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.

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.

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.

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.

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.

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.

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.