Simultaneous quantification of venetoclax, selinexor, voriconazole and fluconazole in human plasma by HPLC-MS/MS and its application in hematological malignancies.

BACKGROUND: Venetoclax and selinexor are essential targeted agents for hematological malignancies, but their clinical application is challenged by pronounced ethnic pharmacokinetic differences-especially in Chinese patients-and CYP3A-mediated drug-drug interactions (DDIs) with azole antifungals (voriconazole, fluconazole). No existing LC-MS/MS method simultaneously quantifies all four drugs, creating an unmet need for therapeutic drug monitoring (TDM). We aimed to develop and validate a rapid, sensitive HPLC-MS/MS method for simultaneous quantification of venetoclax, selinexor, voriconazole, and fluconazole in human plasma. METHODS: Plasma samples underwent protein precipitation pretreatment. Chromatographic separation utilized a C18 column with acetonitrile and 0.1% formic acid gradient elution. Mass detection employed positive-ion multiple reaction monitoring (MRM). The method was validated for specificity, linearity, precision, accuracy, recovery, matrix effect, and stability, then applied to AML patients receiving these medications. RESULTS: Calibration ranges were 40-8000 ng/mL (venetoclax), 5-1000 ng/mL (selinexor), 100-10000 ng/mL (voriconazole), and 320-32000 ng/mL (fluconazole), all with r 2 > 0.9976. Intra-day and inter-day precision and accuracy met acceptance criteria. No significant matrix effect or carry-over was observed. Selinexor exhibited limited stability (<85% remaining after 4 h at room temperature or 24 h at 4 °C); other analytes were stable throughout. The median venetoclax steady-state trough concentration (Css,trough) in Chinese patients was 1025.08 ng/mL. Co-administration with voriconazole increased venetoclax exposure 1.75-14-fold, whereas fluconazole produced a consistent 2.4-2.9-fold increase. Venetoclax 100 mg/d combined with azole antifungals achieved exposures comparable to or exceeding 400 mg/d monotherapy. CONCLUSION: This is the first HPLC-MS/MS method enabling simultaneous quantification of venetoclax, selinexor, voriconazole, and fluconazole, successfully deployed in routine TDM. Voriconazole's marked DDI variability underscores the necessity of TDM-guided dosing, while fluconazole offers a more predictable antifungal prophylaxis alternative. These results deliver population-specific pharmacokinetic data supporting rational co-administration of venetoclax with azole antifungals in Chinese hematologic malignancy patients.