A LC–MS/MS method for therapeutic drug monitoring of sorafenib, regorafenib and their active metabolites in patients with hepatocellular carcinoma

A liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous quantification of sorafenib (SORA), its N-oxide active metabolite and of regorafenib (REGO) and its two active metabolites regorafenib N-oxide and N-desmethyl regorafenib N-oxide in hepatocellular carcinoma patients’ plasma. A proper analytes’ separation was obtained with Synergi Fusion RP column (4 μm, 80 Å, 50 × 2.0 mm) using a gradient elution of 10 mM ammonium acetate with 0.1% formic acid (mobile phase A) and methanol:isopropanol (90:10, v/v, mobile phase B) containing 0.1% formic acid. The analysis was then performed by electrospray ionization in negative mode coupled with a triple quadrupole mass spectrometry, API 4000QT, monitoring two transitions for each analyte, one for the quantification and the other for confirmation. The method could be easily applied to the clinical practice thanks to the short run (7 min), the low amount of patient plasma necessary for the analysis (5 μL) and the fast sample processing based on protein precipitation. The method was therefore fully validated according to FDA and EMA guidelines. The linearity was assessed (R²≥0.998) over the concentration ranges of 50−8000 ng/mL for SORA and REGO, and 30−4000 ng/mL for their metabolites, that appropriately cover the therapeutic plasma concentrations. The presented method also showed adequate results in terms of intra- and inter-day accuracy and precision (CV ≤ 7.2% and accuracy between 89.4% and 108.8%), recovery (≥85.5%), sensitivity, analytes stability under various conditions and the absence of the matrix effect. Once the validation was successfully completed, the method was applied to perform the C_min quantification of SORA, REGO and their metabolites in 54 plasma samples collected from patients enrolled in a clinical study ongoing at the National Cancer Institute of Aviano.