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.