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.
Shandong Institute for Food and Drug Control, Jinan 250101, China.
Molecules. 2022 Jul 1;27(13):4256. doi: 10.3390/molecules27134256.
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 (r2 > 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.
胸腺免疫抑制五肽（TIPP）是一种新型的高效低毒抗炎肽。本研究旨在建立一种选择性的 LC-MS/MS 方法来分析生物样品中的分析物 TIPP，为进一步研究 TIPP 的 PK 和 PD 奠定基础。采用乙腈中加入 2%甲酸和 25mg/mL 二硫苏糖醇作为稳定剂进行蛋白沉淀，然后用二氯甲烷反冲有机相。TIPP 的色谱分离采用 C18 柱，采用梯度洗脱法。在正电喷雾电离下，通过多反应监测分析 TIPP。在鼠浆细胞裂解物、上清液和最终细胞冲洗 PBS 中，TIPP 的浓度与峰面积之间的线性关系分别在 20-5000ng/mL、1-200ng/mL、10-200μg/mL 和 0.1-20ng/mL 范围内建立（r2>0.99）。根据美国 FDA 指南验证，该方法是可接受的。该方法已成功应用于研究 TIPP 在小鼠中的药代动力学，通过皮下注射。TIPP 在小鼠体内的半衰期为 5.987±1.824min，表明 TIPP 在体内迅速消除。使用该方法测定 RBL-2H3 细胞对 TIPP 的摄取量，并通过共聚焦进行了直观验证。此外，通过比较 TIPP 的细胞内浓度和炎症程度，推导出 TIPP 的有效细胞内浓度，为进一步研究 TIPP 的细胞内靶标和机制提供了启示。
