Characterization of fatty acid forms using benchtop NMR in omega-3 oil supplements.

Omega-3 fatty acid supplements, such as fish oil and plant-based oils, have gained popularity because of their potential health benefits. However, the quality and composition of these supplements can vary widely, particularly in terms of the two main forms of omega-3 fatty acids: triacylglycerols (TAGs) and ethyl esters (EEs). TAGs are the natural form found in fish oil but are prone to oxidation, whereas EEs are more stable but less well absorbed by the body. Differentiating between these forms is crucial for assessing the efficacy and tolerance of omega-3 supplements. This article describes a novel approach to differentiate between TAG and EE forms of omega-3 fatty acids in dietary supplements, utilizing a 60-MHz benchtop nuclear magnetic resonance (NMR) spectrometer. The proposed method using 1H and 1H-1H COSY NMR provides a quick and accurate approach to screen the forms of omega-3 fatty acids and evaluate their ratios. The presence of diacylglycerol (DAGs) in some supplements was also highlighted by this method and adds some information about the process used (i.e., esterification/enrichment). The affordability and user-friendliness of benchtop NMR equipment make this method feasible for food processing companies or quality control laboratories. In this study, 24 oil supplements were analyzed using NMR analysis in order to demonstrate the potential of this method for the differentiation of TAG and EE forms in omega-3 supplements.

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.

Separation of unsaturated C18 fatty acids using perfluorinated-micellar electrokinetic chromatography: I. Optimization and separation process.

Ammonium perfluorooctanoate (APFOA) was used as a surfactant for the separation of free unsaturated C18 fatty acids by micellar electrokinetic chromatography. A simple background electrolyte of 50 mM APFOA water/methanol (90:10, v/v) at pH = 10 enabled the repeatable separation of oleic acid, elaidic acid, linoleic acid, and alpha-linolenic acid in less than 20 min. Separation conditions were optimized regarding various parameters (organic solvent, counterion, APFOA concentration, and pH). Because the repulsive interactions between fluorocarbon chains and hydrogenated chains are known to lead to segregation and phase separation, the choice of perfluorinated micelles to separate such perhydrogenated long-chain acids could appear astonishing. Therefore, the critical micelle concentration, the charge density, and the mobility of the micelles have been determined, resulting in a first description of the separation process.

Chemometric Analysis of Low-field 1H NMR Spectra for Unveiling Adulteration of Slimming Dietary Supplements by Pharmaceutical Compounds.

The recent introduction of compact or low-field (LF) NMR spectrometers that use permanent magnets, giving rise to proton (1H) NMR frequencies between 40 and 80 MHz, have opened up new areas of application. The two main limitations of the technique are its insensitivity and poor spectral resolution. However, this study demonstrates that the chemometric treatment of LF 1H NMR spectral data is suitable for unveiling medicines as adulterants of slimming dietary supplements (DS). To this aim, 66 DS were analyzed with LF 1H NMR after quick and easy sample preparation. A first PLS-DA model built with the LF 1H NMR spectra from forty DS belonging to two classes of weight-loss DS (non-adulterated, and sibutramine or phenolphthalein-adulterated) led to the classification of 13 newly purchased test samples as natural, adulterated or borderline. This classification was further refined when the model was made from the same 40 DS now considered as representing three classes of DS (non-adulterated, sibutramine-adulterated, and phenolphthalein-adulterated). The adulterant (sibutramine or phenolphthalein) was correctly predicted as confirmed by the examination of the 1H NMR spectra. A limitation of the chemometric approach is discussed with the example of two atypical weight-loss DS containing fluoxetine or raspberry ketone.

Comparative Chemical Profiling and Monacolins Quantification in Red Yeast Rice Dietary Supplements by 1H-NMR and UHPLC-DAD-MS.

Red yeast rice dietary supplements (RYR DS) are largely sold in Western countries for their cholesterol-lowering/regulating effect due to monacolins, mainly monacolin K (MK), which is, in fact, lovastatin, the first statin drug on the market. 1H-NMR was used as an easy, rapid and accurate method to establish the chemical profiles of 31 RYR DS and to quantify their monacolin contents. Among all the 1H resonances of the monacolins found in RYR, only those of the ethylenic protons of the hexahydronaphthalenic ring at 5.84 and 5.56 ppm are suitable for quantification because they show no overlap with the matrix signals. The total content in monacolins per capsule or tablet determined in 28 DS (the content in 3 DS being below the limit of quantification of the method, ≈ 0.25 mg per unit dose) was close to that measured by UHPLC, as shown by the good linear correlation between the two sets of values (slope 1.00, y-intercept 0.113, r2 0.986). Thirteen of the 31 RYR DS analyzed (i.e., 42%) did not provide label information on the concentration of monacolins and only nine of the 18 formulations with an indication (i.e., 50%) actually contained the declared amount of monacolins.

Identification of altered brain metabolites associated with TNAP activity in a mouse model of hypophosphatasia using untargeted NMR-based metabolomics analysis.

Tissue non-specific alkaline phosphatase (TNAP) is a key player of bone mineralization and TNAP gene (ALPL) mutations in human are responsible for hypophosphatasia (HPP), a rare heritable disease affecting the mineralization of bones and teeth. Moreover, TNAP is also expressed by brain cells and the severe forms of HPP are associated with neurological disorders, including epilepsy and brain morphological anomalies. However, TNAP's role in the nervous system remains poorly understood. To investigate its neuronal functions, we aimed to identify without any a priori the metabolites regulated by TNAP in the nervous tissue. For this purpose we used 1 H- and 31 P NMR to analyze the brain metabolome of Alpl (Akp2) mice null for TNAP function, a well-described model of infantile HPP. Among 39 metabolites identified in brain extracts of 1-week-old animals, eight displayed significantly different concentration in Akp2-/- compared to Akp2+/+ and Akp2+/- mice: cystathionine, adenosine, GABA, methionine, histidine, 3-methylhistidine, N-acetylaspartate (NAA), and N-acetyl-aspartyl-glutamate, with cystathionine and adenosine levels displaying the strongest alteration. These metabolites identify several biochemical processes that directly or indirectly involve TNAP function, in particular through the regulation of ecto-nucleotide levels and of pyridoxal phosphate-dependent enzymes. Some of these metabolites are involved in neurotransmission (GABA, adenosine), in myelin synthesis (NAA, NAAG), and in the methionine cycle and transsulfuration pathway (cystathionine, methionine). Their disturbances may contribute to the neurodevelopmental and neurological phenotype of HPP.

Pulsed Field Gradient NMR with Sigmoid Shape Gradient Sampling To Produce More Detailed Diffusion Ordered Spectroscopy Maps of Real Complex Mixtures: Examples with Medicine Analysis.

NMR diffusion measurements are based on signal attenuation. In the case of complex mixtures for which some molecules are diffusing quickly while others are significantly slower, it is challenging to obtain a diffusion ordered spectroscopy (DOSY)-type 2D map giving reliable information on all molecules. In this paper, we propose a new gradient sampling approach based on a sigmoid shape allowing the acquisition of a significant number of points for both the fast and slow diffusing molecules. We applied this new gradient sampling strategy to deformulate two medicines whose composition was known (Esomeprazole) or unknown (Mebendazole). PFG NMR associated with a sigmoid gradient ramp is an exciting strategy to study drugs as a whole, i.e., the active ingredient(s) and excipients.

Photochemical Degradation of the Anticancer Drug Bortezomib by V-UV/UV (185/254 nm) Investigated by (1)H NMR Fingerprinting: A Way to Follow Aromaticity Evolution.

We have investigated the removal of bortezomib, an anticancer drug prescribed in multiple myeloma, using the photochemical advanced oxidation process of V-UV/UV (185/254 nm). We used two complementary analytical techniques to follow the removal rate of bortezomib. Nuclear magnetic resonance (NMR) is a nonselective method requiring no prior knowledge of the structures of the byproducts and permits us to provide a spectral signature (fingerprinting approach). This untargeted method provides clues to the molecular structure changes and information on the degradation of the parent drug during the irradiation process. This holistic NMR approach could provide information for monitoring aromaticity evolution. We use liquid chromatography, coupled with high-resolution mass spectrometry (LC-MS), to correlate results obtained by (1)H NMR and for accurate identification of the byproducts, in order to understand the mechanistic degradation pathways of bortezomib. The results show that primary byproducts come from photoassisted deboronation of bortezomib at 254 nm. A secondary byproduct of pyrazinecarboxamide was also identified. We obtained a reliable correlation between these two analytical techniques.

Evaluation of a benchtop cryogen-free low-field ¹H NMR spectrometer for the analysis of sexual enhancement and weight loss dietary supplements adulterated with pharmaceutical substances.

Nuclear magnetic resonance (NMR) spectroscopy is a unique tool for detection, structural characterization, and quantification of compounds in complex mixtures. However, due to cost constraints, NMR is rarely used in routine quality control (QC) analysis. The recent release of benchtop cryogen-free low-field NMR spectrometers represents a technological break in the NMR field. In this paper, we evaluated the potential of a benchtop cryogen-free 60 MHz spectrometer for uncovering adulteration of "100% natural" sexual enhancement and weight loss dietary supplements. We demonstrated that the adulterant(s) can readily be detected in ≈20 min of recording after a very simple and rapid sample preparation. We also showed that the quantification by the internal standard method can be done on the low-field NMR spectrometer and leads to results similar to those obtained with high-field NMR. Considering the cost and space efficiency of these spectrometers, we anticipate their introduction in QC laboratories as well as in governmental agencies, especially in the field of fraud detection.

The POWER saga from 2007 to 2022: An example of a sexual enhancement dietary supplement tainted by different adulterants and still on the market.

Ten POWER dietary supplements, chronologically called tabs, pills then caps, and advertised as 100% natural aphrodisiacs, were analyzed by 1H NMR from 2007 to 2022. They were all tainted by PDE-5 inhibitors. Eight different adulterants were identified (sildenafil (1), sildenafil analogues (6), and vardenafil analogue (1)). Their amounts ranged from 15 to 145 mg/capsule. Four supplements contained at least 100 mg/capsule of PDE-5 inhibitor or analogue, the maximal recommended dose of sildenafil. The nature of the adulterant has changed over time, probably to evade its detection by regulatory agencies routine screening tests. Despite several warnings and/or seizures from several European food and/or health authorities, the dietary supplement POWER is still on sale on the Internet, thus demonstrating the impossibility of controlling this market. Faced with this situation, the consumer should be better informed by establishing at the European level a public database of tainted dietary supplements on the model of that of the US Food and Drug Administration. It should indicate the product name, its photo, the adulterant name, and be easily accessible to everyone.

Quality evaluation of berberine food supplements with high-field and compact 1H NMR spectrometers.

Due to their health benefits, including regulating blood sugar and lowering cholesterol, berberine food supplements (FS) are widely used by consumers. This study aims to evaluate the quality of such products by proposing a new analytical methodology based on low-field NMR. Eighteen berberine FS were analyzed with both conventional (500 MHz) and benchtop (60 MHz) NMR spectrometers. Three quantitative 60 MHz 1H NMR methodologies were performed to determine berberine contents that were compared to those obtained at 500 MHz considered as reference measurements. To make the recording time of the spectra acquired at low field compatible with the requirements of a routine control, the quantification was carried out using a calibration curve established under conditions of incomplete relaxation of BrB protons. This methodology, applied to a test sample of 15 mg of FS, allowed to accurately measure a minimum quantity of berberine of ≈ 10 mg/capsule or tablet in 15 min. Regarding the FS, their labels are often unclear and/or incomplete for the consumer. Moreover, only 56 % of the FS analyzed actually contain the claimed quantity of berberine. The amounts of active they supply per day are extremely variable with only 39 % of the FS delivering a sufficient dose to achieve a hypoglycemic or hypolipidemic effect (1000-1500 mg/day based on literature data). These results show that health authorities should institute much stricter control and regulation over the production, labeling and marketing of berberine-based FS.

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.

Inhibition of alkaline phosphatase impairs dyslipidemia and protects mice from atherosclerosis.

Calcium accumulation in atherosclerotic plaques predicts cardiovascular mortality, but the mechanisms responsible for plaque calcification and how calcification impacts plaque stability remain debated. Tissue-nonspecific alkaline phosphatase (TNAP) recently emerged as a promising therapeutic target to block cardiovascular calcification. In this study, we sought to investigate the effect of the recently developed TNAP inhibitor SBI-425 on atherosclerosis plaque calcification and progression. TNAP levels were investigated in ApoE-deficient mice fed a high-fat diet from 10 weeks of age and in plaques from the human ECLAGEN biocollection (101 calcified and 14 non-calcified carotid plaques). TNAP was inhibited in mice using SBI-425 administered from 10 to 25 weeks of age, and in human vascular smooth muscle cells (VSMCs) with MLS-0038949. Plaque calcification was imaged in vivo with 18F-NaF-PET/CT, ex vivo with osteosense, and in vitro with alizarin red. Bone architecture was determined with µCT. TNAP activation preceded and predicted calcification in human and mouse plaques, and TNAP inhibition prevented calcification in human VSMCs and in ApoE-deficient mice. More unexpectedly, TNAP inhibition reduced the blood levels of cholesterol and triglycerides, and protected mice from atherosclerosis, without impacting the skeletal architecture. Metabolomics analysis of liver extracts identified phosphocholine as a substrate of liver TNAP, who's decreased dephosphorylation upon TNAP inhibition likely reduced the release of cholesterol and triglycerides into the blood. Systemic inhibition of TNAP protects from atherosclerosis, by ameliorating dyslipidemia, and preventing plaque calcification.

Metabolomic study of plasma of patients with abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is an important health problem, both because of AAA rupture and death and because of increased cardiovascular mortality. Identification of new biomarkers of AAA may suggest novel pathological mechanisms and targets for new medical treatments to slow AAA progression. Metabolic changes in AAA patients were mainly related to carbohydrate and lipid metabolism and many of these changes can be associated with a situation of insulin resistance (which can be related to metabolic syndrome) together with altered amino acid metabolism. For the first time, metabolites that can be associated with differential metabolism by the gut microflora of AAA patients have also been found. Moreover, aminomalonic acid in plasma has been shown to be the metabolite with the biggest difference between patients suffering from large aneurysm (>5 cm) and controls.

Quality assessment of commercial Magnoliae officinalis Cortex by ¹H-NMR-based metabolomics and HPLC methods.

INTRODUCTION: The quality control of Magnoliae officinalis Cortex, a commonly used traditional Chinese medicine, is currently based on the assay of the two active compounds, honokiol and magnolol, by TLC or HPLC. OBJECTIVE: To compare ¹H-NMR-based metabolomics with the HPLC method for controlling the quality of Magnoliae officinalis Cortex. To identify the metabolites contributing to the differences between the samples and to discriminate different medicinal parts and geographic origins of these samples by ¹H-NMR-based metabolomics. METHODOLOGY: ¹H-NMR and several multivariate analysis techniques were applied to analyse the extracts of 18 batches of Magnoliae officinalis Cortex commercial samples, and the contents of honokiol and magnolol in these samples were determined by HPLC. The correlation analysis between the data from ¹H-NMR and HPLC was performed with the mixOmics software based on an unsupervised method. RESULTS: Honokiol and magnolol were the main compounds responsible for the discrimination of samples from different batches, thus proving that the choice of these two compounds as markers for quality assessment by HPLC is relevant. The two sources of Magnoliae officinalis Cortex recorded in the Chinese Pharmacopoeia, Magnolia officinalis and Magnolia officinalis var. biloba, could be differentiated from ¹H-NMR data, but the pattern recognition analysis by PLS-DA was unsuccessful in discriminating samples from various geographical origins. CONCLUSION: The combination of ¹H-NMR that gives a comprehensive profile of the metabolites and HPLC that targets two biomarkers is an efficient means for a better quality control of Magnoliae officinalis Cortex.

Luminescent lanthanide complexes for reactive oxygen species biosensing and possible application in Alzheimer's diseases.

Histopathological hallmarks of Alzheimer's disease (AD) are intracellular neurofibrillary tangles and extracellular formation of senile plaques composed of the aggregated amyloid-beta peptide along with metal ions (copper, iron or zinc). In addition, oxidative stress is considered as an important factor in the etiology of AD and a multitude of metalloproteins and transporters is affected, leading to metal ion misregulation. Redox-active metal ions (e.g., copper) can catalyze the production of reactive oxygen species (ROS) in the presence of molecular oxygen and a reductant such as ascorbate. The ROS thus produced, in particular the hydroxyl radical which is the most reactive one, may contribute to oxidative stress conditions. Thus, detecting ROS in vivo or in biological models of AD is of interest for better understanding AD etiology. The use of biocompatible and highly specific and sensitive probes is needed for such a purpose, since ROS are transient species whose steady-state concentrations are very low. Luminescent lanthanide complexes are sensitive probes that can meet these criteria. The present review focuses on the recent advances in the use of luminescent lanthanide complexes for ROS biosensing. It shows why the use of luminescent lanthanide complexes is of particular interest for selectively detecting ROS ( O2·- , HO• , 1 O2 , H2 O2 , etc.) in biological samples in the µM-nM range. It particularly focuses on the most recent strategies and discusses what could be expected with the use of luminescent lanthanide complexes for better understanding some of the molecular mechanisms underlying the development of Alzheimer's disease.

Quality assessment of Curcuma dietary supplements: Complementary data from LC-MS and 1H NMR.

Due to the numerous potential health benefits of Curcuma, turmeric dietary supplements (DS) are among the top selling products. To assess the quality of these formulations, thirty Curcuma DS along with five standard Curcuma rhizomes were analyzed with UHPLC-MS and 1H NMR. The chemometric treatment of the UHPLC-MS spectra showed a significant variability of their chemical composition that was confirmed by 1H NMR which allowed the absolute quantification of the Curcuma major bioactive components, i.e. curcuminoids (curcumin, demethoxycurcumin and bisdemethoxycurcumin), and turmerones (aryl-, α- and ß-) as well as piperine, a commonly associated curcumin bioavailability enhancer: respectively 3.5-556, 0-8.6, 0.18-8.1 mg/capsule or tablet. The comparison of the actual and claimed quantities of curcuminoids and piperine showed that 58% of the DS contained the expected amounts of actives.

Synthetic cannabinoids in e-liquids: A proton and fluorine NMR analysis from a conventional spectrometer to a compact one.

The 1H NMR profiles of 13 samples of e-liquids supplied by French customs were obtained with high-field and low-field NMR. The high-field 1H NMR spectra allowed the detection of matrix signals, synthetic cannabinoids, and flavouring compounds. Quantitative results were obtained for the five synthetic cannabinoids detected: JWH-210, 5F-MDMB-PICA, 5F-ADB, 5F-AKB48, and ADB-FUBINACA. Conventional GC-MS analysis was used to confirm compound identification. Fluorine-19 NMR was proposed for the quantification of fluorinated synthetic cannabinoids and was successfully implemented on both 400 MHz and 60 MHz NMR spectrometers. This study based on few examples explored the potentiality of low-field NMR for quantitative and quantitative analysis of synthetic cannabinoids in e-liquids.

Current trends and advances in analytical techniques for the characterization and quantification of biologically recalcitrant organic species in sludge and wastewater: A review.

The study of organic matter in wastewater is a major regulatory and environmental issue and requires new developments to identify non-biodegradable refractory compounds, produced mainly by thermal treatments. Recent advances linking physicochemical properties to spectroscopic analyzes (UV, Fluorescence, IR) have shown that the refractory property is favored by several physicochemical parameters: weight, hydrophobicity, aromaticity and chemical functions. Currently, the most effective developments for the quantification of refractory compounds are obtained with hyphenated methods, based on steric separation of the macromolecular species by steric exclusion chromatography (SEC)/PDA/Fluorescence systems. Hyphenated techniques using High Resolution Mass Spectrometry (HRMS), ultra-high-resolution mass spectrometry with Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and NMR have been developed to analyze macromolecules in wastewater with minor sample preparation procedures. A particular class has been identified, the melanoidins, generated by Maillard reactions between sugars, amino acids, peptides and proteins present in wastewater and sludge, but low molecular weight compounds formed as intermediates, such as ketones, aldehydes, pyrazines, pyridines or furans, are also recalcitrant and are complex to identify in the complex matrices. The lack of available standards for the study of these compounds requires the use of specific techniques and data processing. Advances in chemometrics are obtained in the development of molecular or physicochemical indices resulting from the data generated by the analytical detectors, such as aromaticity calculated by SUVA254 and determined by UV, fluorescence, molar mass, H/C ratio or structural studies (measuring the amount of unsaturated carbon) given by hyphenated techniques with SEC. It is clear that nitrogen compounds are widely involved in refractoriness. New trends in nitrogen containing compounds characterization follow two axes: through SEC/PDA/Fluorescence and HRMS/NMR techniques with or without separation. Other techniques widely used in food or marine science are also being imported to this study, as it can be seen in the use of "omics" methods, high-performance thin layer chromatography (HPTLC) and chromatography at the critical condition, rounding out the important developments around SEC. While improving the performance of stationary phases is one of the challenges, it results in a fundamental understanding of the retention mechanisms that today provide us with more information on the structures identified. The main objective of this review is to present the spectroscopic and physicochemical techniques used to qualify and characterize refractoriness with a specific focus on chemometric approaches.

Isolation and identification of ten new sildenafil derivatives in an alleged herbal supplement for sexual enhancement.

A sexual enhancer dietary supplement in pre-commercialization phase was analyzed. It contained the two phosphodiesterase-5 inhibitors (PDE-5i) sildenafil and methisosildenafil as major adulterants. Fourteen more sildenafil derivatives were detected and after isolation, their structures were elucidated thanks to NMR, high resolution and tandem mass spectrometry, and UV spectroscopy. Ten of them were never described. All these compounds are probably by-products of different reaction steps during the synthesis of the two PDE-5i that were not properly eliminated during the purification procedure. The total amount of sildenafil-related compounds was estimated at 68 mg per capsule, sildenafil and methisosildenafil accounting for 20 mg and 38 mg respectively.