{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T00:44:43Z","timestamp":1760229883903,"version":"build-2065373602"},"reference-count":22,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2022,7,1]],"date-time":"2022-07-01T00:00:00Z","timestamp":1656633600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Natural Science Foundation of China","award":["NO. 81872788"],"award-info":[{"award-number":["NO. 81872788"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Thymus immunosuppressive pentapeptide (TIPP) is a novel anti-inflammatory peptide with high efficacy and low toxicity. This study aims to establish a selective LC-MS\/MS method for analyzing the analyte TIPP in biological samples, laying the foundation for further PK and PD studies of TIPP. Protein precipitation was conducted in acetonitrile supplemented with 2% formic acid and 25 mg\/mL dithiothreitol as a stabilizer, which was followed by backwashing the organic phase using dichloromethane. The chromatographic separation of TIPP was achieved on a C18 column with a gradient elution method. During positive electrospray ionization, TIPP was analyzed via multiple-reaction monitoring. The linear relationships between the concentration of TIPP and peak area in murine plasma cell lysates, supernatants, and the final cell rinse PBS were established within the ranges of 20\u20135000 ng\/mL, 1\u2013200 ng\/mL, 10\u2013200 \u03bcg\/mL, and 0.1\u201320 ng\/mL, respectively (r2 &gt; 0.99). Validated according to U.S. FDA guidelines, the proposed method was proved to be acceptable. Such a method had been successfully applied to investigate the pharmacokinetics of TIPP in mice via subcutaneous injection. The plasma half-life in mice was 5.987 \u00b1 1.824 min, suggesting that TIPP is swiftly eliminated in vivo. The amount of TIPP uptake by RBL-2H3 cells was determined using this method, which was also visually verified by confocal. Furthermore, the effective intracellular concentration of TIPP was deduced by comparing the intracellular concentration of TIPP and degrees of inflammation, enlightening further investigation on the intracellular target and mechanism of TIPP.<\/jats:p>","DOI":"10.3390\/molecules27134256","type":"journal-article","created":{"date-parts":[[2022,7,4]],"date-time":"2022-07-04T20:59:18Z","timestamp":1656968358000},"page":"4256","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The Pharmacokinetics in Mice and Cell Uptake of Thymus Immunosuppressive Pentapeptide Using LC-MS\/MS Analysis"],"prefix":"10.3390","volume":"27","author":[{"given":"Shang","family":"Chen","sequence":"first","affiliation":[{"name":"Key Laboratory of Chemical Biology (Ministry of Education), NMPA Key Laboratory for Quality Research and Evaluation of Carbohydrate-based Medicine, Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chenyan","family":"Ren","sequence":"additional","affiliation":[{"name":"Key Laboratory of Chemical Biology (Ministry of Education), NMPA Key Laboratory for Quality Research and Evaluation of Carbohydrate-based Medicine, Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9709-7512","authenticated-orcid":false,"given":"Yuan","family":"Ji","sequence":"additional","affiliation":[{"name":"Shandong Institute for Food and Drug Control, Jinan 250101, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dongke","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Chemical Biology (Ministry of Education), NMPA Key Laboratory for Quality Research and Evaluation of Carbohydrate-based Medicine, Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xinke","family":"Zhang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Chemical Biology (Ministry of Education), Department of Pharmacology, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fengshan","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Chemical Biology (Ministry of Education), NMPA Key Laboratory for Quality Research and Evaluation of Carbohydrate-based Medicine, Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China"},{"name":"NMPA Key Laboratory for Quality Research and Evaluation of Carbohydrate-based Medicine, National Glycoengineering Research Center, Shandong University, Jinan 250012, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,7,1]]},"reference":[{"key":"ref_1","unstructured":"(2018). The Global Asthma Report 2018, Global Asthma Network."},{"key":"ref_2","unstructured":"Global Initiative for Asthma (2021, December 25). Global Strategy for Asthma Management and Prevention. Available online: www.ginasthma.org."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"15025","DOI":"10.1038\/nrdp.2015.25","article-title":"Asthma","volume":"1","author":"Holgate","year":"2015","journal-title":"Nat. Rev. Dis. Primers."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1016\/j.jconrel.2016.04.039","article-title":"Porous antioxidant polymer microparticles as therapeutic systems for the airway inflammatory diseases","volume":"233","author":"Jeong","year":"2016","journal-title":"J. Control. Release"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2252","DOI":"10.3390\/ijms16012252","article-title":"Inhibition of the IgE-mediated activation of RBL-2H3 cells by TIPP, a novel thymic immunosuppressive pentapeptide","volume":"16","author":"Lian","year":"2015","journal-title":"Int. J. Mol. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"256","DOI":"10.1016\/j.intimp.2014.12.019","article-title":"A novel pentapeptide originated from calf thymus named TIPP shows an inhibitory effect on lung allergic inflammation","volume":"24","author":"Lian","year":"2015","journal-title":"Int. Immunopharmacol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1016\/j.drudis.2014.10.003","article-title":"Peptide therapeutics: Current status and future directions","volume":"20","author":"Fosgerau","year":"2015","journal-title":"Drug Discov. Today."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/j.jchromb.2011.09.030","article-title":"LC\u2013MS\/MS for protein and peptide quantification in clinical chemistry","volume":"883\u2013884","author":"Rauh","year":"2012","journal-title":"J. Chromatogr. B Anal. Technol. Biomed. Life Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1002\/mas.20120","article-title":"Mass spectrometry for the quantification of bioactive peptides in biological fluids","volume":"26","author":"Tamvakopoulos","year":"2007","journal-title":"Mass Spectrom. Rev."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1379","DOI":"10.4155\/bio.11.112","article-title":"Bioanalytical approaches to analyzing peptides and proteins by LC-MS\/MS","volume":"3","author":"Ewles","year":"2011","journal-title":"Bioanalysis"},{"key":"ref_11","unstructured":"Lian, Q.Q. (2015). Study on the Inhibitory Effects of Thymic Immunosuppressive Pentapeptide on Airway Inflammation of Asthma and Its Action Mechanism. [Ph.D. Thesis, Shandong University]."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"818","DOI":"10.1038\/sj.bjp.0706240","article-title":"Inhibition of the antigen-induced activation of rodent mast cells by putative Janus kinase 3 inhibitors WHI-P131 and WHI-P154 in a Janus kinase 3-independent manner","volume":"145","author":"Linwong","year":"2005","journal-title":"Br. J. Pharmacol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"975","DOI":"10.1016\/j.immuni.2019.03.018","article-title":"The Cytokines of Asthma","volume":"50","author":"Lambrecht","year":"2019","journal-title":"Immunity"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/j.jchromb.2013.04.030","article-title":"Bioanalytical LC-MS\/MS of protein-based biopharmaceuticals","volume":"929","author":"Broek","year":"2013","journal-title":"J. Chromatogr. B Anal. Technol. Biomed. Life Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.jpha.2012.10.006","article-title":"Quantitation of bivalirudin, a novel anticoagulant peptide, in human plasma by LC-MS\/MS: Method development, validation and application to pharmacokinetics","volume":"3","author":"Li","year":"2013","journal-title":"J. Pharm. Anal."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1016\/j.ejpb.2018.12.011","article-title":"Bioanalytical method development and validation for determination of fibroblast growth factor peptide and its application to pharmacokinetic studies","volume":"135","author":"Prasanchaimontri","year":"2019","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5080","DOI":"10.1021\/acs.analchem.5b04368","article-title":"Characterization of IgG2 Disulfide Bonds with LC\/MS\/MS and Postcolumn Online Reduction","volume":"88","author":"Liu","year":"2016","journal-title":"Anal. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1186\/1559-0275-11-7","article-title":"Optimization for peptide sample preparation for urine peptidomics","volume":"11","author":"Sigdel","year":"2014","journal-title":"Clin. Proteom."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"809","DOI":"10.4155\/bio.12.28","article-title":"Recent advances in column switching sample preparation in bioanalysis","volume":"4","author":"Kataoka","year":"2012","journal-title":"Bioanalysis"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2902","DOI":"10.1016\/j.jchromb.2011.08.021","article-title":"An LC\u2013MS\/MS assay to determine plasma pharmacokinetics of cyclic thymic hexapeptide (cTP6) in rhesus monkeys","volume":"879","author":"Meng","year":"2011","journal-title":"J. Chromatogr. B Anal. Technol. Biomed. Life Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1016\/j.jpba.2018.06.057","article-title":"Quantitative analysis of the cyclic peptide GG-8-6 in rat plasma using LC-MS\/MS and its application to a pharmacokinetic study","volume":"159","author":"Chen","year":"2018","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_22","unstructured":"US FDA (2018, August 15). Bioanalytical Method Validation Guidance for Industry, Available online: https:\/\/www.fda.gov\/files\/drugs\/published\/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/27\/13\/4256\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:41:48Z","timestamp":1760139708000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/27\/13\/4256"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,7,1]]},"references-count":22,"journal-issue":{"issue":"13","published-online":{"date-parts":[[2022,7]]}},"alternative-id":["molecules27134256"],"URL":"https:\/\/doi.org\/10.3390\/molecules27134256","relation":{},"ISSN":["1420-3049"],"issn-type":[{"type":"electronic","value":"1420-3049"}],"subject":[],"published":{"date-parts":[[2022,7,1]]}}}