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Characterization of in vivo metabolites in rat urine following an oral dose of masitinib by liquid chromatography tandem mass spectrometry.

ABSTRACT: Masitinib (MST) is an orally administered drug that targets mast cells and macrophages, important cells for immunity, by inhibiting a limited number of tyrosine kinases. It is currently registered in Europe and USA for the treatment of mast cell tumors in dogs. AB Science announced that the European Medicines Agency has accepted a conditional marketing authorization application for MST to treat amyotrophic lateral sclerosis. In our work, we focused on studying in vivo metabolism of MST in Sprague-Dawley rats. Single oral dose of MST (33 mg kg-1) was given to Sprague-Dawley rats (kept in metabolic cages) using oral gavage. Urine was collected and filtered at 0, 6, 12, 18, 24, 48, 72 and 96 h from MST dosing. An equal amount of ACN was added to urine samples. Both organic and aqueous layers were injected into liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect in vivo phase I and phase II MST metabolites. The current work reports the identification and characterization of twenty in vivo phase I and four in vivo phase II metabolites of MST by LC-MS/MS. Phase I metabolic pathways were reduction, demethylation, hydroxylation, oxidative deamination, oxidation and N-oxide formation. Phase II metabolic pathways were the direct conjugation of MST, N-demethyl metabolites and oxidative metabolites with glucuronic acid. Part of MST dose was excreted unchanged in urine. The literature review showed no previous articles have been made on in vivo metabolism of MST or detailed structural identification of the formed in vivo phase I and phase II metabolites.

SUBMITTER: Kadi AA

PROVIDER: S-EPMC5953916 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UNLABELLED: BACKGROUND: Anabolic androgenic steroids, such as stanozolol, are typically misused by athletes during preparation for competition. Out-of-competition testing presents a unique challenge in the current anti-doping detection system owing to logistic reasons. Analysing hair for the presence of a prohibited drug offers a feasible solution for covering the wider window in out-of-competition testing. To assist in vivo studies aiming to establish a relationship between drug levels detected in hair, urine and blood, sensitive methods for the determination of stanozolol and its major metabolite 3'-hydroxystanozolol were developed in pigmented hair, urine and serum, using brown Norway rats as a model system and liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: For method development, spiked drug free rat hair, blood and urine samples were used. The newly developed method was then applied to hair, urine and serum samples from five brown Norway rats after treatment (intraperitoneal) with stanozolol for six consecutive days at 5.0?mg/kg/day. The assay for each matrix was linear within the quantification range with determination coefficient (r2) values above 0.995. The respective assay was capable of detecting 0.125?pg/mg stanozolol and 0.25?pg/mg 3'-hydroxystanozolol with 50?mg hair; 0.063?ng/mL stanozolol and 0.125?ng/mL 3'-hydroxystanozolol with 100 ?L of urine or serum. The accuracy, precision and extraction recoveries of the assays were satisfactory for the detection of both compounds in all three matrices. The average concentrations of stanozolol and 3'-hydroxystanozolol, were as follows: hair?=?70.18?±?22.32?pg/mg and 13.01?±?3.43?pg/mg; urine?=?4.34?±?6.54?ng/mL and 9.39?±?7.42?ng/mL; serum?=?7.75?±?3.58?ng/mL and 7.16?±?1.97?ng/mL, respectively. CONCLUSIONS: The developed methods are sensitive, specific and reproducible for the determination of stanozolol and 3'-hydroxystanozolol in rat hair, urine and serum. These methods can be used for in vivo studies further investigating stanozolol metabolism, but also could be extended for doping testing. Owing to the complementary nature of these tests, with urine and serum giving information on recent drug use and hair providing retrospective information on habitual use, it is suggested that blood or urine tests could accompany hair analysis and thus avoid false doping results.

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Characterization of in vivo metabolites in rat urine following an oral dose of masitinib by liquid chromatography tandem mass spectrometry.

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