (NMR) spectrometers utilizing permanent magnets have emerged as versatile tools with applications across various fields, including food and pharmaceuticals. Their efficacy is further enhanced when coupled with chemometric methods. This study presents an innovative approach to leveraging a compact benchtop NMR spectrometer coupled with chemometrics for screening honey-based food supplements adulterated with active pharmaceutical ingredients. Initially, fifty samples seized by French customs were analyzed using a 60 MHz benchtop spectrometer. The investigation unveiled the presence of tadalafil in 37 samples, sildenafil in 5 samples, and a combination of flibanserin with tadalafil in 1 sample. After conducting comprehensive qualitative and quantitative characterization of the samples, we propose a chemometric workflow to provide an efficient screening of honey samples using the NMR dataset. This pipeline, utilizing partial least squares discriminant analysis (PLS-DA) models, enables the classification of samples as either adulterated or non-adulterated, as well as the identification of the presence of tadalafil or sildenafil. Additionally, PLS regression models are employed to predict the quantitative content of these adulterants. Through blind analysis, this workflow allows for the detection and quantification of adulterants in these honey supplements.

Isolation and characterization of a novel tadalafil analogue adulterant, N-cyclohexyl nortadalafil, in a dietary supplement.

A novel tadalafil analogue was detected during routine screening of dietary supplements suspected to be adulterated with an erectile dysfunction drug(s) by using ultra-high-performance liquid chromatography coupled to Quadrupole-Exactive Orbitrap mass spectrometry (UPLC­Q­Exactive Orbitrap/MS). The MS2 spectrum of the compound was highly similar to that of tadalafil within the range of < 300 m/z. The UV spectrum of the compound was almost identical to that of tadalafil. The analogue was purified by semi-preparative HPLC and structurally elucidated by FT-IR, MS and NMR analysis. The analogue was finally determined as N-cyclohexyl nortadalafil, featuring the cyclohexyl group instead of the N-methyl group on the piperazinedione ring of tadalafil.

Green & Sensitive pH-dependent Spectrofluorimetric Assay of Tamsulosin Hydrochloride and Tadalafil in their New Combined Formulation for Benign Prostatic Hyperplasia: Application to Spiked Human Plasma.

Sensitive and green spectrofluorimetric methods were utilized for Tamsulosin Hydrochloride (TAM) and Tadalafil (TDL) assessment in bulk and their newly available combined mixture for benign prostatic hyperplasia and erectile dysfunction. The technique relies on measuring native fluorescence of TAM in 0.1 N HCl at 324 nm and TDL in 0.1 N NaOH at 348 nm due to their different fluorimetric behavior in acidic and basic media where TAM has no fluorescence in basic medium and vice versa. To achieve better regression, the spectra were derivatized allowing determination of TAM at 314 nm and TDL at 320 and 380 nm (peak to peak) by applying third and first derivative, respectively. In addition, pH-dependent "constant-wavelength synchronous" spectrofluorimetry was applied where TAM and TDL were determined at 218 nm in acidic medium and at 268 nm in basic medium, respectively. Finally, derivatizing the latter emission spectra allowed determination of TAM and TDL at 232 nm and at 262 and 278 nm (peak to peak), respectively. Acidic and basic emission spectra where scanned at λexc = 225 nm (for TAM assay) and at λexc = 247 nm (for TDL assay), respectively. Fluorescence-concentration plots were linear and the proposed methods were used for analysis of TAM and TDL combined laboratory prepared formulation. These procedures are green, sensitive and of low cost which make them suitable for quality control analysis of the two drugs. In addition, the high selectivity of the proposed methods was tested by successfully applying them for TAM and TDL assay in plasma samples.

Assay of new combination of tamsulosin and tadalafil using synchronous spectrofluorimetric method coupled with mathematical tools.

A fixed-dose combination therapy of tamsulosin andtadalafilis now available for treatmentof lower urinary tract symptoms associated with benign prostatic hyperplasia. The decrease in sexual performance which is a side-effect of tamsulosin can be compensated by using tadalafil. This study is the first to develop and optimize a synchronous spectrofluorimetric method coupled with derivative and derivative ratio mathematical tools for the determination of tamsulosin andtadalafil in their newly released dosage form. The method successed in quantification of tadalafil by measuring the second-order derivative synchronous spectrofluorimetric amplitude at 278 nm (corresponding to zero-crossing of tamsulosin) using Δƛ=11 nm. On the other hand, first derivative ratio synchronous spectrofluorimetric peak amplitude was measured at 260 nm for determination of tamsulosin, using Δƛ=15 nm and divisor concentration of 0.1 µg mL-1. The method validation was performed using ICH guidelines. The linear responses of tamsulosin andtadalafil were from 0.04 to 0.5 and 0.05-0.5 µg mL-1, respectively. High sensitivity was achieved as represented by slope values of 0.32 and 608.4 for tamsulosin andtadalafil, respectively. The method is sensitive enough to detect concentration as low as 0.024 for both drugs. The proposed integrated spectrofluorimetric method showed good simplicity, selectivity and greenness. It can be successfully applied for analysis of both drugs in dosage form.

Spray-dried co-amorphous Tadalafil ternary mixtures: a promising platform towards the enhancement of solubility and bioavailability

Abstract Tadalafil (Tad) is a poorly water-soluble drug (BCS class II) that is used for the treatment of erectile dysfunction. An enhancement of aqueous solubility is vital to accelerate its onset of action and subsequently enhance its therapeutic effect. Binary and ternary mixtures of Tad with different amino acids (histidine, valine, alanine or arginine) and other excipients (mannitol and SLS) were prepared and then spray dried. The solubilizing efficiency and physicochemical characterization of all spray dried mixtures of Tad were studied. The optimum formulation was investigated in male rats to determine the onset of erection and the pharmacokinetic parameters of Tad. In general terms, the drug solubility of spray-dried formulae was enhanced compared to the crystalline form of the drug as a result of the formation of co-amorphous structures. The final result revealed that the Tad/alanine/mannitol spray-dried mixture (F10) showed the highest solubility and an improvement in its physicochemical characteristics. Moreover, F10 showed a significantly faster erection in rats with an improvement in Tad pharmacokinetic parameters when compared to the crystalline drug. Thus, F10 is selected as a promising formulation that successfully enhanced the bioavailability and the therapeutic efficacy of Tad.

Tadalafil and Sildenafil illicit association: Stability- indicating HPLC method, photodegradation kinetic and toxicological studies

Abstract The illicit market of counterfeit medicines containing sildenafil and tadalafil has been causing serious public health problems. Thus, further studies on this illicit association are needed. A stability-indicating HPLC method was developed for simultaneous determination of tadalafil (TAD) and sildenafil (SIL) using a C18 column (250 x 4.6 mm, 5 µm). Detection was achieved at 284 nm, for TAD, and 292 nm, for SIL. The method was considered to be specific, linear, precise, accurate, robust, and sensitive. In the photodegradation kinetic studies, the drugs showed a first-order reaction rate when isolated, and zero-order when associated. Toxicological assays demonstrated that the photodegraded drugs decreased cell viability in compared to non- degraded drugs, suggesting cytotoxic activity. Additional, mutagenic activity was not observed under the tested conditions. Photodegraded drugs, in association, depicted DNA damage index, suggesting genotoxic effects. The obtained results will be able to support the forensic intelligence laboratories, as well as to alert the population about the risk inherent to consuming counterfeit products.

Detection of 94 compounds related to sexual enhancement including sildenafil, tadalafil, vardenafil and their analogues in various formulations of dietary supplements and food samples using HPLC and LC-MS/MS.

With an increase in the detection of structural and functional analogues of phosphodiesterase type 5 inhibitors (PDE-5i) in dietary supplements (DS) and foods, public health is threatened. Some products advertise natural ingredients despite containing PDE-5i that can cause serious adverse effects on human health. To avoid detection during routine screening, novel PDE-5i have been synthesised and added to DS and foods. The purpose of this study was to detect, identify, and quantify 94 PDE-5i and related compounds in DS and foods. Furthermore, the study investigated the detection cases and compared them by sample type, formulation, and compounds. The HPLC and LC-MS/MS methods were validated for limit of detection (LOD), limit of quantification (LOQ), linearity, and recovery in solid and liquid type samples. Both HPLC and LC-MS/MS showed satisfactory results, which were in conformance with the ICH guidelines. A total of 404 samples, including DS (99), and foods (305) were purchased from online and offline markets. Samples divided into 5 types of formulation were analysed; tablet, capsule, pilula (herbal medicine pill), powder and liquid type. Of these 130 samples (47 of 99 DS, and 83 of 305 foods) contained one or more PDE-5i or related compounds. Among the five types of formulation, the tablet type showed the highest detection rate (61.1%) in DS, whereas the capsule type showed the highest detection rate (53.8%) in food samples. This study will be helpful for monitoring illegal ED-related products, providing information to consumers, and ultimately contributing to protecting public health.

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.

Fluorescence based immunochromatographic sensor for rapid and sensitive detection of tadalafil and comparison with a gold lateral flow immunoassay.

Tadalafil (TDL) is an illegal additive drug found in drinks and functional foods that could threaten public health. There was a great concern whether the adulteration occurred in coffee added with similar type of herbs. Here we have developed a rapid, simple, sensitive, and semi-quantitative lateral flow immunoassay (LFIA) based on gold and fluorescence labelled monoclonal antibody (mAb) for detection of TDL in coffee sample. Under optimal conditions, the cut off limits using gold nanoparticles labelled mAb (GLM) was found to be 250 ng/mL and 100 ng mL using fluorescent labelled mAb (FLM) in coffee samples. The coffee samples were spiked with TDL, and the LFIA with GLM gave average recoveries of 92-105.3% (intra-assay) and 96.6-105.9% (inter-assay), meanwhile with FLM gave recoveries 97.9-107.3% (intra-assay) and 98.3-108.9% (inter-assay). Results gave LFIA with FLM more sensitive than with GLM and all the test can be completed within 10 min, which would be an option for convenient and rapid assay of TDL detection.

UHPLC for quality evaluation of genuine and illegal medicines containing sildenafil citrate and tadalafil.

One of the highest incidences of illegal drug products is related to phosphodiesterase-5 inhibitors, used in treatment of erectile dysfunction, including those containing sildenafil citrate and tadalafil. In this context, comprehensive evaluation of the quality of genuine and illegal medicines was performed. A simple and rapid ultra-high performance liquid chromatography (UHPLC-UV) method to quantify sildenafil and tadalafil in the presence of six degradation products was developed and validated. Sildenafil and tadalafil were submitted to forced degradation. The separation was carried out on a Kinetex C18 (50 × 2.1 mm; 1.7 µm) column with mobile phase composed of acetonitrile and aqueous triethylamine solution. The calibration curves were linear in the range of 14-126 µg mL-1 for sildenafil citrate and 4-36 µg mL-1 for tadalafil and the method proved to be selective, precise, accurate and robust. Sildenafil degraded in oxidative media, whereas tadalafil degraded in acidic, alkaline and oxidative environment. The chemical structures and the mechanisms for the formation of the main degradation products were proposed by UHPLC coupled to tandem mass spectrometry. The UHPLC-UV method was applied in the pharmaceutical analysis of genuine and seized medicines. Some of them did not meet quality standards, mainly due to contents below specifications and the large variation on contents between units within a batch.

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.

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.

Novel HPTLC densitometric methods for determination of tamsulosin HCl and tadalafil in their newly formulated dosage form: Comparative study and green profile assessment.

A promising combination of tamsulosin HCl and tadalafil has recently been introduced for treating two prevalent and associated urological disorders: benign prostate hyperplasia and erectile dysfunction. Novel HPTLC methods were designed and validated for assaying the cited drugs in their challenging combined formulation. Separation was achieved using HPTLC silica gel 60 F254 plates as a stationary phase with a densitometric measurement at 280 nm. The proposed methods with two different chromatographic systems were successfully applied: a conventional mixture (method I) of ethyl acetate-toluene-methanol-ammonia (5:3:2:0.5, by volume) and a greener one (method II) with ethyl acetate-ethanol-ammonia (8:2:0.1, by volume). The two methods were evaluated through a comparative study in terms of selectivity, tailing factor, developing time and concentration ranges. The greenness profile for each method was then appraised with several green guides, namely GlaxoSmithKline solvent sustainability guide, Environmental, Health and Safety (EHS) tool, National Environmental Method Index (NEMI) and Eco-scale. Moreover, method specificity and peak homogeneity were evaluated by peak purity assessment using the winCATS® software spectral correlation tool. The methods have potential for being simple, fast, economic and selective, and the greener one could be a good option for sustainable analysis of the drugs.

D-optimal design as a useful tool response surface methodology for the optimization of signals from synchronous fluorescence prior to simultaneous determination of avanafil and tadalafil.

A rapid, smart and sensitive first derivative spectrofluorimetric method has been carried out for the simultaneous estimation of avanafil and tadalafil either in their pure form, tablet dosage form or spiked human plasma. The measurements of normal emission spectra or synchronous fluorescence intensity of both drugs show severe overlap which hindered their determination using normal fluorescence or synchronous intensity. Therefore, a highly sensitive first derivative synchronous fluorescence procedure was used to resolve this overlap. The method is based upon measurement of the amplitude of the first derivative of synchronous fluorescence intensity of both drugs at Δλ = 70 nm and at suitable wavelength of 396 nm and 364 nm for avanafil and tadalafil, respectively. Under the optimum conditions, the linear determination ranges are 50-1800 and 5-400 ng mL-1 with a detection limit of 12.93 and 1.46 ng mL-1 for avanafil and tadalafil, respectively. A response surface methodology was used for optimization using D-optimal design which can be used for determination of the exact optimum parameters specifically designed for this method. In addition; it is a good way to graphically clarify the relationship between various experimental variables and the synchronous fluorescence intensity.

Ultraviolet cutoff area and predictive ability of partial least squares regression method: A pharmaceutical case study.

UV cutoff area (COA) is known to be the wavelength band where solvents used for analysis can absorb radiation and accordingly affect the absorption spectra of drugs of interest being analyzed, even if blank experiments are done to eliminate solvent interference. However, this area may show peaks of significance for some drugs, and accordingly some researchers tend to include it in analysis. This study is presenting the importance of avoiding using COA, where it may represent significant negative effect on predictive ability of some linear chemometric methods like partial least squares regression PLSR. The presented study is using previously analyzed pharmaceutical mixtures of Dapoxetine Hydrochloride (DAP) and Tadalafil (TAD) as a case study, whether in pure forms or in dosage form, where the study uses two datasets for analysis, the first aims to include COA and the second dataset avoids it, then a statistical comparison is conducted for training sets, test sets and dosage form datasets to see how far COA may interfere with analysis results. Generally, the results show significant difference in datasets for t and F statistics for analysis of dosage form sets; which reflects changes in predictive ability of used chemometric method upon inclusion of COA in absorbance datasets, and accordingly unsuitability of using COA especially for routine quality control analysis of pharmaceutical mixtures.

A Validated Green HPTLC Method for Quantitative Determination of Dapoxetine Hydrochloride and Tadalafil in Bulk and Pharmaceutical Formulations.

Dapoxetine hydrochloride (DAP) and Tadalafil (TAD) were separated and determined quantitatively using a validated green high-performance thin layer chromatographic (HPTLC) method in their binary mixtures either as raw materials or in pharmaceutical formulations. The concentration ranges were 0.1-1.6 and 0.2-2.5 µg/band for dapoxetine and tadalafil, respectively, with accuracies of 98.93% ± 0.62 and 99.26% ± 1.39, respectively. Silica gel HPTLC F254 plates were used to carry out the separation. The mobile phase used was a mixture of ethanol-ethyl acetate (1:9 by volume), which is environmentally green and harmless. Densitometric scanning with UV detector was used to detect the separated peaks at 222 nm. ICH guidelines were followed to validate the suggested method, and the results prove that they can be used for regular analysis in quality control laboratories with compatible results.

Falsified vardenafil tablets available online.

It is often reported that falsified medicines have harmful effects on patients both Japan and abroad. In this study, we purchased vardenafil tablets on the internet and investigated their quality and authenticity using visual observations, authenticity investigations, non-destructive tests (handheld NIR and Raman spectroscopy), and quality analyses (active ingredient content and tablet dissolution rate). We used genuine 20-mg Levitra tablets that were sold in Japan and tablets from Bayer AG (Germany) as controls. In April 2015, we obtained 28 samples from 15 websites on the internet. Our authenticity investigations revealed that 11 (40%) were genuine products and 17 (60%) were falsified products. Handheld NIR and Raman results revealed that the falsified products had different spectra to the genuine products. Principal component analysis of the NIR and Raman spectra showed variation among the falsified products. The 11 genuine products were of good quality, and the 17 falsified products were of poor quality. The falsified products contained sildenafil (the active ingredient of Viagra) or tadalafil (the active ingredient of Cialis) instead of vardenafil. Our results show that falsified Vardenafil tablets are sold on the internet and that it is important to prevent illegal internet sales and increase consumer awareness of the presence of falsified medicines.

Smart spectrophotometric assessment of tamsulosin hydrochloride and tadalafil in their new pharmaceutical formulation for treatment of benign prostatic hyperplasia and erectile dysfunction.

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.

Enhancing counterfeit and illicit medicines grouping via feature selection and X-ray fluorescence spectrometry.

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.

Elucidation of the absolute configuration of a tadalafil analogue found as adulterant in a health supplement by mass spectrometry, chiroptical methods and NMR spectroscopy.

The chirality of a dipropylaminopretadalafil stereoisomer isolated from a health supplement has been studied. Under high resolution mass spectrometry (HRMS) study, this unknown compound seems to be one of the trans configuration tadalafil analogues i.e. (6S, 12aR) or (6R, 12aS), owing to the same precursor ion at m/z 492 with mass errors within ±2 ppm tolerance and very close retention times. Moreover, the MS2 fragmentation pattern is also very similar to the two trans isomers. Fortunately, the unknown compound can be distinguished from the two trans isomers by enantioselective separation with the use of a chiral column. Further comparison studies with a series of homologous compounds without a diketopiperazine ring on ellipticity and optical rotation support the unknown compound to be in the cis-(6R, 12aR) configuration. The nuclear magnetic resonance (NMR) in one dimensional (1D) and nuclear overhauser effect spectroscopy (NOESY) have affirmed the abovementioned configuration.