Coordination chemistry applied to anti-doping analysis: Cobalt quantification as its diethyldithiocarbamate complex by liquid chromatography coupled to high resolution tandem mass spectrometry.

Cobalt was included on the World Anti-Doping Agency Prohibited List in 2015 due to its effect on stimulus of erythropoiesis via stabilization of hypoxia-inducible factor. Although it has proven benefits for performance enhancement, the unavailability of inductively coupled plasma-mass spectrometry on routine of the accredited laboratories is a factor that reduces its applicability in anti-doping analysis. Therefore, an analytical method for quantification of urinary cobalt as its diethyldithiocarbamate complex by liquid chromatography coupled with high-resolution tandem mass spectrometry was developed and validated. Palladium was proposed as internal standard and rhodium as a complexation control. A microwave-assisted acid digestion of the urine samples was essential, not only to eliminate the matrix effect but mainly to avoid the non-specific bond of cobalt to endogenous molecules. A linear method was obtained over the studied range from a negative urine control to a spiked concentration of 25 ng/mL, with an estimated limit of quantification of 2.5 ng/mL, and an adequate combined standard uncertainty of 11.4%. Considering that all reagents are commercially available, the proposed strategy is feasible to be included on routine sample preparation. Monitoring urinary cobalt concentrations globally opens the perspective to support the anti-doping system to define a suitable threshold value and to understand its potential misuse by athletes seeking for performance improvement.

Assessing limits of detection in qualitative methods: A simple implementation of logistic regression in a web-based R Shiny application environment and its potential in toxicology and doping control.

The estimation of limits of detection (LOD) for solely qualitative methods in analytical chemistry may prove challenging because all the approaches with which chemists are familiar require some type of numeric data input. The best model to describe the binary response in these methods (detected/not detected) is a logistic model; however, these models are not easily handled by most of the laboratories and generally demand expensive statistical software packages. In this work, the advantages of applying this approach are discussed and its implementation using commercial spreadsheet software is demonstrated. A free online application based on the R environment using shinyapps was developed and its application was validated and discussed with a dataset of 57 different target compounds analyzed in urine according to the requirements of the World Anti-Doping Agency (WADA). This tool allows free, extremely quick, and easy determinations of LOD in qualitative analyses as well as the determination of the probabilities of detection in any given concentration.

A simple quinoline salt derivative is active in vitro against Plasmodiumfalciparum asexual blood stages and inhibits the development of cerebral malaria in murine model.

Chloroquine (CQ) was the most effective and widely used drug for the prophylaxis and treatment of severe and non-severe malaria. Although its prophylactic use has led to resistance to P. falciparum in all endemic countries, CQ still remains the drug of choice for the treatment of vivax malaria. Otherwise, the speed in which parasite resistance to available antimalarials rises and spreads in endemic regions points to the urgent need for the development of new antimalarials. Quinoline derivatives have been used as a tool in the search for new drugs and were investigated in the present study in an attempt to produce a HIT compound to avoid the cerebral malarial (CM). Seven compounds were synthesized, including three quinoline derivate salts. The cytotoxicity and antiplasmodial activity were assayed in vitro, highlighting compound 3 as a HIT, which also showed interaction with ferriprotoporphyrin IX similarly to CQ. Physicochemical and pharmacokinetic properties of absorption were found to be favorable when analyzed in silico. The in vivo assays, using the experimental cerebral malaria (ECM) model, showed important values of parasite growth inhibition on the 7th day-post infection (Q15 15 mg/kg: 76.9%, Q30 30 mg/kg: 90,1% and Q50 50 mg/kg: 92,9%). Compound 3 also showed significant protection against the development of CM, besides hepatic and renal parameters better than CQ. In conclusion, this quinoline derivative demonstrated promising activity for the treatment of malaria and was able to avoid the development of severe malaria in mice.

Variable Data Independent Acquisition and Data Mining Exploring Feature-Based Molecular Networking Analysis for Untargeted Screening of Synthetic Cannabinoids in Oral Fluid.

Novel psychoactive substances (NPS) are constantly emerging in the drug market, and synthetic cannabinoids (SCs) are included in this NPS family. Forensic laboratories often struggle with these continually emerging SCs, forcing them to develop an untargeted workflow to incorporate these psychoactive drugs in their procedures. Usually, forensic laboratories select analytical methods based on targeted mass spectrometry (MS) technologies for strictly tracking already known NPS. The appropriate way to tackle unknown substances is to develop pipelines for untargeted analysis that include LC-HRMS analytical methods and data analysis. Once established, this strategy would allow drug testing laboratories to be always one step ahead of the new trends concerning the "designer drugs" market. To address this challenge an untargeted workflow based on mass spectrometry data acquisition and data analysis was developed to detect SCs in oral fluid (OF) samples at a low concentration range. The samples were extracted by mixed-mode solid-phase extraction and analyzed by Liquid Chromatography - High-Resolution Mass Spectrometry (LC-HRMS). Tandem mass spectra (MS2) were recorded performing a variable isolation width across a mass range of all theoretical precursor ions (vDIA) after the chromatographic separation. After raw data processing with the MSDial software, the deconvoluted features were sent to GNPS for Feature-Based Molecular Networking (FBMN) construction for nontargeted data mining. The FBMN analysis created a unique integrated network for most of the SCs assessed in the OF at a low level (20 ng/mL). These results demonstrate the potential of an untargeted approach to detect different derivatives of SCs at trace levels for forensic applications.

In vitro and in vivo antiplasmodial activity of novel quinoline derivative compounds by molecular hybridization.

Chloroquine (CQ) has been the main treatment for malaria in regions where there are no resistant strains. Molecular hybridization techniques have been used as a tool in the search for new drugs and was implemented in the present study in an attempt to produce compound candidates to treat malarial infections by CQ-resistant strains. Two groups of molecules were produced from the 4-aminoquinoline ring in conjugation to hydrazones (HQ) and imines (IQ). Physicochemical and pharmacokinetic properties were found to be favorable when analyzed in silico and cytotoxicity and antiplasmodial activity were assayed in vitro and in vivo showing low cytotoxicity and selectiveness to the parasites. Candidates IQ5 and IQ6 showed important values of parasite growth inhibition in vivo on the 5th day after infection (IQ5 15 mg/kg = 72.64% and IQ6 15 mg/kg = 71.15% and 25 mg/kg = 93.7%). IQ6 also showed interaction with ferriprotoporphyrin IX similarly to CQ. The process of applying condensation reactions to yield imines is promising and capable of producing molecules with antiplasmodial activity.

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.

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.

DNA typing: an accessory evidence in doping control.

A clear positive case for anabolic steroids doping was confounded by alleged urine tampering during doping control procedures. Review of the chain of custody showed no flaws, but nevertheless the athlete was adamant that the urine sample should be analyzed for DNA in order to support her contention that she was not the donor of the sample. The results obtained showed that the urine sample that scored positive for steroids contained nuclear DNA that could not be matched to the DNA obtained from the athlete's blood. On the other hand, the same urine sample contained mitochondrial DNA whose nucleotide sequences spanning the hyper variable regions HV1 and HV2 proved to be identical to those determined in mitochondrial DNA amplified from the athlete's blood. The occurrence of an extraneous genotype is compatible with exogenous nuclear DNA admixture to the athlete's urine. Alternatively, taking in consideration the mitochondrial DNA, we could not exclude that a sibling or a maternal relative of the athlete could have acted as a donor of the urine utilized for doping control and DNA analysis. Both situations point to possible tampering of the urine by the athlete. Adjudication at CAS maintained previous national and international federation decision that there was no proof of a chain of custody flaw to justify the athlete's allegation of urine substitution after collection.