Jiahui Lin ^1,2 Aiting Jiang ¹ Juntao Zheng ¹ Jingjing Wu ¹ Hao Li ^1,2 Shirong Cai ³ Yu-Long He ³ Xiao Chen ¹ Zhong Guoping ⁴ Xinhua Zhang ^3* Ke-jing Tang ^1* Yanzhe Xia ^1*

• ¹ Department of Pharmacy, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
• ² School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong Province, China
• ³ Department of Gastrointestinal Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
• ⁴ Institute of Clinical Pharmacology, Sun Yat-sen University, Guangzhou, China

Background: Ripretinib, a broad-spectrum tyrosine kinase inhibitor, has been approved for the treatment of advanced gastrointestinal stromal tumors in adult patients. Clinical studies have shown that higher in vivo exposure of ripretinib correlates with improved efficacy, highlighting the potential clinical significance of therapeutic drug monitoring. In this study, a simple and stable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was attempted to be established and validated for pharmacokinetic studies of ripretinib and its metabolite DP-5439 and therapeutic drug monitoring in human plasma.Method: Ripretinib and DP-5439 were separated by chromatography using a Thermofisher Hypersil GOLD TM C18 HPLC column. The mobile phase for gradient elution is composed of 0.1% formic acid in water and acetonitrile. Multiple reaction monitoring was implemented along with electrospray ionization positive mode for detection. The ion pairs of ripretinib, DP-5439 and internal standard D8- ripretinib were m/z 510.1→m/z 417, m/z 496.11→m/z 402.9 and m/z 518.15→m/z 420, respectively. Plasma samples from ripretinib-treated patients of our hospital were collected for pharmacokinetic analysis.Results: Ripretinib and DP-5439 demonstrated a strong linear relationship over 10 to 5000 μg/L (R 2 > 0.99). Accuracy, precision, specificity, recoveries, matrix effect, stability, and dilution effect were all validated and found to meet the required criteria. Following validation, the method was utilized to determine plasma samples from patients treated with ripretinib. The median steady-state trough concentrations (Cmin, range) were 398.50 (66.98~1458.91) μg/L for ripretinib and 654.74 (30.71~1522.48) μg/L for DP-5439, with a total median concentration of 1129.46 (140.95~2981.39) μg/L in patients receiving ripretinib at 150 mg once daily. Meanwhile, using the established methods, the study conducted pharmacokinetics studies on four patients with ripretinib and DP-5439.This study developed and validated a robust LC-MS/MS method for determining ripretinib and its metabolite DP-5439 in human plasma. Furthermore, the practicality of this method in clinical sample analysis was demonstrated. This approach can serve as an effective tool for the pharmacokinetics analysis and therapeutic drug monitoring in patients treated with ripretinib.