Untargeted metabolomics in doping control : detection of new markers of testosterone misuse by ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry

The use of untargeted metabolomics for the discovery of markers is a promising and virtually unexplored tool in the doping control field. Hybrid quadrupole time-of-flight (QTOF) and hybrid quadrupole Orbitrap (Q Exactive) mass spectrometers, coupled to ultrahigh pressure liquid chromatography, are excellent tools for this purpose. In the present work, QTOF and Q Exactive have been used to look for markers for testosterone cypionate misuse by means of untargeted metabolomics. Two different groups of mine samples were analyzed, collected before and after the intramuscular administration of testosterone cypionate. In order to avoid analyte losses in the sample treatment, samples were just 2-fold diluted with water and directly injected into the chromatographic system. Samples were analyzed in both positive and negative ionization modes. Data from both systems were treated under untargeted metabolomic strategies using XCMS application and multivariate analysis. Results from the two mass spectrometers differed in the number of detected features, but both led to the same potential marker for the particular testosterone ester misuse. The in-depth study of the MS and MS/MS behavior of this marker allowed for the establishment of 1-cydopentenoylglycine as a feasible structure. The putative structure was confirmed by comparison with synthesized material. This potential marker seems to come from the metabolism of the cypionic acid release after hydrolysis of the administered ester. Its suitability for doping control has been evaluated

Involvement of Mechanical Cues in the Migration of Cajal-Retzius Cells in the Marginal Zone During Neocortical Development

Emerging evidence points to coordinated action of chemical and mechanical cues during brain development. At early stages of neocortical development, angiogenic factors and chemokines such as CXCL12, ephrins, and semaphorins assume crucial roles in orchestrating neuronal migration and axon elongation of postmitotic neurons. Here we explore the intrinsic mechanical properties of the developing marginal zone of the pallium in the migratory pathways and brain distribution of the pioneer Cajal-Retzius cells. These neurons are generated in several proliferative regions in the developing brain (e.g., the cortical hem and the pallial subpallial boundary) and migrate tangentially in the preplate/marginal zone covering the upper portion of the developing cortex. These cells play crucial roles in correct neocortical layer formation by secreting several molecules such as Reelin. Our results indicate that the motogenic properties of Cajal-Retzius cells and their perinatal distribution in the marginal zone are modulated by both chemical and mechanical factors, by the specific mechanical properties of Cajal-Retzius cells, and by the differential stiffness of the migratory routes. Indeed, cells originating in the cortical hem display higher migratory capacities than those generated in the pallial subpallial boundary which may be involved in the differential distribution of these cells in the dorsal-lateral axis in the developing marginal zone

Famílies botàniques de plantes medicinals

Facultat de Farmàcia, Universitat de Barcelona. Ensenyament: Grau de Farmàcia, Assignatura: Botànica Farmacèutica, Curs: 2013-2014, Coordinadors: Joan Simon, Cèsar Blanché i Maria Bosch.Els materials que aquí es presenten són els recull de 175 treballs d’una família botànica d’interès medicinal realitzats de manera individual. Els treballs han estat realitzat per la totalitat dels estudiants dels grups M-2 i M-3 de l’assignatura Botànica Farmacèutica durant els mesos d’abril i maig del curs 2013-14. Tots els treballs s’han dut a terme a través de la plataforma de GoogleDocs i han estat tutoritzats pel professor de l’assignatura i revisats i finalment co-avaluats entre els propis estudiants. L’objectiu principal de l’activitat ha estat fomentar l’aprenentatge autònom i col·laboratiu en Botànica farmacèutica

A first update on mapping the human genetic architecture of COVID-19

peer reviewe

Quantitative determination of paroxetine and its 4-hydroxy-3-methoxy metabolite in plasma by high-performance liquid chromatography/electrospray ion trap mass spectrometry: application to pharmacokinetic studies

7 pages, 4 figures, 3 tables.-- PMID: 12820211 [PubMed].-- Printed version published Jul, 22, 2003.A high-performance liquid chromatography (HPLC) method with tandem mass spectrometric detection is described for the determination of paroxetine, an antidepressant drug, and its metabolite (3S,4R)-4-(4-fluorophenyl)-3-(4-hydroxy-3-methoxyphenoxymethyl)piperidine (HM paroxetine) in human plasma. Plasma samples were hydrolysed with hydrochloric acid and then analytes were extracted with ethyl acetate at alkaline pH. Extracts were analysed by HPLC coupled to an atmospheric pressure ionisation-electrospray (ESI) interface and an ion trap mass spectrometer. Chromatography was performed on a reversed-phase column using acetonitrile/0.02% formic acid (66:34, v/v) as a mobile phase. The mass spectrometer was operated in the multiple reaction monitoring mode. The method was validated over concentration ranges of 0.75-100 g/L and 5-100 g/L for paroxetine and HM paroxetine, respectively. Mean recoveries of 77% for paroxetine and 76% for HM paroxetine were found, with precision always better than 15%. The limits of detection and quantification were 0.20 and 0.70 g/L for paroxetine, and 0.70 and 2.20 g/L for its metabolite. The method was applied to the analysis of plasma samples obtained from nine healthy male volunteers administered with a single oral dose of 20 mg paroxetine. After the 20-mg dose, the mean peak plasma concentration was 8.60 g/L for paroxetine and 92.40 g/L for HM paroxetine showing a tenfold ratio between the metabolite and the parent drug along the entire time-concentration curve.This study was supported by Area Progetto Droga from Istituto Superiore di Sanità, Roma (Italy), Fondo de Investigación Sanitaria, Madrid, Spain (grants FIS 97/1198, FIS 98/0181, FIS 00/0777 and FIS 01/1336) and Generalitat de Catalunya, Barcelona, Spain (GENCAT-CIRIT, grant 2001SGR00407).Peer reviewe

Stereochemical analysis of 3,4-methylenedioxymethamphetamine and its main metabolites by gas chromatography/mass spectrometry

7 pages, 3 figures, 3 tables.-- PMID: 12569443 [PubMed].-- Printed version published Feb 28, 2003.3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is consumed as the racemate but some metabolic steps are enantioselective. In addition, chiral properties are preserved during MDMA biotransformation. A quantitative analytical methodology using gas chromatography/mass spectrometry (GC/MS) to determine enantioselective disposition in the body of MDMA and its main metabolites including 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), and 4-hydroxy-3-methoxyamphetamine (HMA) was developed. Plasma and urine samples were collected from a male volunteer. The analysis of MDMA, MDA, and 4-hydroxy-3-methoxy metabolites by GC/MS required a two-step derivatization procedure. The first step consisted of derivatization of the amine with enantiomerically pure Mosher's reagent ((R)-MTPCl). Triethylamine was used as a base to neutralize hydrochloric acid formed during the reaction allowing quantitative derivatization, which resulted in a substantial improvement in the sensitivity of the method compared with other previously described techniques. Further treatment with ammonium hydroxide was required since both amine and hydroxyl groups underwent derivatization in the reaction. Ammonium hydroxide breaks bonds formed with hydroxyl groups without affecting amine derivatives. The second derivatization step using hexamethyldisilazane was needed for metabolites containing phenol residues. This derivatization method permitted the stereochemically specific study of MDMA and its main monohydroxylated metabolites by GC/MS. A detailed study of the chemical reactions involved in the derivatization steps was indispensable to develop a straightforward, sensitive, and reproducible method for the analysis of the parent drug compound and its metabolites.This study was funded by research grants FIS 98/0181, FIS 01/1336, and CIRIT 2001SGR00407.Peer reviewe

Determination of recent growth hormone abuse using a single dried blood spot

BACKGROUND: Although it is being increasingly applied, blood collection for drug testing in sport presents some logistic issues that complicate full applicability on a large scale. The use of dried blood spots (DBS) could benefit compliant blood testing considerably owing to its simplicity, minimal invasiveness, analyte stability, and reduced costs. The aim of this study was to evaluate the applicability of DBS to the methodology approved by the World Anti-Doping Agency (WADA) for detection of doping by recombinant human growth hormone (rhGH) in serum. METHODS: A protocol for a single DBS analysis using the hGH isoforms differential immunoassays (kit 1 and kit 2) was developed and validated. A clinical study with healthy volunteers injected for 3 consecutive days with a low subcutaneous dose (0.027 mg · kg(-1) · day(-1) · person(-1)) of rhGH was conducted. Finger prick DBS and paired-time serum samples from arm venipuncture were compared. RESULTS: The analysis of the DBS-based protocol indicated that with only a single blood spot it was possible to detect positivity for growth hormone abuse. In spite of the low rhGH dose administered and independently of the kit used, the window of detection for DBS was confirmed in all analyzed samples up to 8 h after rhGH administration and extended up to 12 h in 50% of the cases. Serum positivity was detected in all studied samples for 12 h after administration. CONCLUSIONS: These results support the usefulness of DBS as a biological matrix for testing recent growth hormone abuse.Funding: Spanish Ministerio de Economia y Competitividad (MINECO) (DEP2012-32048), International Olympic Committee (IOC)-2014 IOC Anti-Doping Research Fund, and Italian Istituto Superiore di Sanit

Determination of recent growth hormone abuse using a single dried blood spot

BACKGROUND: Although it is being increasingly applied, blood collection for drug testing in sport presents some logistic issues that complicate full applicability on a large scale. The use of dried blood spots (DBS) could benefit compliant blood testing considerably owing to its simplicity, minimal invasiveness, analyte stability, and reduced costs. The aim of this study was to evaluate the applicability of DBS to the methodology approved by the World Anti-Doping Agency (WADA) for detection of doping by recombinant human growth hormone (rhGH) in serum. METHODS: A protocol for a single DBS analysis using the hGH isoforms differential immunoassays (kit 1 and kit 2) was developed and validated. A clinical study with healthy volunteers injected for 3 consecutive days with a low subcutaneous dose (0.027 mg · kg(-1) · day(-1) · person(-1)) of rhGH was conducted. Finger prick DBS and paired-time serum samples from arm venipuncture were compared. RESULTS: The analysis of the DBS-based protocol indicated that with only a single blood spot it was possible to detect positivity for growth hormone abuse. In spite of the low rhGH dose administered and independently of the kit used, the window of detection for DBS was confirmed in all analyzed samples up to 8 h after rhGH administration and extended up to 12 h in 50% of the cases. Serum positivity was detected in all studied samples for 12 h after administration. CONCLUSIONS: These results support the usefulness of DBS as a biological matrix for testing recent growth hormone abuse.Funding: Spanish Ministerio de Economia y Competitividad (MINECO) (DEP2012-32048), International Olympic Committee (IOC)-2014 IOC Anti-Doping Research Fund, and Italian Istituto Superiore di Sanit