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The Brazilian National Metrology Institute produced a suite of certified reference materials (CRMs) intended as internal standards (ISs) for quantitative nuclear magnetic resonance (qNMR). Being a ratio primary method, the use of qNMR in organic chemistry has already crossed the borders of research laboratories, despite the cost of instrumentation. The International Bureau of Weights and Measures (BIPM) proposed eight potential qNMR ISs. Four candidate materials were selected for their solubility in various solvents and distinct chemical shifts, making them suitable for qNMR analysis of diverse analytes. The certification process compared orthogonal primary methods such as mass balance, qNMR, freezing-point depression, and coulometry, to ensure independent value assignment. Different approaches were compared to assess batch homogeneity and stability. While directly comparing the main compound's chromatographic area proved to be a quick and fit-for-purpose approach, the determination of individual impurities provided lower uncertainties but required more laborious work. CRM batches of maleic acid, dimethyl sulfone, potassium hydrogen phthalate, and dimethyl terephthalate were delivered with over 999.8 mg g -1 purity and uncertainty in the range of 0.6 to 3 mg g -1 (k = 2). The literature shows certification procedures for qNMR ISs whose traceability chain is exclusively based on qNMR measurements. As opposed to that, the methodology presented here provides robust certified values assigned by methods independent of qNMR, in accordance with BIPM recommendations and less prone to qNMR biases. The CRMs developed in this work have already been used for SI-traceable purity evaluation of compounds such as drugs and pesticides, by laboratories in Brazil and abroad., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Certified reference materials (CRM) of amphetamine derivatives were produced through a simple, rapid and efficient synthesis in both batch and continuous-flow conditions, accompanied by the development of a comprehensive certification protocol for this class of substances. Our chemistry enabled the synthesis of MDA, MDMA, PMA and PMMA in two steps from safrole and estragole with overall yields of 38-61 % in 48 hours under batch conditions and 61-65 % in 65 minutes under continuous-flow conditions, followed by the development of a certification protocol for these materials through identity checking, homogeneity, stability, and characterization studies. Furthermore, as result of this work, a very pure CRM of MDA.HCl with 99.1±1.4 g/100 g of certified characterization value was produced. Considering the importance of supplying amphetamine calibrants for public security efforts in Forensic Chemistry, the potential therapeutical applications, and responding to the rising demand for the synthesis of CRM, this work presents a pioneering approach for the production of amphetamine and related compounds., (© 2023 Wiley-VCH GmbH.)

The estimation of limits of detection (LOD) for solely qualitative methods in analytical chemistry may prove challenging because all the approaches with which chemists are familiar require some type of numeric data input. The best model to describe the binary response in these methods (detected/not detected) is a logistic model; however, these models are not easily handled by most of the laboratories and generally demand expensive statistical software packages. In this work, the advantages of applying this approach are discussed and its implementation using commercial spreadsheet software is demonstrated. A free online application based on the R environment using shinyapps was developed and its application was validated and discussed with a dataset of 57 different target compounds analyzed in urine according to the requirements of the World Anti-Doping Agency (WADA). This tool allows free, extremely quick, and easy determinations of LOD in qualitative analyses as well as the determination of the probabilities of detection in any given concentration., (© 2022 John Wiley & Sons Ltd.)

Introduction: Data Fusion-based Discovery (DAFdiscovery) is a pipeline designed to help users combine mass spectrometry (MS), nuclear magnetic resonance (NMR), and bioactivity data in a notebook-based application to accelerate annotation and discovery of bioactive compounds. It applies Statistical Total Correlation Spectroscopy (STOCSY) and Statistical HeteroSpectroscopy (SHY) calculation in their data using an easy-to-follow Jupyter Notebook., Method: Different case studies are presented for benchmarking, and the resultant outputs are shown to aid natural products identification and discovery. The goal is to encourage users to acquire MS and NMR data from their samples (in replicated samples and fractions when available) and to explore their variance to highlight MS features, NMR peaks, and bioactivity that might be correlated to accelerated bioactive compound discovery or for annotation-identification studies., Results: Different applications were demonstrated using data from different research groups, and it was shown that DAFdiscovery reproduced their findings using a more straightforward method., Conclusion: DAFdiscovery has proven to be a simple-to-use method for different situations where data from different sources are required to be analyzed together., (© 2022 John Wiley & Sons Ltd.)

Introduction: In this era of 'omics' technology in natural products studies, the complementary aspects of mass spectrometry (MS)- and nuclear magnetic resonance (NMR)-based techniques must be taken into consideration. The advantages of using both analytical platforms are reflected in a higher confidence of results especially when using replicated samples where correlation approaches can be used to statistically link results from MS to NMR., Objectives: Demonstrate the use of Statistical Total Correlation (STOCSY) for linking results from MS and NMR data to reach higher confidence in compound identification., Methodology: Essential oil samples of Melaleuca alternifolia and M. rhaphiophylla (Myrtaceae) were used as test objects. Aliquots of 10 samples were collected for GC-MS and NMR data acquisition [proton ( 1 H)-NMR, and carbon-13 ( 13 C)-NMR as well as two-dimensional (2D) heteronuclear single quantum correlation (HSQC), heteronuclear multiple-bond correlation (HMBC), and HSQC-total correlated spectroscopy (TOCSY) NMR]. The processed data was imported to Matlab where STOCSY was applied., Results: STOCSY calculations led to the confirmation of the four main constituents of the sample-set. The identification of each was accomplished using; MS spectra, retention time comparison, 13 C-NMR data, and scalar correlations of the 2D NMR spectra., Conclusion: This study provides a pipeline for high confidence in compound identification using a set of essential oils samples as test objects for demonstration., (© 2022 John Wiley & Sons, Ltd.)

Comparability of measurement results and their metrological traceability to the International System of Units (SI) are fundamental tools to ensure reliable decisions in the social sphere, commerce, and science. The use of appropriate references in analytical chemistry, such as certified reference materials (CRMs) of high purity substances, is one of the required procedures to obtain traceable measurements. When commercial standards with non-certified purity values are used, traceability must be achieved by determining the purity of the standard using a potential primary reference measurement procedure or other appropriate methods. Quantitative nuclear magnetic resonance (qNMR) is a technique with the potential to be used in primary measurement procedures. This work presents the determination of purity by 1 H qNMR for nitrofuran metabolites 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AHD). Furthermore, a recent qNMR method developed by our group to improve the quantitative performance of measurements using 13 C nucleus was used to determine the purity of semicarbazide (SEM) nitrofuran metabolite. Purity values obtained by qNMR for AOZ, AMOZ, and AHD standards were compared to values obtained by the mass balance approach using a suite of analytical methods: Karl Fischer (KF) coulometric titration and thermogravimetry (TG) for the determination of water and residual solvents, gas and liquid chromatography for the determination of impurities structurally related to the metabolites. The results obtained by qNMR and mass balance were consistent.Graphical abstract.

Unlabelled: The growing consumption of nutritional supplements might represent a problem, given the concern about the quality of these supplements. One of the most used supplements is whey protein (WP); because of its popularity, it has been a target of adulteration with substitute products, such as cheaper proteins with lower biological value. To investigate this type of adulteration, this study used shotgun proteomics analyses by MS(E) (multiplexed, low- and high-collision energy, data-independent acquisition) of WP-based supplements. Seventeen WP-based supplement samples were evaluated. Chicken, maize, rice, potato, soybean, and wheat proteins were considered as probable sources of bovine whey adulteration. Collectively, 523 proteins were identified across all 16 samples and replicates, with 94% of peptides inside a normal distribution within 10ppm of maximum error. In 10 of the 16 samples analyzed, only proteins from bovine whey could be detected, while in the other samples several other protein sources were detected in high concentrations, especially soybean, wheat, and rice. These results point out a probable adulteration and/or sample contamination during manufacturing that could only be detected using this proteomic approach., Significance: The present work shows how shotgun proteomics can be used to provide reliable answers in quality control matters, especially focusing on Whey Protein nutritional supplements which are a very popular subject in food and nutrition. In order to achieve an appropriate methodology, careful evaluation was performed applying extremely rigorous quality criteria, established for the proteomic analysis. These criteria and the methodological approach used in this work might serve as a guide for other authors seeking to use proteomics in quality control., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Verapamil and naproxen Parallel Artificial Membrane Permeability Assay (PAMPA) permeability was studied using lipids not yet reported for this model in order to facilitate the quantification of drug permeability. These lipids are 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and an equimolar mixture of DMPC/DSPC, both in the absence and in the presence of 33.3 mol% of cholesterol. PAMPA drug permeability using the lipids mentioned above was compared with lecithin-PC. The results show that verapamil permeability depends on the kind of lipid used, in the order DMPC > DMPC/DSPC > DSPC. The permeability of the drugs was between 1.3 and 3.5-times larger than those obtained in lecithin-PC for all the concentrations of the drug used. Naproxen shows similar permeability than verapamil; however, the permeability increased with respect to lecithin-PC only when DMPC and DMPC/DSPC were used. This behavior could be explained by a difference between the drug net charge at pH 7.4. On the other hand, in the presence of cholesterol, verapamil permeability increases in all lipid systems; however, the relative verapamil permeability respect to lecithin-PC did not show any significant increase. This result is likely due to the promoting effect of cholesterol, which is not able to compensate for the large increase in verapamil permeability observed in lecithin-PC. With respect to naproxen, its permeability value and relative permeability respect lecithin-PC not always increased in the presence of cholesterol. This result is probably attributed to the negative charge of naproxen rather than its molecular weight. The lipid systems studied have an advantage in drug permeability quantification, which is mainly related to the charge of the molecule and not to its molecular weight or to cholesterol used as an absorption promoter.

Quantitative nuclear magnetic resonance measurements have become more popular over the last decade. The introduction of new methods and experimental parameters has been of fundamental importance in the development of new applications. Amongst these new developments is the introduction of electronic referencing for quantifications. The use of electronic referencing eliminates errors in the analyses as a result of weighting of internal standards as well as undesired problems as a result of the solubility of the standards in the analyte solution and chemical interactions between the analyte and the internal standard. In this work, we have studied the quantification of a very important analyte in a food matrix, benzoic acid in orange juice, as a model to the validation and measurement uncertainty estimation of electronic referencing using (1)H NMR in food analyses. The referencing method applied was the pulse length-based concentration measurement. Method was validated and showed good results for the precision and accuracy parameters evaluated. A certified reference material and a reference material candidate were analyzed, and extremely good results were obtained. Reported relative expanded uncertainties are in the 1.07-1.39% range that can be considered an extremely good performance for the analysis of a food complex matrix. Measurement uncertainty was evaluated by two different approaches, and the pulse calibrations for the samples and for the reference have been shown to account for approximately 80% of the total uncertainty of the measurement., (Copyright © 2014 John Wiley & Sons, Ltd.)

Salbutamol is commonly used in asthma treatment, being considered a short-effect bronchodilator. This drug poses special interest in certain fields of chemical analysis, such as food, clinical and doping analyses, in which it needs to be analyzed with quantitative precision and accuracy. Salbutamol, however, is known to degrade under certain conditions and this is critical if quantitative results must be generated. The present work aimed to investigate salbutamol extraction from urine samples, to determine whether salbutamol is unstable in other solvents as well as in urine samples, to elucidate the structures of the possible degradation products and to validate an analytical method using the extraction procedure evaluated. Stability investigations were performed in urine at different pH values, in methanol and acetone at different temperatures. Semi-preparative liquid chromatography was performed for the isolation of degradation products, and gas chromatography coupled to mass spectrometry as well as nuclear magnetic resonance were used for identification. Three unreported methylation products were detected in methanolic solutions and had their structures elucidated. Urine samples showed a reduction in salbutamol concentration of up to 25.8% after 5 weeks. These results show that special care must be taken regarding salbutamol quantitative analyses, since degradation either in standard solutions or in urine could lead to incorrect values., (Copyright © 2013 John Wiley & Sons, Ltd.)

Certified reference materials (CRMs) are essential tools to guarantee the metrological traceability of measurement results to the International System of Units (SI), which means the accuracy and comparability of results over time and space. In the pharmaceutical area, only a few CRMs are available and the use of (non-certified) reference materials is a much more common practice. In this paper, the studies on a new candidate CRM of sodium diclofenac based on the ISO Guides 34:2009 and 35:2005 are described. The project steps included characterization, homogeneity test, stability studies, and uncertainties estimation. In the characterization, the mass fractions of organic, inorganic, and volatile impurities were determined, and the results were cross-checked by independent reference methods or interlaboratorial study. The API mass fraction was calculated by mass balance and cross-checked by quantitative proton nuclear magnetic resonance (¹H qNMR). The paper also presents a Monte Carlo simulation to estimate the measurement uncertainty as an approach to validate the GUM results in ¹H qNMR. The homogeneity between batch units was verified, and the candidate CRM stability under transport and storage conditions was evaluated in short- and long-term stability studies. The CRM certified property value and corresponding expanded uncertainty, obtained from the combined standard uncertainty multiplied by the coverage factor (k=2), for a confidence level of 95%, was (999.76+0.10) mg g⁻¹., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The use of "nutritional supplements" containing unapproved substances has become a regular practice in amateur and professional athletes. This represents a dangerous habit for their health once no data about toxicological or pharmacological effects of these supplements are available. Most of them are freely commercialized online and any person can buy them without medical surveillance. Usually, the steroids intentionally added to the "nutritional supplements" are testosterone analogues with some structural modifications. In this study, the analyzed product was bought online and a new anabolic steroid known as methylstenbolone (2,17α-dimethyl-17β-hydroxy-5α-androst-1-en-3-one) was detected, as described on label. Generally, anabolic steroids are extensively metabolized, thus in-depth knowledge of their metabolism is mandatory for doping control purposes. For this reason, a human excretion study was carried out with four volunteers after a single oral dose to determine the urinary metabolites of the steroid. Urine samples were submitted to enzymatic hydrolysis of glucuconjugated metabolites followed by liquid-liquid extraction and analysis of the trimethylsilyl derivatives by gas chromatography coupled to tandem mass spectrometry. Mass spectrometric data allowed the proposal of two plausible metabolites: 2,17α-dimethyl-16ξ,17β-dihydroxy-5α-androst-1-en-3-one (S1), 2,17α-dimethyl-3α,16ξ,17β-trihydroxy-5α-androst-1-ene (S2). Their electron impact mass spectra are compatible with 16-hydroxylated steroids O-TMS derivatives presenting diagnostic ions such as m/z 231 and m/z 218. These metabolites were detectable after one week post administration while unchanged methylstenbolone was only detectable in a brief period of 45 h., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Exemestane is an aromatase enzyme complex inhibitor. Its metabolism in humans is not fully described and there is only one known metabolite: 17β-hydroxyexemestane. In this work, excretion studies were performed with four volunteers aiming at the detection of new exemestane metabolites in human urine by gas chromatography coupled to mass spectrometry (GC-MS) after enzymatic hydrolysis and liquid-liquid extraction. Urine samples collected from four volunteers were analyzed separately. The targets of the study were mainly the 6-exomethylene oxidized metabolites. Two unreported metabolites were identified in both free and glucuconjugated urine fractions from all four volunteers, both of them were the result of the 6-exomethylene moiety oxidation: 6ξ-hydroxy-6ξ-hydroxymethylandrosta-1,4-diene-3,17-dione (metabolite 1) and 6ξ-hydroxyandrosta-1,4-diene-3,17-dione (metabolite 2). Furthermore, only in glucoconjugated fractions from all volunteers, one metabolite arising from the A-ring reduction was identified as well, 3ξ-hydroxy-5ξ-androst-1-ene-6-methylene-17-one (metabolite 3). The molecular formulae of all these metabolites were ascertained by the determination of exact masses using gas chromatography coupled to high resolution mass spectrometry (GC-HRMS). Moreover, all metabolites were confirmed using an alternative derivatization with methoxyamine and MSTFA/TMS-imidazole., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Trimethylsilylation of anabolic agents and their metabolites is frequently achieved by using the derivatization mixture N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)/NH(4)I/2-mercaptoethanol. Nevertheless, artifacts were formed when this mixture was employed in the monitoring of exemestane and its main metabolite 17β-hydroxyexemestane prior to GC-MS analysis. These artifacts were identified as the N-methyltrifluoroacetamide (MTFA) and trimethylsiloxyethylmercapto products of the respective trimethylsilyl (TMS) derivatives. Furthermore, artifact formation was evaluated taking the structure (1,4-diene-3-keto-6-exomethylene) of the compounds into account. Although these artifacts are relevant for investigations regarding the derivatization process and may be of interest in many fields, they are detrimental to cope with the requirements of the World Anti-Doping Agency (WADA) in terms of the limits of detection (LODs) required. To overcome this issue, a method using an alternative derivatization was proposed: formation of methyloxime-TMS derivatives through double derivatization using O-methylhydroxylamine/pyridine and MSTFA/TMS imidazole after enzymatic hydrolysis and liquid-liquid extraction. Samples from an excretion study after administration of exemestane to healthy volunteers were analyzed by the proposed method and detection of both exemestane and its main metabolite was possible. This method showed excellent results for both analytes meeting the LODs required for antiestrogenic agents (50  ng/mL) established by WADA. The method was validated for the main metabolite, it was robust and cost-effective for qualitative and quantitative purposes, with LOD and LOQ of 10  ng/mL and 25  ng/mL, respectively., (2010 John Wiley & Sons, Ltd.)