Quantification of Venetoclax and Navitoclax in Liquor and Serum

One hundred µL of liquor was mixed with an equal volume of methanol, containing Navitoclax as internal standard (1 ng/mL). For serum, 100 µL were mixed with 900 µL methanol, containing 10 ng/mL Navitoclax. The samples were centrifuged, and the supernatant was measured by LC-MS. Analysis was done on an Agilent 1290 HPLC coupled to a QTOF 5600 mass spectrometer (AB Sciex). Samples were separated on a Zorbax SB-C18 column (2.1 × 50 mm; Agilent) using a gradient as follows: 0 min (70% solvent B: Methanol), 2.5 min (90% B), 3 min (90% B), 3.1 min (70% B), 5.5 (70% B). Solvent A was 5 mM ammonium acetate. The flow rate was 0.6 mL/min. Navitoclax and Venetoclax were detected in product ion scan mode (positive ionization) and by monitoring the corresponding MSMS fragment (974 -> 742 and 868 -> 321, respectively). For quantification, a respective 6-point calibration curve was used for each sample type (liquor: 2–50 ng mL⁻¹ and serum 0.1–10 µg mL⁻¹). Calibration samples were prepared by spiking the appropriate amount of Venetoclax in drug-free liquor and serum, respectively. Data analysis was performed using Analyst TF 1.7.1 and MultiQuant 3.0.2 (AB Sciex).