DEFAR - Departamento de Farmácia
URI permanente desta comunidadehttp://www.hml.repositorio.ufop.br/handle/123456789/530
Navegar
4 resultados
Resultados da Pesquisa
Item Evaluation of dermorphin metabolism using zebrafish water tank model and human liver microsomes.(2021) Castro, Juliana de Lima; Pereira, Henrique Marcelo Gualberto; Sousa, Valéria P. de; Martucci, Maria Elvira PoletiBackground: Dermorphin is a heptapeptide with an analgesic potential higher than morphine that does not present the same risk for the development of tolerance. These pharmacological features make dermorphin a potential doping agent in competitive sports and it is already prohibited for racehorses. For athletes, the development of an efficient strategy to monitor for its abuse necessitates an investigation of the metabolism of dermorphin in humans. Methods: Here, human liver microsomes and zebrafish were utilized as model systems of human metabolism to evaluate the presence and kinetics of metabolites derived from dermorphin. Five hours after its administration, the presence of dermorphin metabolites could be detected in both models by liquid chromatography coupled to high- resolution mass spectrometry. Results: Although the two models showed common results, marked differences were also observed in relation to the formed metabolites. Six putative metabolites, based on their exact masses of m/z 479.1915, m/z 501.1733, m/z 495.1657, m/z 223.1073, m/z 180.1017 and m/z 457.2085, are proposed to represent the metabolic pattern of dermor- phin. The major metabolite generated from the administration of dermorphin in both models was YAFG-OH (m/z 457.2085), which is the N-terminal tetrapeptide previously identified from studies on rats. Conclusion: Its extensive characterization and commercial availability suggest that it could serve as a primary target analyte for the detection of dermorphin misuse. The metabolomics approach also allowed the assignment of other confirmatory metabolites.Item A high throughput approach for determination of dermorphin in human urine using LC-HRMS and LC-MS/MS for doping control purposes.(2020) Castro, Juliana de Lima; Martucci, Maria Elvira Poleti; Pereira, Henrique Marcelo Gualberto; Sousa, Valéria P. deDermorphin is a peptide with analgesic actions similar to morphine, but with greater effect and less potential to cause tolerance. The use of dermorphin has been documented in race horses, and its use in humans has already been reported. Considering the potential advantages from the use of dermorphin over morphine, a method to monitor it, and its main metabolite dermorphin (1-4), in humans becomes necessary for doping control. Here, we present two orthogonal methods for this purpose: a high-throughput liquid chromatography coupled to high resolution mass spectrometry (HRMS) as an Initial Testing Procedure and liquid chromatography-tandem mass spectrometry (MS/MS) in the Selected Reaction Monitoring (SRM) acquisition mode for a Confirmation Procedure. For urine samples pre-treatment through a mixed-mode weak cation exchange solid phase extraction (WCX-SPE) emerged as an effective approach to extract peptides from the biological sample. For the HRMS analysis, a Full-MS scan acquisition mode was selected to detect the exact masses of dermorphin and dermorphin (1-4) at m/z 803.37226 and 457.20816, respectively. The SRM method used in the MS/MS confirmation protocol presented high specificity and sensitivity. The selected product ions for dermorphin were 602.2, 202.1 and 574.3 and for dermorphin (1-4) were 207.1, 223.1 and 235.1. Both methods were evaluated for specificity, repeatability, carryover, matrix effects and recovery. No carryover and matrix effects were detected. The Limit of Detection for Initial Testing Procedure and the Limit of Identification for Confirmation Procedure was 2,5 ng/mL. Also, specificity and robustness were acceptable for the application. Together, the developed methods proved to be efficient for the analysis of dermorphin and metabolite for human doping control purpose.Item Pharmacokinetic study of xylazine in a zebrafsh water tank, a human‑like surrogate, by liquid chromatography Q‑Orbitrap mass spectrometry.(2020) Mato, Rebecca Rodrigues; Martucci, Maria Elvira Poleti; Anselmo, Carina de Souza; Aquino Neto, Francisco Radler de; Pereira, Henrique Marcelo Gualberto; Sardela, Vinícius FigueiredoPurpose: This study aims to investigate a zebrafish (Danio Rerio) water tank (ZWT) as an alternative model for the study of the metabolism of xylazine. Methods: The ZWT approach for the study of metabolism consisted of two aquariums, where 18 fish and xylazine were added into the first tank. The second one, with 18 fish without drug, served as a negative control. The samples were submitted to a comprehensive analytical method developed for doping control purposes by liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS) operating in five different acquisition modes. Glycoconjugate metabolites were evaluated indirectly by extracting the samples with and without the enzymatic hydrolysis step using β-glucuronidase. Results: In total, 11 phase I and II metabolites were detected and characterized, of which four were previously described for humans and two for horses, and five metabolites were described for the first time. The main metabolites were 4-hydroxylated (M2) and oxygenated (M1) derivatives. Both metabolites were suggested as analytical targets for xylazine misuse. Around 79% of para- and meta-hydroxylated derivatives were in glycoconjugate form, whereas for oxo-hydroxylated and sulfone-hydroxylated derivatives of xylazine, around 83% and 70% were metabolized to the glycoconjugate form, respectively. Conclusions: Xylazine was the subject of extensive metabolism in zebrafish. 4-Hydroxylated (M2) and oxygenated (M1) derivatives were the most abundant phase I metabolites as the main targets for doping control.Item Comprehensive analysis by liquid chromatography Q‐Orbitrap mass spectrometry : fast screening of peptides and organic molecules.(2018) Sardela, Vinícius Figueiredo; Martucci, Maria Elvira Poleti; Araujo, Amanda Lessa Dutra de; Leal, E. C.; Oliveira, D. S.; Carneiro, Gabriel Reis Alves; Deventer, K.; Eenoo, P. Van; Pereira, Henrique Marcelo Gualberto; Aquino Neto, Francisco Radler deThe 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.