The number of substances nominally listed in the prohibited list of the World Anti‐Doping Agency increases each year. Moreover, many of these substances do not have a single analytical target and ...must be monitored through different metabolites, artifacts, degradation products, or biomarkers. A new analytical method was developed and validated for the simultaneous analysis of peptides and organic molecules using a single sample preparation and LC‐Q‐HRMS detection. The simultaneous analysis of 450 target molecules was performed after cleanup on a mixed‐mode solid‐phase extraction cartridge, combined with untreated urine. The cleanup solvent and reconstitution solvent were the most important parameters for achieving a comprehensive sample preparation approach. A fast chromatographic run based on a multistep gradient was optimized under different flows; the detection of all substances without isomeric coelution was achieved in 11 minutes, and the chromatographic resolution was considered a critical parameter, even in high‐resolution mass spectrometry detection. The mass spectrometer was set to operate by switching between positive and negative ionization mode for FULL‐MS, all‐ion fragmentation, and FULL‐MS/MS2. The suitable parameters for the curved linear trap (c‐trap) conditions were determined and found to be the most important factors for the development of the method. Only FULL‐MS/MS2 enables the detection of steroids and peptides at concentrations lower than the minimum required performance levels set by World Anti‐Doping Agency (1 ng mL−1). The combination of the maximum injection time of the ions into the c‐trap, multiplexing experiments, and loop count under optimized conditions enabled the method to be applied to over 10 000 samples in only 2 months during the 2016 Rio Summer Olympic and Paralympic Games. The procedure details all aspects, from sample preparation to mass spectrometry detection. FULL‐MS data acquisition is performed in positive and negative ion mode simultaneously and can be applied to untargeted approaches.
•Analytical methods to identify when inclusion complexes are formed.•Complexes prepared with monoterpenes and different cyclodextrins.•Freeze-drying is the best option to form inclusion complexes of ...borneol.
Monoterpenes are non-polar secondary metabolites widely used by industry due to their excellent therapeutic, food-ingredient and cosmetic properties. However, their low solubility in water limits their use. In this sense, cyclodextrins (CDs) have been widely used to solve these technological challenges. Thus, this study aims to use (–)-borneol as a monoterpene model to prepare inclusion complexes between β-CD and hydroxypropyl-β-CD (HP-β-CD) through different ways and characterize them in order to choose the best inclusion method to improve physicochemical properties of monoterpenes. To achieve this goal, the samples were prepared by physical mixture (PM), paste complex (PA) and freeze-drying complex (FD) and then, extensively characterized by thermal analysis, Fourier-transform infrared spectroscopy, scanning electron microscopy, size particle, X-ray diffraction and nuclear magnetic resonance. The physicochemical results showed that freeze-drying was more effective to form inclusion complexes between (–)-borneol with both CDs. This research highlights the importance of recognizing the best method to prepare inclusion complexes, including food additives as (–)-borneol, to achieve better results in food preparations.
The environment is currently exposed to a large variety of man-made chemicals (e.g. for industrial, medicinal use) which have potential adverse effects to its ecological status. In addition, the ...densely populated areas represent local high emissions of those chemicals leading to more aggravating consequences. Estrogenic compounds that end-up in environmental water directly affect living organisms by interfering with their endocrine metabolism. The assessment of their presence in the environment requires sensitive and selective analytical methods. Nineteen estrogenic compounds belonging to different classes (5 free estrogens, 6 conjugated estrogens, 3 progestogens and 5 phytoestrogens) have been studied. The analytical methodology developed is based on solid phase extraction followed by liquid chromatography tandem mass spectrometry and has been applied to study the occurrence of the above mentioned analytes in environmental waters from the state of Rio de Janeiro (Brazil). Due to insufficient infra-structure in this region, waste waters are released onto the environment without or with incomplete previous treatment. The results show that high levels of the phytoestrogens daidzein, coumestrol and genistein of up to 366 ng/L and progesterone of up to 47 ng/L could be found in river water. Estrogens and their conjugated derivatives were detected in the lower ng/L range up to 7 ng/L. The main estrogens estrone, estradiol and the synthetic ethinyl estradiol could not be detected. The developed method showed overall good performance with recoveries above 80% (with one exception), limits of detection ≤
2 ng/L, good linearity and reproducibility.
Formoterol is a long acting β2-agonist and has proven to be a very effective bronchodilating agent. Hence it is frequently applied therapeutically for the treatment of asthma. Because β2-agonists ...might be misused in sports for the stimulatory effects and for growth-promoting action their use is restricted. Since January 2012, formoterol is prohibited in urinary concentrations higher than 30ng/mL. The objective of this study was to develop and validate a simple and robust ultra high performance liquid chromatographic–tandem mass spectrometric (UHPLC–MS/MS) method for the direct quantification of formoterol in urine. Sample preparation was limited to an enzymatic hydrolysis step after which 2μL was injected in the chromatographic system. Chromatography was performed on a C8-column using gradient conditions. The mobile phase consisted of water/methanol (H2O/MeOH) both containing 0.1% acetic acid (HOAc) and 1mM ammonium acetate (NH4OAc). Calibration curve were constructed between 15 and 60ng/mL. Validation data showed bias of 1.3% and imprecision of 5.4% at the threshold. Ion suppression/enhancement never exceeded 7%. Calculating measurement uncertainty showed proof of applicability of the method. Stability of formoterol was also investigated at 56°C (accelerated stability test) at pH 1.0/5.2/7.0 and 9.5. At the physiological pH values of 5.2 and 7.0, formoterol showed good stability. At pH 1.0 and 9.5 significant degradation was observed.
Doping control screening based on the enhanced resolution of comprehensive two-dimensional (2D) gas chromatography hyphenated to time of flight mass spectrometer was investigated. The identification ...of anabolic agents (clenbuterol, norandrosterone, epimetendiol, two methyltestosterone metabolites and 3′-hydroxystanozolol) contained in a spiked urine sample (2
ng/ml) was demonstrated. Special emphasis was given to 3′-hydroxystanozolol, mainly considering the difficulty in its detection. In contrast to conventional GC–MS approaches that must use single-ion monitoring, the GC
×
GC–TOFMS method enabled the identification of that metabolite through the deconvolution of the full mass spectrum and also resolved the co-eluted peaks of 3′-hydroxystanozolol and an endogenous component.
A method for identifying the metabolites of sibutramine 1-(4(chlorophenyl)-N,N-dimethyl-α-(2-methylpropyl))cyclobutanemethanamine) in urine, utilizing a double derivatization strategy, with ...N-methyl-N-(trimethylsilyl)-trifluoroacetamide and N-methyl-bis-(trifluoroacetamide), in gas chromatography/mass spectrometry is proposed. This methodology results in mass spectra with at least three fragments in abundance superior to 20%, attending the World Anti-Doping Agency identification criteria for qualitative assays. The characterization of the derivatives was obtained through two ionization modes: Chemical Ionization and Electron Impact ionization, both in full scan mode. Sibutramine was administered to 5 (five) volunteers and the excretion profile followed for 92
h. Routine analytical, hydroxy-cyclobutane-bis-nor-sibutramine which becomes the more abundant metabolite in the first 10
h and hydroxy-isopropyl-bis-nor-sibutramine which becomes the most abundant after 40
h, were proposed for doping monitoring.
The compound 3,4-dimethyl-5-phenyl-1,3-oxazolidine can appear as an artifact during the gas chromatographic analysis of ephedrines. Its presence is a risk for doping control and forensic analyses. An ...evaluation about the consequences of its formation showed the possibility of a false positive for ephedrine, a false negative for pseudophedrine and increased uncertainty in the quantitative approach. Misinterpretations can be avoided with the observation of fragments
m/
z 56 and 71 in the ephedrine mass spectrum during GC–MS analysis and also by the formation of N-TFA-O-TBDMS derivatives prior to GC analysis. These N-TFA-O-TBDMS derivatives lead to an increase in the number and mass of diagnostic ions, meet the identification criteria, and provide an improvement in chromatographic resolution, allowing the separation of the ephedrines.
Aims: The chemical composition of ethanol extracts from a Brazilian (Et‐Bra) and a Bulgarian (Et‐Blg) propolis, and their activity against the protozoan Trypanosoma cruzi, several fungi and bacteria ...species were determined.
Methods and Results: The chemical composition was determined by high temperature high resolution gas chromatography coupled to mass spectrometry. Microbiological activity was assayed in vitro against T. cruzi, Candida albicans, Sporothrix schenckii, Paracoccidioides brasiliensis, Neisseria meningitidis, Streptococcus pneumoniae and Staphylococcus aureus.
Conclusions: Et‐Bra and Et‐Blg, although with totally distinct compositions, were active against T. cruzi and the three species of fungi. Et‐Blg was more effective than Et‐Bra against bacteria, particularly N. meningitidis and Strep. pneumoniae.
Significance and Impact of the Study: Although with different classes of components, both propolis extracts showed microbicidal activity. For the bactericidal activity it was possible to establish a positive correlation with the high content of flavonoids of the Bulgarian extract.
Purine inborn errors of metabolism (IEM) are serious hereditary disorders, which should be suspected in any case of neonatal fitting, failure to thrive, recurrent infections, neurological deficit, ...renal disease, self-mutilation and other manifestations. Investigation usually starts with uric acid (UA) determination in urine and plasma. UA, the final product of purine metabolism in humans, may be altered not only in purine IEM, but also in other related pathologies and clinical conditions. However, data and information about abnormal UA levels are scattered in the literature, often being controversial and confusing. A comprehensive overview has been elaborated, according to abnormal UA levels in urine and plasma, which associates these alterations with purine IEM. Other possible diseases, clinical conditions, diet and drug intake, related to the metabolism of uric acid, are also presented. The article includes tables that classify the disorders according to different patterns of UA alterations, with pertinent enzymes, clinical symptoms, inheritance and comments. Additionally, summarized pathophysiological mechanisms of important disorders are described. The overview is intended to assist in the interpretation of the results of UA analyses. It demonstrates that variation of UA concentrations in urine and plasma may constitute an effective tool in screening for purine IEM and other related pathological conditions.
Limb‐girdle muscular dystrophies (LGMD) are a group of genetically heterogeneous disorders characterized by predominantly proximal muscle weakness. We aimed to characterize epidemiological, clinical ...and molecular data of patients with autosomal recessive LGMD2/LGMD‐R in Brazil. A multicenter historical cohort study was performed at 13 centers, in which index cases and their affected relatives' data from consecutive families with genetic or pathological diagnosis of LGMD2/LGMD‐R were reviewed from July 2017 to August 2018. Survival curves to major handicap for LGMD2A/LGMD‐R1‐calpain3‐related, LGMD2B/LGMD‐R2‐dysferlin‐related and sarcoglycanopathies were built and progressions according to sex and genotype were estimated. In 370 patients (305 families) with LGMD2/LGMD‐R, most frequent subtypes were LGMD2A/LGMD‐R1‐calpain3‐related and LGMD2B/LGMD‐R2‐dysferlin‐related, each representing around 30% of families. Sarcoglycanopathies were the most frequent childhood‐onset subtype, representing 21% of families. Five percent of families had LGMD2G/LGMD‐R7‐telethonin‐related, an ultra‐rare subtype worldwide. Females with LGMD2B/LGMD‐R2‐dysferlin‐related had less severe progression to handicap than males and LGMD2A/LGMD‐R1‐calpain3‐related patients with truncating variants had earlier disease onset and more severe progression to handicap than patients without truncating variants. We have provided paramount epidemiological data of LGMD2/LGMD‐R in Brazil that might help on differential diagnosis, better patient care and guiding future collaborative clinical trials and natural history studies in the field.
