Comprehensive analysis by liquid chromatography Q-Orbitrap mass spectrometry: Fast screening of peptides and organic molecules.

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.

Development and validation of a ultra high performance liquid chromatography-tandem mass spectrometric method for the direct detection of formoterol in human urine.

Formoterol is a long acting ß(2)-agonist and has proven to be a very effective bronchodilating agent. Hence it is frequently applied therapeutically for the treatment of asthma. Because ß(2)-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 30 ng/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 2 µL was injected in the chromatographic system. Chromatography was performed on a C(8)-column using gradient conditions. The mobile phase consisted of water/methanol (H(2)O/MeOH) both containing 0.1% acetic acid (HOAc) and 1mM ammonium acetate (NH(4)OAc). Calibration curve were constructed between 15 and 60 ng/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.

Consequences of the formation of 3,4-dimethyl-5-phenyl-1,3-oxazolidine on the analysis of ephedrines in urine by gas chromatography and a new method for confirmation as N-trifluoroacetyl-O-t-butyldimethylsilyl ether derivatives.

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.

Analysis of phytoestrogens, progestogens and estrogens in environmental waters from Rio de Janeiro (Brazil).

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 < or =2 ng/L, good linearity and reproducibility.

Analytical challenges in doping control: Comprehensive two-dimensional gas chromatography with time of flight mass spectrometry, a promising option.

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 (2ng/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 x 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.

Uric acid changes in urine and plasma: an effective tool in screening for purine inborn errors of metabolism and other pathological conditions.

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.

Chemical composition and microbicidal activity of extracts from Brazilian and Bulgarian propolis.

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.

Further lipophilic flavonols in Vellozia graminifolia (Velloziaceae) by high temperature gas chromatography: quick detection of new compounds.

Further lipophilic flavonols present in Vellozia graminifolia have been determined by high temperature high resolution gas chromatography (HTHRGC) and by HTHRGC coupled to mass spectrometry (MS). These methods resulted in the detection, isolation and characterisation of a monoisoprenylated flavonol 3,5,4'-trimethoxy-3'-hydroxy-6,7-(2"-isopropenyldihydrofurano)flavone from the ethyl acetate extract of the whole plant. The structural elucidation was accomplished using spectral data, including two-dimensional NMR, and on chemical transformations. Both HTHRGC and HTHRGC-MS were shown to be alternative and extremely valuable methods for the quick screening of flavonoid aglycones and other chemical metabolites of the Velloziaceae.

Seasonal variation of the chemical constituents from Croton species.

The terpenes, sterols, alkaloid (glaucine) and alpha-tocopherol show seasonal variation for Croton hemiargyreus hemiargyreus and Croton echinocarpus. The amounts of triterpenes are higher during the tropical summer and in most samples the major sesquiterpene was characterized as caryophyllene. The seasonal variation of glaucine showed a maximum between June and October for C. hemiargyreus, and was present only in January and June in C. echinocarpus.

Rapid screening of polar compounds in Brazilian propolis by high-temperature high-resolution gas chromatography-mass spectrometry.

Methanol extracts of propolis from six different places, five in Rio de Janeiro state and one in São Paulo state, both in the Southeast of Brazil, were investigated using high-temperature high-resolution gas chromatography (HT-HRGC) and HT-HRGC-mass spectometry. The main purpose of the study was to establish the applicability of HT-HRGC as an analytical method for systematic studies of polar propolis fractions. Several compounds, including carbohydrates, phenolic acid derivatives, and high molecular weight compounds (e.g., wax esters of long chain fatty alcohols) could be readily characterized in the crude extracts by HT-HRGC-MS. HT-HRGC and HT-HRGC-MS were shown to be quick and informative tools for rapid analysis of crude polar extracts without cleanup.

Rapid screening of natural products by high-resolution high-temperature gas chromatography.

The crude ethanol extracts from the leaves of three Croton hemiargyrus hemiargyreus plants are fractionated by thin-layer chromatography, yielding five fractions. The fractions and the crude extract are analyzed by high-temperature high-resolution gas chromatography coupled with mass spectrometry (HT-HRGC-MS). Several natural products, including thermolabile components, can be characterized directly in the samples, such as alkaloids, terpenes, flavonoids, acids, alcohols, etc. The cold on-column technique proves to be appropriate for the injection of these thermolabile compounds. HT-HRGC-MS is shown to be a valuable tool and an alternative technique to classical phytochemical procedures for the simple and fast routine analysis of natural products in crude extracts.

Systematic study of high molecular weight compounds in Amazonian plants by high temperature gas chromatography-mass spectrometry.

The fractions of hexane and dichloromethane extraction from marupá (Simaruba amara) and (Bertholletia excelsa) leaves were analyzed by HT-HRGC (high temperature high resolution gas chromatography) and HT-HRGC coupled to mass spectrometry (HT-HRGC-MS). Several compounds can be characterized including unusual high molecular weight compounds.

Analysis and quantitation of rotenoids and flavonoids in Derris (Lonchocarpus urucu) by high-temperature high-resolution gas chromatography.

A glass capillary column coated with PS-086 (15% phenyl-80% methylpolysiloxane, 15 m x 0.30-mm i.d., 0.1-microm film thickness) is used to analyze extracts from Lonchocarpus urucu (Derris urucu). Several secondary metabolites (8 flavonoids, 10 rotenoids) are characterized without derivatization, and the rotenoids are quantitated by high-temperature high-resolution gas chromatography (HTHRGC) and HTHRGC coupled with mass spectrometry (HTHRGC-MS). The limit of detection in flame ionization detection of rotenone is approximately 0.5 microg/mL, and the limit of quantitation was 2 microg/mL. Derris urucu bark is an excellent source of rotenone isomers (80 mg/g), deguelin (30 mg/g), and rotenolone (26 mg/g). Single solvent extractions (hexane, methylene dichloride, acetone, or methanol) are not able to fully extract the flavonoids and rotenoids. Complete extraction is achieved using a mixture of methanol-methylene dichloride (1:1), indicating a complex association of these compounds with the plant tissue. HTHRGC and HTHRGC-MS are shown to be quick and informative tools for the rapid analysis of crude extracts without the need for prior derivatization and fractionation.

Application of high-temperature gas chromatography-mass spectrometry to the investigation of glycosidically bound components related to cashew apple (Anacardium occidentale L. Var. Nanum) volatiles.

Free and bound volatile components of a Brazilian cashew apple variety (Anacardium occidentale L. var. nanum) were obtained by simultaneous distillation-extraction (SDE) and XAD-2 adsorption. According to gas chromatography-mass spectrometry (GC-MS) analyses and retention indices, 62 free volatile constituents were characterized and quantified. They were esters (40%), terpenes (20%), hydrocarbons (14%), fatty acids (9%), aldehydes (8%), alcohols (3%), lactones (3%), ketones (1%), phenols (1%), and norisoprenoids (1%). The glycosidically bound volatile precursors were analyzed by high-temperature GC-MS, after room temperature silylation. Several conjugated alcohols and cinnamic acids were detected and reported as cashew apple glycosyl constituents for the first time.

Improvement of enantioselective syntheses and chiral high resolution gas chromatographic analyses of (+)-2-allyl-2-carboethoxy-cyclopentanol.

The improvement of the biocatalytic reduction of 2-allyl-carboethoxy-cyclopentanone (2) to the corresponding cyclopentanol derivative (+)-(1R,2R)-(1) was accomplished employing baker's yeast in organic media. This chiral cyclopentanol derivative (1), analyzed by high resolution gas chromatography performed over beta-cyclodextrin stationary phase, was obtained in 38% yield (> 99% e.e.).