{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,14]],"date-time":"2026-02-14T02:56:04Z","timestamp":1771037764882,"version":"3.50.1"},"reference-count":33,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,26]],"date-time":"2025-12-26T00:00:00Z","timestamp":1766707200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82173894"],"award-info":[{"award-number":["82173894"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Beijing Natural Science Foundation of China","award":["7232246"],"award-info":[{"award-number":["7232246"]}]},{"name":"Beijing Natural Science Foundation of China","award":["L202042"],"award-info":[{"award-number":["L202042"]}]},{"DOI":"10.13039\/501100012166","name":"National Key R&D Program of China","doi-asserted-by":"publisher","award":["2023YFC2706100"],"award-info":[{"award-number":["2023YFC2706100"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background\/Objectives: Given the clinical limitations of azathioprine (AZA) in treating inflammatory bowel disease, this study developed an AZA-loaded microbiota-modulating and colon-targeted nanoparticle constructed from pectin, Zein, and Eudragit\u00aeS100 (APZE), which was hypothesized to enhance efficacy while reducing toxicity. A liquid chromatography-tandem mass spectrometry (LC-MS\/MS) method was established to simultaneously quantify AZA and its metabolites, enabling the investigation of the pharmacokinetic and microbial metabolism differences between APZE and AZA suspension (AZAS). Methods: APZE was characterized, and an LC-MS\/MS method was developed for quantifying AZA and its metabolites in multiple matrices. Given the potential of APZE for colon targeting and modulation of the microbiota, which may affect drug absorption, distribution, and microbiota-mediated metabolism, we determined analyte concentrations in rat plasma, tissues, and microbial cultures at different time points following administration of APZE or AZAS. Results: AZA, 6-mercaptopurine (6-MP), 6-methylmercaptopurine (6-MMP), and 6-thioguanine (6-TG) were quantified in positive ion mode, and 6-thiouric acid (6-TU) in negative ion mode. The assay demonstrated excellent accuracy, precision, and stability over the concentration range of 5\u20131000 ng\/mL. Orally administered APZE exhibited higher bioavailability, improved intestinal absorption, and reduced formation of the inactive metabolite 6-TU compared to AZAS. In microbial cultures, AZA was metabolized primarily to 6-MP, and APZE underwent more extensive metabolism to 6-MP than AZAS. Conclusions: This method provides accurate and precise quantification of physiologically relevant concentrations of AZA and its metabolites (6-MP, 6-MMP, 6-TG, and 6-TU), offering a bioanalytical tool for the pharmacokinetic and gut microbiota metabolism studies of AZA formulations. These findings suggest that APZE is a promising drug delivery formulation.<\/jats:p>","DOI":"10.3390\/ph19010058","type":"journal-article","created":{"date-parts":[[2025,12,29]],"date-time":"2025-12-29T07:52:59Z","timestamp":1766994779000},"page":"58","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development and Application of an LC-MS\/MS Method for Simultaneous Quantification of Azathioprine and Its Metabolites: Pharmacokinetic and Microbial Metabolism Study of a Colon-Targeted Nanoparticle"],"prefix":"10.3390","volume":"19","author":[{"given":"Jingjing","family":"Zhang","sequence":"first","affiliation":[{"name":"Clinical Research Center, Beijing Children\u2019s Hospital, Capital Medical University, National Center for Children\u2019s Health, Beijing 100045, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiaqi","family":"Han","sequence":"additional","affiliation":[{"name":"Clinical Research Center, Beijing Children\u2019s Hospital, Capital Medical University, National Center for Children\u2019s Health, Beijing 100045, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ning","family":"Sun","sequence":"additional","affiliation":[{"name":"Clinical Research Center, Beijing Children\u2019s Hospital, Capital Medical University, National Center for Children\u2019s Health, Beijing 100045, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuhan","family":"Zhu","sequence":"additional","affiliation":[{"name":"Clinical Research Center, Beijing Children\u2019s Hospital, Capital Medical University, National Center for Children\u2019s Health, Beijing 100045, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dong","family":"Mei","sequence":"additional","affiliation":[{"name":"Clinical Research Center, Beijing Children\u2019s Hospital, Capital Medical University, National Center for Children\u2019s Health, Beijing 100045, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4968-6045","authenticated-orcid":false,"given":"Libo","family":"Zhao","sequence":"additional","affiliation":[{"name":"Clinical Research Center, Beijing Children\u2019s Hospital, Capital Medical University, National Center for Children\u2019s Health, Beijing 100045, China"},{"name":"Department of Pharmacy, Peking University Third Hospital, Beijing 100191, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Cai, Z., Wang, S., and Li, J. 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