The Pharmacokinetics in Mice and Cell Uptake of Thymus Immunosuppressive Pentapeptide Using LC-MS/MS Analysis
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 p...
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MDPI AG
2022-07-01
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| Online Access: | https://www.mdpi.com/1420-3049/27/13/4256 |
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| author | Shang Chen Chenyan Ren Yuan Ji Dongke Liu Xinke Zhang Fengshan Wang |
| author_facet | Shang Chen Chenyan Ren Yuan Ji Dongke Liu Xinke Zhang Fengshan Wang |
| author_sort | Shang Chen |
| collection | DOAJ |
| description | 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 20–5000 ng/mL, 1–200 ng/mL, 10–200 μg/mL, and 0.1–20 ng/mL, respectively (<i>r</i><sup>2</sup> > 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 ± 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. |
| first_indexed | 2024-03-09T04:00:19Z |
| format | Article |
| id | doaj.art-4df4bed28a52454f994fe5aee8403426 |
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| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-09T04:00:19Z |
| publishDate | 2022-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-4df4bed28a52454f994fe5aee84034262023-12-03T14:14:08ZengMDPI AGMolecules1420-30492022-07-012713425610.3390/molecules27134256The Pharmacokinetics in Mice and Cell Uptake of Thymus Immunosuppressive Pentapeptide Using LC-MS/MS AnalysisShang Chen0Chenyan Ren1Yuan Ji2Dongke Liu3Xinke Zhang4Fengshan Wang5Key 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, ChinaKey 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, ChinaShandong Institute for Food and Drug Control, Jinan 250101, ChinaKey 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, ChinaKey Laboratory of Chemical Biology (Ministry of Education), Department of Pharmacology, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, ChinaKey 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, ChinaThymus 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 20–5000 ng/mL, 1–200 ng/mL, 10–200 μg/mL, and 0.1–20 ng/mL, respectively (<i>r</i><sup>2</sup> > 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 ± 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.https://www.mdpi.com/1420-3049/27/13/4256LC-MS/MSbioanalytical method developmentallergic asthmapharmacokineticscell uptake |
| spellingShingle | Shang Chen Chenyan Ren Yuan Ji Dongke Liu Xinke Zhang Fengshan Wang The Pharmacokinetics in Mice and Cell Uptake of Thymus Immunosuppressive Pentapeptide Using LC-MS/MS Analysis Molecules LC-MS/MS bioanalytical method development allergic asthma pharmacokinetics cell uptake |
| title | The Pharmacokinetics in Mice and Cell Uptake of Thymus Immunosuppressive Pentapeptide Using LC-MS/MS Analysis |
| title_full | The Pharmacokinetics in Mice and Cell Uptake of Thymus Immunosuppressive Pentapeptide Using LC-MS/MS Analysis |
| title_fullStr | The Pharmacokinetics in Mice and Cell Uptake of Thymus Immunosuppressive Pentapeptide Using LC-MS/MS Analysis |
| title_full_unstemmed | The Pharmacokinetics in Mice and Cell Uptake of Thymus Immunosuppressive Pentapeptide Using LC-MS/MS Analysis |
| title_short | The Pharmacokinetics in Mice and Cell Uptake of Thymus Immunosuppressive Pentapeptide Using LC-MS/MS Analysis |
| title_sort | pharmacokinetics in mice and cell uptake of thymus immunosuppressive pentapeptide using lc ms ms analysis |
| topic | LC-MS/MS bioanalytical method development allergic asthma pharmacokinetics cell uptake |
| url | https://www.mdpi.com/1420-3049/27/13/4256 |
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