Identification and Characterization of Rotigotine Degradation Products by HPLC Coupled DAD and CAD Detectors and HRMS Through Q-Orbitrap and Electrospray Ionization.

Rotigotine (RTG) is a dopamine agonist used in the treatment of Parkinson's disease. As it is susceptible to oxidation, stability studies must be carefully designed for the identification and characterization of all possible degradation products. Here, RTG degradation was evaluated according to the International Conference on Harmonization guidelines under various stress conditions, including acidic and basic hydrolysis, oxidative, metallic, photolytic, and thermal conditions. Additionally, more severe stress conditions were applied to induce RTG degradation. Significant degradation was only observed under oxidative and photolytic conditions. The samples were analyzed by high performance liquid chromatography coupled to photodiode array detectors, charged aerosol, and high-resolution mass spectrometry. Chromatographic analyses revealed the presence of eight substances related to RTG, four of which were already described and were qualified impurities (impurities B, C, K and E) and four new degradation products (DP-1 - DP-4), whose structures were characterized by high-resolution mass spectrometry through Q-Orbitrap and electrospray ionization. In the stress testing of the active pharmaceutical ingredient in solid form, significant RTG degradation was observed in the presence of the oxidative matrix. The results corroborate the literature that confirm the high susceptibility of RTG to oxidation and the importance of using different detectors to detect degradation products in forced degradation studies.

Is zebrafish (Danio rerio) water tank model applicable for the assessment of glucocorticoids metabolism? The budesonide assessment.

Knowledge of the metabolic profile is essential for doping control analysis in sport since most drugs are excreted after an elaborate biotransformation process. Currently, Zebrafish Water Tank (ZWT) model has been applied to investigate the metabolism of different doping agents. Nevertheless, the class of glucocorticoids has not been subjected to this model for metabolism studies. In the present work, budesonide (BUD) was applied as a pilot to investigate the metabolic pathways of glucocorticoids in the ZWT model. The BUD biotransformation in ZWT model was compared to the described metabolism in humans. Samples from ZWT experiments were collected after BUD administration and analyzed by Liquid Chromatography coupled to High Resolution Mass Spectrometry (LC-HRMS). Following the identification and characterization of all significant metabolites described for humans, it was observed that the ZWT was able to produce in a relevant amount the main target for doping control purposes: the 6ß-hydroxy BUD. In addition, prior knowledge about the lack of butyrylcholinesterase activity in the zebrafish organism was considered for the evaluation for the formation of the 16α-hydroxy prednisolone, the most intense BUD metabolite in human urine. Biotransformation of BUD by ZWT focused on metabolites with the acetal fraction preserved, including the intermediate metabolite for the 16α-hydroxy prednisolone pathway. However,analternative metabolic pathway for the complete biotransformation of the 16α-hydroxy prednisolone intermediate was not observed, leading to the absence of the major human metabolite in the ZWT model. The findings reported in this study elucidate for the first time the application and limitations of the ZWT model to evaluate the metabolism of other glucocorticoids.

Phase II stanozolol metabolism study using the zebrafish water tank (ZWT) model.

Stanozolol (STAN) is an androgen anabolic steroid often misused in sports competitions and prohibited at all times by the World Anti-Doping Agency (WADA). It can be long term detected by the analysis of human urine for traces of intact glucuronide metabolites. The Zebrafish Water Tank (ZWT) experimental setup can produce phase I STAN metabolites. In the present study, we investigated the in vivo phase II metabolism of STAN through the ZWT model to determine whether the ZWT produces metabolites relevant for doping control. We added STAN to a 200 mL recipient containing eight fish at 32 ± 1 °C. We analyzed the noninvasive samples (recipient water) both with and without pretreatment using Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC-HRMS/MS) in positive ionization mode. Our data show that four hydroxylated-sulfate and four hydroxylated-glycoconjugate metabolites were formed, two of the last ones being 3'OH-STAN-Glucuronide and 16ß-OH-STAN-Glucuronide. Additionally, two STAN-Glucuronide derivatives were produced: one was confirmed to be 17epi-STAN-N-Glucuronide, and the other was presumed to be STAN-O-Glucuronide. After eight hours of the experiment, STAN-O-Glucuronide was the most intense phase II metabolite produced. The accumulation curves suggest that high concentrations of fish and substrate in water are required to form phase II metabolites.

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.

Development and validation of a dissolution test for lutein tablets and evaluation of intestinal permeability.

Lutein is a carotenoid with antioxidant activity that is present in various dosage forms. The bioavailability of carotenoid from oral dosage formulations depends on their release, dissolution and its permeability through the gastrointestinal tract. Here, a dissolution test was developed for evaluating formulations and the bioavailability was assessed. The test utilized a USP-apparatus II with rotations of 50, 75 and 100rpm in water with P80 at 1, 2 and 5% (w/v). A non-everted rat intestinal sac model was used in conjunction to assess the intestinal permeability. The most discriminative conditions were 100rpm in water with 2% polysorbate 80, which showed profile differences between two formulations. The intestinal permeation studies showed a lag-time and apparent permeability coefficient that were characteristic of highly permeable drugs. We suggest that a dissolution test can be an essential quality control tool for formulations containing compounds as lutein, although not mandatory by the regulation agencies.