Asymmetric bioreduction of ß-ketoesters derivatives by Kluyveromyces marxianus: influence of molecular structure on the conversion and enantiomeric excess.

This study presents the bioreduction of six ß-ketoesters by whole cells of Kluyveromyces marxianus and molecular investigation of a series of 13 ß-ketoesters by hologram quantitative structure-activity relationship (HQSAR) in order to relate with conversion and enantiomeric excess of ß-stereogenic-hydroxyesters obtained by the same methodology. Four of these were obtained as (R)-configuration and two (S)-configuration, among them four compounds exhibited >99% enantiomeric excess. The ß-ketoesters series LUMO maps showed that the ß-carbon of the ketoester scaffold are exposed to undergo nucleophilic attack, suggesting a more favorable ß-carbon side to enzymatic reduction based on adopted molecular conformation at the reaction moment. The HQSAR method was performed on the ß-ketoesters derivatives separating them into those provided predominantly (R)- or (S)-ß-hydroxyesters. The HQSAR models for both (R)- and (S)-configuration showed high predictive capacity. The HQSAR contribution maps suggest the importance of ß-ketoesters scaffold as well as the substituents attached therein to asymmetric reduction, showing a possible influence of the ester group carbonyl position on the molecular conformation in the enzyme catalytic site, exposing a ß-carbon side to the bioconversion to (S)- and (R)-enantiomers.

Thermodynamic-based retention time predictions of endogenous steroids in comprehensive two-dimensional gas chromatography.

This work evaluates the application of a thermodynamic model to comprehensive two-dimensional gas chromatography (GC × GC) coupled with time-of-flight mass spectrometry for anabolic agent investigation. Doping control deals with hundreds of drugs that are prohibited in sports. Drug discovery in biological matrices is a challenging task that requires powerful tools when one is faced with the rapidly changing designer drug landscape. In this work, a thermodynamic model developed for the prediction of both primary and secondary retention times in GC × GC has been applied to trimethylsilylated hydroxyl (O-TMS)- and methoxime-trimethylsilylated carbonyl (MO-TMS)-derivatized endogenous steroids. This model was previously demonstrated on a pneumatically modulated GC × GC system, and is applied for the first time to a thermally modulated GC × GC system. Preliminary one-dimensional experiments allowed the calculation of thermodynamic parameters (ΔH, ΔS, and ΔC p ) which were successfully applied for the prediction of the analytes' interactions with the stationary phases of both the first-dimension column and the second-dimension column. The model was able to predict both first-dimension and second-dimension retention times with high accuracy compared with the GC × GC experimental measurements. Maximum differences of -8.22 s in the first dimension and 0.4 s in the second dimension were encountered for the O-TMS derivatives of 11ß-hydroxyandrosterone and 11-ketoetiocholanolone, respectively. For the MO-TMS derivatives, the largest discrepancies were from testosterone (9.65 ) for the first-dimension retention times and 11-keto-etiocholanolone (0.4 s) for the second-dimension retention times.

Effective treatment and prevention of attempted suicide, anxiety, and aggressiveness with fluoxetine, despite proven use of androgenic anabolic steroids.

The treatment of a man who attempted suicide after experiencing symptoms of anxiety and aggressiveness associated with the use of androgenic-anabolic steroids (AAS) is described. This report includes 30 days of inpatient treatment and a 6-month follow-up. Regular use of fluoxetine apparently prevented the onset of anxiety, depression, aggressiveness, and suicide ideation, even with the concurrent use of AAS. The urinary concentration of androgens, metabolites of AAS, and fluoxetine were monitored through analysis of urinary samples by the Brazilian Laboratory of Doping Control. Our results are congruent with previous findings describing the risk of suicide prompted by AAS use as well as the efficacy of fluoxetine in the treatment of mood disorders associated with the use of anabolic steroids.

Chemical and statistical analyses of blotter paper matrix drugs seized in the State of Rio de Janeiro.

Drugs of abuse are psychoactive substances illicitly distributed and used worldwide. In Rio de Janeiro, Brazil, they represent a public health issue and are directly related to several social problems. The recent increase in appearances of new psychoactive substances (NPS), derived from structural modifications of existing psychoactive substances, poses a threat to public health and forensic laboratories worldwide, as little is known about these substances. This study aimed to chemically and geographically map drugs of abuse from blotter papers seized by the Civil Police of Rio de Janeiro State (PCERJ) between 2006 and 2019. High-performance analytical techniques, such as gas chromatography-mass spectrometry (GC-MS) and Orbitrap mass spectrometry (Orbitrap-MS), combined with statistical analyses, were employed to characterize the seized samples. The most common chemical compounds in NPS found in this study were synthetic phenethylamines, i.e., molecules from the 25I-NBOH (2-(((4-iodo-2,5-dimethoxyphenethyl)amino)methyl)phenol) and 25I-NBOMe (2-(4-iodo-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine) families. Prior to 2014, the majority of seized blotter papers contained lysergic acid diethylamide (LSD) and were concentrated in the Metropolitan region. An upsurge in blotter paper seizures was observed from 2014 to 2017; the most common substances during this time were from the NBOMe family. NBOH compounds emerged in 2016 in coastal regions with high tourism, reaching over 1300 items only in 2017. Only one synthetic cannabinoid was found among the blotter papers seized in Rio de Janeiro between 2006 and 2019. The assembled chemical data and statistical analyses allowed the mapping and monitoring of the chemical profiles of the seized blotter papers, providing a strong foundation for the understanding of the origins and movement of these drugs around the RJ State.

Analysis of exemestane and 17ß-hydroxyexemestane in human urine by gas chromatography/mass spectrometry: development and validation of a method using MO-TMS derivatives.

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 17ß-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.

Comprehensive Zebrafish Water Tank Experiment for Metabolic Studies of Testolactone.

Testolactone is a potent steroid aromatase (CYP19A1) inhibitor, and its main effect is a reduction in estradiol and estrone and an increase in testosterone and androstenedione levels. In this work, we evaluated a zebrafish water tank (ZWT) as a model to investigate testolactone biotransformation and the possibility to increase knowledge regarding the applicability of the ZWT on steroid hormone elimination research, as well as on the impact of steroid hormones on the endogenous metabolism of zebrafish. High-resolution mass spectrometry combined with SIEVE software was used to discriminate the peaks of interest based on significant changes in the relative signal intensity of the m/z values between different ZWT experiments. The metabolites, 4,5-dihydrotestolactone and 1,2,4,5-tetrahydrotestolactone, the same metabolites as those described in humans, were detected in ZWT, both in quite similar proportions. The presence of testolactone in the ZWT caused a rise in testosterone and androstenedione in the water tank, similar to that in human serum. These data suggest that, while the concentration of testolactone was high enough to inhibit the aromatase enzyme, an accumulation of androgens in the water occurred, indicating that the ZWT can be considered a model to investigate the impact of steroids on live organisms.

Delirium following ingestion of marijuana present in chocolate cookies.

OBJECTIVE: To describe a case of marijuana-induced delirium and the techniques used to detect psychoactive agents and metabolites in urine. METHODS: A case of delirium following involuntary ingestion of cannabis is described. A urine sample was analyzed applying various chemical procedures, using high-resolution gas chromatography coupled with mass spectrometry technique. 11-Nor-delta9-tetrahydrocannabinol-9-carboxylic acid was quantified by the methodology for steroids and cannabis. RESULTS: A 26-year-old man was hospitalized with severe agitation and psychotic behavior 36 hours after suspected involuntary ingestion of cannabis contained in a brigadeiro, a typical Brazilian cookie, consumed during a party. Treatment with neuroleptics resulted in complete regression of the symptoms over 2 weeks. The urine sample revealed marijuana ingestion, demonstrated by the presence of its metabolite, 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid in high concentration. No other investigated substance was found in the patient's urine. CONCLUSION: Severe delirium can follow oral ingestion of cannabis integrated into homemade cookies.

Treatment of an industrial stream containing vinylcyclohexene by the H2O2/UV process.

Petrochemical industries generate wastewaters containing pollutants that can severely impact the biological treatment systems. Some streams from specific production units may contain nonbiodegradable or toxic compounds that impair the performance of the wastewater treatment plant and should be segregated and treated by specific techniques. In this work, the utilization of chemical oxidation (H2O2/UV) was investigated for removing 4-vinylcyclohexene (VCH) from a liquid stream coming from the production of hydroxylated liquid polybutadiene (HLPB). Besides VCH, this stream also contains ethanol, hydrogen peroxide, and many other organic compounds. Experiments were carried out in a small-scale photochemical reactor (0.7 L) using a 25-W low-pressure mercury vapor lamp. The photochemical reactor was operated in batch, and the reaction times were comprised between 10 and 60 min. Assays were also performed with a synthetic medium containing VCH, H2O2, and ethanol to investigate the removal of these substances in a less complex aqueous matrix. By-products formed in the reaction were identified by gas chromatography and mass spectroscopy (GC-MS). VCH was significantly removed by the oxidation process, in most assays to undetectable levels. Ethanol removal varied from 16 to 23 % depending on the reaction conditions. Acetic acid, acetaldehyde, and diols were detected as by-products of the industrial wastewater stream oxidation. A drop on the toxicity of the industrial stream was also observed in assays using the organism Artemia salina.

Systematic analysis of glycerol: colourimetric screening and gas chromatography-mass spectrometric confirmation.

Glycerol is a naturally occurring polyol in the human body, essential for several metabolic processes. It is widely used in the food, pharmaceutical, and medical industries and in clinical practice as a plasma volume expander (PVE). Athletes, however, may use glycerol to mask the presence of forbidden substances or to enhance performance, inclusively through hyperhydration achieved by glycerol ingestion with added fluid. These practices are considered doping, and are prohibited by the World Anti-Doping Agency (WADA). Therefore, glycerol was introduced in the prohibited list. Doping through glycerol ingestion can readily be identified by detection of elevated glycerol concentrations in urine. In this paper, a protocol for the fast detection of glycerol in urine is proposed. It consists of a previous visual colourimetric screening, followed by a quantitative/qualitative confirmation analysis by mass spectrometry. The screening procedure involves a reaction in which polyhydric alcohols are oxidized by periodate to formic acid and formaldehyde, which is detected by the addition of a fuchsin solution. For the subsequent qualitative/quantitative confirmation analysis, a gas chromatography-mass spectrometry based approach with a non-deuterated internal standard and a drying step of only 10 min is proposed. The linear correlation was demonstrated within WADA´s threshold range. The calculated RSD were 2.1% for within-day precision and 2.8% for between-day precision. The uncertainty estimation was calculated, and a value of 2.7% was obtained. The procedure may also be used for the analysis of other polyols in urine, as for example the PVE mannitol.

Three series of high molecular weight alkanoates found in Amazonian plants.

Electron impact mass spectra were measured by high temperature high resolution gas chromatography-mass spectrometry (HT-HRGC-MS) for three homologous series of high molecular weight compounds present in the Amazonian plants Marupá (Simaruba amara) and Brazil nut (Bertholettia excelsa). Based on their mass spectra, the compounds were identified as three wax ester series of alpha-tocopherol (vitamin E), beta-tocopherol and phytol (2,6,10,14-tetramethylhexadec-14-en-16-ol). The interpretations are supported by high resolution mass spectrometry and GC retention indices of authentic standards.

Chiral separation of gamma-butyrolactone derivatives by gas chromatography on 2,3-di-O-methyl-6-O-tert.-butyldimethylsilyl-beta-cyclodextrin.

The chiral GC separation of 2-alkyl-2-keto-gamma-butyrolactone derivatives and their alcohol analogs using 2,3-di-O-methyl-6-O-tert.-butyldimethylsilyl-beta-cyclodextrin (DIMETBCD) as chiral selector was discussed. The results, supported by the ketone preliminary molecular modelling calculations, suggest that the chiral recognition for DIMETBCD depends more on the geometry than on the polarity of the alkyl substituents on the butyrolactones. Hydrogen bonds and alkyl group steric effects should be an important function of the alcohol chiral recognition for DIMETBCD. Comparison of the retention times of the alcohol derivatives, in achiral and chiral stationary phases, suggests a specific structural effect for the cyclodextrin selctor.

Validation of the determination of oxymetholone in human plasma analysis using gas chromatography-mass spectrometry. Application to pharmacokinetic studies.

A simple and rapid procedure for extraction of oxymetholone in human plasma using gas chromatography coupled with quadrupole mass spectrometric was evaluated. The method involves analyte extraction with tert.-butylmethylether after alkalinization of the plasma and derivatization with MSTFA-NH(4)I-2-mercaptoethanol before the high resolution gas chromatographic-mass spectrometry separation. Methyltestosterone was used as internal standard. The calibration curves were linear, with typical r(2) values >0.995 and F(table)>F(calculated) (alpha=0.05). Recovery from plasma proved to be more than 70%. The method was accurate and reproducible, and was successfully applied to determine the pharmacokinetic parameters of oxymetholone for healthy volunteers after oral administration of 50 mg of the compound. The (C(max)) and (T(max)) were 18.8 +/- 0.4 ng/ml and 210 +/- 42.4 min, respectively.

Linalool from Lippia alba: study of the reproducibility of the essential oil profile and the enantiomeric purity.

A new chemotype of the aromatic Verbenaceae species Lippia alba Mill. N. E. Br. from southeastern Brazil has recently been shown to have a high content of linalool in the leaf essential oil. Vegetative propagation of this chemotype was conducted at six different locations in Brazil, and the variation of the content and the optical purity of linalool in the oils were verified. Yields (0.6-0.9%, hydrodistillation), chemical composition, linalool content, and optical purity of the oils from all the plants were compared, using GC-FID, GC-MS, chiral chromatography, and retention index calculation. No plant exceeded the matrix in linalool content (46.5 to 90.7%), and the chemical profile of the oils was the same for all the samples. Purification of linalool to a content close to 100% was effected by vacuum distillation of the crude oil. Chiral analysis showed exclusively the presence of S-linalool in all the crude oils and in the distilled samples.

Lupeol alkanoates in Brazilian propolis.

High temperature high resolution gas chromatography coupled to mass spectrometry (HT-HRGC-MS) isa powerful analytical tool. In this work we applied this technique to the study of crude extracts of propolis collected near the city of Uberlândia-Minas Gerais State. Eucalyptus trees and native plants from "cerrado" (savannah) were the material sources disposable for the Apis mellifera bees. A lot of known propolis constituents were identified, however, several high molecular weight compounds including lupeol alkanoates were identified for first time in propolis.

Identification of sympathomimetic alkylamine agents in urine using liquid chromatography-mass spectrometry and comparison of derivatization methods for confirmation analyses by gas chromatography-mass spectrometry.

The detection of 11 sympathomimetic alkylamines in urine was presented with a focus on human doping control is proposed using liquid chromatography tandem mass spectrometry (LC-QqQ) and high resolution mass spectrometry (LC-HRMS) as a screening tool after a dilute-and-shoot (DS) approach. For the LC-HRMS analyses, several compounds exhibited better limits of detection (L.O.D.) than the LC-QqQ. However, due to their small differences in structure, co-elution among the alkylamines was observed. Therefore, the chemical conversion of the alkylamines into an appropriate derivative for the confirmation analyses using gas chromatography-mass spectrometry (GC-MS) was evaluated. Five derivatization approaches were evaluated in an attempt to increase the analytical response and the confidence of the identification. The choice of the appropriated derivative for each alkylamine makes their spectra more easily interpretable, fulfills the WADA's rather strict identification criteria and enables the unequivocal identification of alkylamines in urine.

Asymmetric bioreduction of ketoesters derivatives by Kluyveromyces marxianus: influence of molecular structure on the conversion and enantiomeric excess

ABSTRACT This study presents the bioreduction of six β-ketoesters by whole cells of Kluyveromyces marxianus and molecular investigation of a series of 13 β-ketoesters by hologram quantitative structure-activity relationship (HQSAR) in order to relate with conversion and enantiomeric excess of β-stereogenic-hydroxyesters obtained by the same methodology. Four of these were obtained as (R)-configuration and two (S)-configuration, among them four compounds exhibited >99% enantiomeric excess. The β-ketoesters series LUMO maps showed that the β-carbon of the ketoester scaffold are exposed to undergo nucleophilic attack, suggesting a more favorable β-carbon side to enzymatic reduction based on adopted molecular conformation at the reaction moment. The HQSAR method was performed on the β-ketoesters derivatives separating them into those provided predominantly (R)- or (S)-β-hydroxyesters. The HQSAR models for both (R)- and (S)-configuration showed high predictive capacity. The HQSAR contribution maps suggest the importance of β-ketoesters scaffold as well as the substituents attached therein to asymmetric reduction, showing a possible influence of the ester group carbonyl position on the molecular conformation in the enzyme catalytic site, exposing a β-carbon side to the bioconversion to (S)- and (R)-enantiomers.

Plasma volume expanders: use in medicine and detecting misuse in sports.

Plasma volume expanders comprise a heterogeneous group of substances used in medicine that are intravenously administered in cases of great blood loss owing to surgery or medical emergency. These substances, however, can also be used to artificially enhance performance of healthy athletes in sport activities, and to mask the presence of others substances. These practices are considered doping, and are therefore prohibited by the International Olympic Committee and the World Antidoping Agency. Consequently, drug testing procedures are essential. The present work provides an overview of plasma volume expanders, assembling pertinent data such as chemical characteristics, physiological aspects, adverse effects, doping and analytical detection methods, which are currently dispersed in the literature.

Study of the endogenous steroid profile of male athletes from the Brazilian National Soccer Championship 2009.

Changes in the endogenous profile of androgenic anabolic steroids (AAS) may be interpreted as markers of doping. The objective of this study was to evaluate the endogenous profile of AAS in male athletes of the 2009 Brazilian National Soccer Championship, in normal conditions, particularly in the light of the revision of World Anti-Doping Agency's (WADA) Technical Document on the Interpretation of Endogenous AAS in athletes for doping control drafted in that year, as well as comparing these results to profiles already published in the literature. The upper limit of the 95% central reference interval of the following parameters for the studied population were estimated to be significantly higher than WADA's criteria, with a confidence of 90%: DHEA (about 2.3 times higher), Adiol (1.2 times higher), Bdiol (2.7 times higher), and Adiol/E (6 times higher). These findings seem to imply that WADA's criteria proposed in 2009 for DHEA, Adiol, Bdiol, and Adiol/E may not have been applicable to the studied population. Moreover, their comparison to previously published studies pointed to the need to evaluate in detail the appropriateness of adopting these criteria as universal, since there seems to be variations among different populations of athletes.

Determination of stavudine in human serum by on-line solid-phase extraction coupled to high-performance liquid chromatography with electrospray ionization tandem mass spectrometry: application to a bioequivalence study.

A method based on solid-phase extraction (SPE) coupled to high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the determination of stavudine in human serum, using didanosine as internal standard. The acquisition was performed in multiple reaction monitoring (MRM) mode. The method was linear over the studied range (10-2000 ng/mL), with r(2) > 0.99, and the run time was 4 min. The intra- and inter-assay precisions (%) were in the ranges 0.1-13.6 and 2.6-9.9, respectively, and the intra- and inter-assay accuracies were >92%. The absolute recoveries were approximately 100% (10 ng/mL), 98% (30 ng/mL), 105% (750 ng/mL) and 105% (1500 ng/mL). The limits of detection and quantitation were 4 and 10 ng/mL, respectively. The analytical method was applied to a bioequivalence study, in which 24 healthy adult volunteers (12 men) received single oral doses (40 mg) of reference and two test stavudine formulations, in an open, three-period, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration), AUC(0-10) and AUC(0-inf) (areas under the serum concentration vs. time curve from time zero to 10 h and to infinity, respectively), were in the range 80-125%, which supports the conclusion that the two test formulations are bioequivalent to the reference formulation with respect to the rate and extent of stavudine absorption.