Use of quantitative 1H and 13C NMR to determine the purity of organic compound reference materials: a case study of standards for nitrofuran metabolites.

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 1H 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 13C 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.

Implementation and Performance of the Gas Chromatography/Combustion/Isotope Ratio Mass Spectrometry-Based Method for the Confirmatory Analysis of Endogenous Anabolic Steroids during the Rio de Janeiro Olympic and Paralympic Games 2016.

Carbon isotope ratio (CIR) confirmation is one of the most complex and delicate analyses in the doping control field, due to the nature of the molecules to be confirmed, normally present in urinary samples as a consequence of an endogenous production. The requirements for method validation established by the World Anti-Doping Agency (WADA) have been pushing the accredited laboratories to improve their methods. The choice of the method is always a cost benefit ratio involving a hard-working and time-consuming analysis and the guarantee of reporting of reliable results. This work presents the method fully validated by the Brazilian Doping Control Laboratory as part of the preparation for the Rio de Janeiro Summer Olympic and Paralympic Games 2016. Sample preparation encompassed solid-phase extraction, liquid-liquid extraction, enzymatic hydrolysis, acetylation, and purification by preparative high-performance liquid chromatography, and analyses were performed by gas chromatography/combustion/isotope ratio mass spectrometry. This proved to be a robust method to CIR confirmation in a big event, as demonstrated by the analysis of 179 samples during the Games 2016, from clearly negative results and adverse findings for testosterone (T) and related substances, boldenone and its metabolite, 19-norandrosterone and formestane. Two atypical findings were also reported for T and metabolites.

Qualitative transformations of street-seized ecstasy over a decade: A case study in Rio de Janeiro (Brazil).

The illegal drug market is constantly evolving, with new drugs being created and existing ones being modified. Adulterants are often added to the mix, and the primary substance may be secretly replaced by a new one. Once-known tablets can now be vastly different from what they are sold as, all due to the pursuit of profit and evasion of current drug regulations. These alterations in drug composition pose a threat to society, as their effects are still not well understood. Therefore, it is crucial for police intelligence and public health development to obtain the chemical profiles of illicit drugs. This study presents the chemical fingerprinting of ecstasy tablets seized in the state of Rio de Janeiro (Brazil) between 2012 and 2021. The tablet samples were weighed, extracted, diluted with methanol, and acidified before analysis using gas chromatography high-resolution mass spectrometry and attenuated total reflection Fourier transform infrared spectroscopy. The major constituents found were MDMA and clobenzorex, with fewer occurrences of MDA, MDEA, and 2C-B. The results also indicate that the occurrence of mega-events in the study location impacted the chemical fingerprints of ecstasy. A total of 27 combinations of cutting agents, including caffeine, ephedrine, and anesthetics, were identified. Samples composed of clobenzorex were observed throughout the evaluated period in areas near highways, suggesting that this product is mainly used by truck drivers. These findings can help police intelligence units anticipate the behavior of the illicit market during major events, identify traffic routes, and support public health initiatives.

Salbutamol extraction from urine and its stability in different solutions: identification of methylation products and validation of a quantitative analytical method.

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.

Hansen Solubility Parameters Applied to the Extraction of Phytochemicals.

In many analytical chemical procedures, organic solvents are required to favour a better global yield upon the separation, extraction, or isolation of the target phytochemical analyte. The selection of extraction solvents is generally based on the solubility difference between target analytes and the undesired matrix components, as well as the overall extraction procedure cost and safety. Hansen Solubility Parameters are typically used for this purpose. They are based on the product of three coordinated forces (hydrogen bonds, dispersion, and dipolar forces) calculated for any substance to predict the miscibility of a compound in a pure solvent, in a mixture of solvents, or in non-solvent compounds, saving time and costs on method development based on a scientific understanding of chemical composition and intermolecular interactions. This review summarises how Hansen Solubility Parameters have been incorporated into the classical and emerging (or greener) extraction techniques of phytochemicals as an alternative to trial-and-error approaches, avoiding impractical experimental conditions and resulting in, for example, saving resources and avoiding unnecessary solvent wasting.

Gene doping and genomic science in sports: where are we?

The misuse of sport-related gene transfer methods in elite athletes is a real and growing concern. The success of gene therapy in the treatment of hereditary diseases has been most evident since targets in gene therapy products can be used in healthy individuals to improve sports performance. Performing these practices threatens the sporting character of competitions and may pose potential health hazards. Since the World Anti-Doping Agency pronouncement on the prohibition of such practices in 2003, several researchers have been trying to address the challenge of developing an effective method for the detection of genetic doping. This review presents an overview of the published methods developed for this purpose, the advantages and limitations of technologies and the putative target genes. At last, we present the perspective related to the application of the detection methods in the doping control field.

Analysis of underivatised low volatility compounds by comprehensive two-dimensional gas chromatography with a short primary column.

Comprehensive two-dimensional gas chromatography (GC×GC) approaches with cryogenic modulation were developed for the qualitative analysis of selected low volatility compounds in raw coffee bean extracts, without derivatisation. The approaches employed short first (1D) and second (2D) dimension columns, specifically a 1D 65% phenyl methyl siloxane column (11m) and a 2D 5% phenyl methyl siloxane column (1m), which allowed elution of high molar mass compounds (e.g.>600Da). Solutes included hydrocarbons, fatty acids, diterpenes, tocopherols, sterols, diterpene esters, and di- and triacylglycerides. An oven temperature program up to 370°C was employed. The effects of experimental conditions were investigated, revealing that the GC×GC results strongly depended on the cryogenic trap T, and oven T program. An appropriate condition was selected and further applied for group type analysis of low volatility compounds in green Arabica coffee beans. Retention indices were compiled for 1D GC analysis and were similar for the composite column data in GC×GC. The elution of some compounds was confirmed by use of authentic standards. The approach allowed direct analysis of coffee extract in ethyl acetate solution, with improved analyte peak capacity (approximately 200 compounds were detected) without prior fractionation or pre-treatment of the sample. This avoided potential hydrolysis of high molar mass conjugate esters as well as degradation of thermally labile compounds such as the derivatives of the diterpenes cafestol and kahweol.

Non-targeted acquisition strategy for screening doping compounds based on GC-EI-hybrid quadrupole-Orbitrap mass spectrometry: A focus on exogenous anabolic steroids.

This is a first look at a non-targeted screening method based on Orbitrap gas chromatography-mass spectrometry (GC-MS) technology for a large number of banned compounds in sports found in urine, including exogenous anabolic steroids, ß-agonists, narcotics, stimulants, hormone modulators, and diuretics. A simple sample preparation was processed in four steps: an enzymatic hydrolysis, liquid-liquid extraction, evaporation, and trimethylsilylation. All compounds were able to meet the World Anti-Doping Agency's sensitivity criteria with mass accuracies less than 1 ppm and with sufficient points across the peak by running the Orbitrap GC-MS in full-scan mode. In addition, we discuss our initial findings of using a full-scan selected ion monitoring-tandem mass spectrometry (SIM-MS/MS) approach as a way to obtain lower detection limits and reach desirable selectivity for some exogenous anabolic steroids.

Zebrafish (Danio rerio) water tank model for the investigation of drug metabolism: Progress, outlook, and challenges.

Zebrafish (Danio rerio) water tank (ZWT) approach was investigated as an alternative model for metabolism studies based on six different experiments with four model compounds. Sibutramine was applied for the multivariate optimization of ZWT conditions, also for the comparison of the metabolism among ZWT, humans and mice, beyond for the role of CYP2B6 in ZWT. After the optimization, 18 fish and 168 hours of experiments is the minimum requirement for a relevant panel of biotransformation products. A comparison among the species resulted in the observation of the same hydroxylated metabolites, with differences in metabolites concentration ratio. However, the ZWT allowed tuning of the conditions to obtain a specific metabolic profile, depending on the need. In addition, by utilizing CYP2B6 inhibition, a relevant ZWT pathway for the demethylation of drugs was determined. The stereospecificity of the ZWT metabolism was investigated using selegiline and no racemization or inversion transformations were observed. Moreover, the investigation of metabolism of cannabimimetics was performed using JWH-073 and the metabolites observed are the same described for humans, except for the hydroxylation at the indol group, which was explained by the absence of CYP2C9 orthologs in zebrafish. Finally, hexarelin was used as a model to evaluate studies by ZWT for drugs with low stability. As a result, hexarelin displays a very fast metabolization in ZWT conditions and all the metabolites described for human were observed in ZWT. Therefore, the appropriate conditions, merits, and relevant limitations to conduct ZWT experiments for the investigation of drug metabolism are described.

Analysis of synthetic 19-norsteroids trenbolone, tetrahydrogestrinone and gestrinone by gas chromatography-mass spectrometry.

Tetrahydrogestrinone, gestrinone and trenbolone are synthetic 19-norsteroids with androgenic properties sharing a labile conjugated ketotrienyl motif. Their LC-MS analyses tend to overcome classical derivatization problems, a shortcoming to the use of GC-MS. Therefore, alternative derivatization procedures were evaluated. The procedure with methoxylamine: pyridine followed by TMSImid: MSTFA gave the best results. This is attributed to the stability of the MO-TMS derivatives hindering the formation of artifacts and tautomerism. A full method is presented including SPE, hydrolysis and liquid-liquid extraction. It was possible to confirm the analytes below 2 ng/mL in urine, being the method robust and cost effective also for screening proposes.

Is zebrafish (Danio rerio) a tool for human-like metabolism study?

One of the greatest challenges in anti-doping science is the large number of substances available and the difficulty in finding the best analytical targets to detect their misuse. Therefore, metabolism studies involving prohibited substances are fundamental. However, metabolism studies in humans could face an important ethical bottleneck, especially for non-approved substances. An emerging model for metabolism assessment is the zebrafish, due to its genetic similarities with humans. In the present study, the ability of adult zebrafish to produce metabolites of sibutramine and stanozolol, substances with a well-known metabolism that are widely used as doping agents in sports, was evaluated. They represent 2 of the most abused classes of doping agents, namely, stimulants and anabolic steroids. These are classes that have been receiving attention because of the upsurge of synthetic analogues, for which the side effects in humans have not been assessed. The samples collected from the zebrafish tank water were hydrolysed, extracted by solid-phase extraction, and analysed by liquid chromatography with high resolution mass spectrometry (LC-HRMS). Adult zebrafish could produce several sibutramine and stanozolol metabolites, including demethylated, hydroxylated, dehydroxylated, and reduced derivatives, all of which have already been detected in human urine. This study demonstrates that adult zebrafish can absorb, oxidise, and excrete several metabolites in a manner similar to humans. Therefore, adult zebrafish seem to be a very promising tool to study human-like metabolism when aiming to find analytical targets for doping control. Copyright © 2017 John Wiley & Sons, Ltd.

Doping control analysis at the Rio 2016 Olympic and Paralympic Games.

This paper summarises the results obtained from the doping control analyses performed during the Summer XXXI Olympic Games (August 3-21, 2016) and the XV Paralympic Games (September 7-18, 2016). The analyses of all doping control samples were performed at the Brazilian Doping Control Laboratory (LBCD), a World Anti-Doping Agency (WADA)-accredited laboratory located in Rio de Janeiro, Brazil. A new facility at Rio de Janeiro Federal University (UFRJ) was built and fully operated by over 700 professionals, including Brazilian and international scientists, administrative staff, and volunteers. For the Olympic Games, 4913 samples were analysed. In 29 specimens, the presence of a prohibited substance was confirmed, resulting in adverse analytical findings (AAFs). For the Paralympic Games, 1687 samples were analysed, 12 of which were reported as AAFs. For both events, 82.8% of the samples were urine, and 17.2% were blood samples. In total, more than 31 000 analytical procedures were conducted. New WADA technical documents were fully implemented; consequently, state-of-the-art analytical toxicology instrumentation and strategies were applied during the Games, including different types of mass spectrometry (MS) analysers, peptide, and protein detection strategies, endogenous steroid profile measurements, and blood analysis. This enormous investment yielded one of the largest Olympic legacies in Brazil and South America. Copyright © 2017 John Wiley & Sons, Ltd.

Multiresidue method for simultaneous analysis of aflatoxin M1, avermectins, organophosphate pesticides and milbemycin in milk by ultra-performance liquid chromatography coupled to tandem mass spectrometry.

A method developed for the simultaneous analysis of aflatoxin M1, abamectin, doramectin, eprinomectin, ivermectin, moxidectin, acephate, azinphos-ethyl, azinphos-methyl, diazinon, methamidophos, methidathion, mevinphos, pirimiphos-ethyl and pirimiphos-methyl in whole raw milk, based on the QuEChERS method for extraction and clean-up, with detection and quantification by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) is described. The method was validated according to parameters of the Analytical Quality Assurance Manual from the Brazilian Ministry of Agriculture and Commission Decision 2002/657/EC, and proved suitable for analysis of these analytes within the proposed working range, with recovery values between 77% and 110%, a standard deviation lower than 20%, limits of detection between 0.05 and 0.99 µg l(-)(1), and limits of quantification between 0.15 and 1.98 µg l(-1). Samples from animals treated with abamectin, doramectin, ivermectin and diazinon were analysed by the validated method. Residues of aflatoxin M1 were also found in field samples at levels below the established maximum residue limit.

Optimization of an online heart-cutting multidimensional gas chromatography clean-up step for isotopic ratio mass spectrometry and simultaneous quadrupole mass spectrometry measurements of endogenous anabolic steroid in urine.

Measuring carbon isotope ratios (CIRs) of urinary analytes represents a cornerstone of doping control analysis and has been particularly optimized for the detection of the misuse of endogenous steroids. Isotope ratio mass spectrometry (IRMS) of appropriate quality, however, necessitates adequate purities of the investigated steroids, which requires extensive pre-analytical sample clean-up steps due to both the natural presence of the target analytes and the high complexity of the matrix. In order to accelerate the sample preparation and increase the automation of the process, the use of multidimensional gas chromatography (MDGC) prior to IRMS experiments, was investigated. A well-established instrumental configuration based on two independent GC ovens and one heart-cutting device was optimized. The first dimension (1D) separation was obtained by a non-polar column which assured high efficiency and good loading capacity, while the second dimension (2D), based on a mid-polar stationary phase, provided good selectivity. A flame ionization detector monitored the 1D, and the 2D was simultaneously recorded by isotope ratio and quadrupole mass spectrometry. The assembled MDGC set-up was applied for measuring testosterone, 5α- and 5ß-androstanediol, androsterone, and etiocholanolone as target compounds and pregnanediol as endogenous reference compound. The urine sample were pretreated by conventional sample preparation steps comprising solid-phase extraction, hydrolysis, and liquid-liquid extraction. The extract obtained was acetylated and different aliquots were injected into the MDGC system. Two high performance liquid chromatography steps, conventionally adopted prior to CIR measurements, were replaced by the MDGC approach. The obtained values were consistent with the conventional ones. Copyright © 2016 John Wiley & Sons, Ltd.

Electrospray ionization mass and tandem mass spectra of a series of N-pyrazolylmethyl and N-triazolylmethyl N-phenylpiperazines: new dopaminergic ligands with potential antipsychotic properties.

Recently, two analogous series of N-pyrazolylmethyl and N-triazolylmethyl N-phenylpiperazines have been prepared and found to be potential antipsychotic drugs acting as new selective ligands of the dopamine D2 receptor. Herein we report a systematic study of their high-resolution electrospray ionization mass and tandem mass spectra in which the main dissociation routes of their protonated molecules are determined and rationalized. The ESI-MS/MS data is very characteristic for both series allowing straightforward isomeric differentiation. A single and dominant fragment ion for the pyrazole series and four major fragment ions for the triazole series are useful for selective reaction MS monitoring of these potential drugs in biological fluids.

New approaches on the analyses of thermolabile coffee diterpenes by gas chromatography and its relationship with cup quality.

A new gas chromatography method using pulsed split injector (PS-GC) was validated to quantify thermolabile diterpenes cafestol, kahweol and isokahweol in methanolysed Arabica coffee oils. Linearity was 0.99 from 8 to 69mgmL(-1), recovery ranged from 99% to 101% and precision of less than 4% was obtained. Besides, Soxhlet extraction time was evaluated and Tukey׳s test indicated that the mass of diterpenes obtained in 4h is equivalent to a 16h period, which represents a space-time yield four times higher. The microwave assisted methanolysis proved to be efficient to quantitatively convert the natural diterpene esters in their respective alcohols and fatty acid methyl esters, accompanied by PS-GC. Also, the intact diterpene esters were analyzed by GC for the first time by the comparison between cold on-column (COC) and PS injection techniques. In all these stages, the molecular integrity of the thermolabile furokaurane diterpenes was maintained. The methanolysed oils from 13 samples of green Brazilian Arabica coffees were analyzed by PS-GC and the diterpenes composition varied from 8 to 12% w/w in oil and 0.7-1% in coffee beans. The ratio between cafestol and kahweol was successfully used to predict the quality of coffee even before the roasting and brewing processes.

Improvement in steroid screening for doping control with special emphasis on stanozolol.

The Medical Commission of the International Olympic Committee forbids the use of anabolic androgenic steroids and beta2-agonists to improve athletic performance. In this work we have selected examples of anabolic androgenic compounds and their metabolites to evaluate the GC-MS analysis of some trimethylsilyl derivatives. The aim is to set the best GC conditions to improve the detection within the whole range of analyte elution temperatures. The initial column temperature was changed to 105 or 140 degrees C followed by 40 degrees C min(-1) to 200 degrees C and then 15 degrees C min(-1) to 300 degrees C. Using 140 degrees C as the initial oven temperature it was possible to obtain narrower initial analyte distributions for the compounds that elutes at the beginning of the chromatogram as clenbuterol, mabuterol, epimethylenediol and norandrosterone, without loss of derivatized metabolites signal. Later. eluting analytes, such as the stanozolol metabolites, furazabol and oxandrolone were not affected. Temperatures below 140 degrees C. resulted in partial derivatization for some analytes mainly stanozolol related structures. Therefore evaluation of derivatization conditions as occurring in three steps, the vial, vaporization chamber and capillary column, was thoroughly assessed. The new program temperature improves the signal-to-noise ratio for some compounds and shows adequate resolution for endogenous compounds. Some of the difficult key separations necessary for doping control enforcement were also obtained with the proposed method.

Chemical composition of Tipuana tipu, a source for tropical honey bee products.

Tipuana tipu (Benth.) Kuntze is a tree from the leguminosae family (Papilionoideae) indigenous in Argentina and extensively used in urbanism, mainly in Southern Brazil. The epicuticular waxes of leaves and branch, and flower surface were studied by high temperature high resolution gas chromatography. Several compounds were characterized, among which the aliphatic alcohols were predominant in branch, leaves and receptacle. Alkanes were predominant only in the petals and the aliphatic acids were predominant in stamen. In branches and leaf epicuticular surfaces, six long chain wax esters series were characterized, as well as lupeol and b-amyrin hexadecanoates.

Distribution of quinic acid derivatives and other phenolic compounds in Brazilian propolis.

The quinic acid derivatives (including 4-feruoyl quinic and 5-ferruoyl quinic acids characterized for first time in propolis samples) and other phenolic compounds were quantified in thirteen Brazilian propolis samples by HPLC analysis. For chemometrical analysis, the distribution of quinic acid derivatives and other phenolic compounds were considered. The results suggest that the Brazilian propolis with floral origin from Citrus sp. have the highest concentration of the quinic acid derivatives (between 11.0 to 58.4 mg/mg of the dried crude hydroalcoholic extract) and therefore would probably show a more effective hepatoprotective activity.

[Comments on studies of industrial and non-industrial environments in Brazil: a comparative approach]. / Considerações sobre estudos de ambientes industriais e não industriais no Brasil: uma abordagem comparativa.

Air quality in industrial environments has been studied since the second half of the 20th century. Well-documented results show that exposure to chemical pollutants through their consumption, production, and storage can damage workers' health. New research focusing on non-industrial environments has shown unexpected correlations between air quality and health effects. Artificial ventilation systems have been related to health-related complaints and a high rate of absenteeism. Symptoms related to air quality in non-industrial environments are recognized by the World Health Organization and encompassed under the so-called sick building syndrome (SBS). In Brazil there are few studies comparing indoor air quality and health. The present study is intended to map the studies already conducted in Brazil in order to encourage further research in this area, due to its importance for public health.