Polar rp column

Chromatographic analysis was performed using a Shimadzu LC-20AD HPLC system (Shimadzu, Kyoto, Japan) and equipped with the CTC HTS-XT PAL Leap autosampler (Carrboro, NC, USA). The separation column was Synergi Polar-RP column (50 × 2.0 mm, 4 μm, Phenomenex, Torrance, CA). Mobile phase A was water with 0.1 % v/v formic acid, 31 % v/v acetonitrile, 31 % v/v methanol and mobile phase B was methanol with 0.5 % v/v formic acid at a flow rate of 0.5 ml/min according to the following linear gradient: 0–2.50 min, 55 % B; 2.51–3.50 min, 95 % B; 3.51–4.00 min, 55 % B. The injection volume was 20 μL.

The experiment was performed as described⁶⁶ . Briefly, 0.4 mg male or female pooled HLM was incubated with 100 µM clozapine with the presence or absence of 2 µM ketoconazole (CYP3A4 inhibitor). After 30 min of incubation, the reaction was stopped by adding ice cold acetonitrile. Protein was precipitated twice, and the collected supernatant was stored at −20 °C. Lastly, a 10 µl portion of the supernatant was injected into the LC-MS system to determine the concentration of parent compound and metabolites⁶⁷ . The detection and quantification of clozapine and metabolites was performed using high-performance liquid chromatography (Agilent 1200 Series, Santa Clara CA) coupled with mass spectrometry (TSQ Quantum triple stage quadrupole mass spectrometer; Thermo-Electron, San Jose, CA). Chromatographic separation was performed with a Phenomenex Polar RP column, 75 × 2.0 mm, 4.0 micron, (Torrance, CA) with a mobile phase containing (60:40) DI water with 0.1% formic acid: Acetonitrile with 0.1% formic acid, at a flow rate of 500 µL/min, with the column temperature set at 30 °C. The experiment was repeated independently 3 times. Data acquisition was performed with Xcalibur Version 2.07 (Thermo-Electron). Statistical analyses were performed using the student t test. P value <0.05 was defined as a statistically significant threshold.

An extraction protocol was established to quantify concentrations of venetoclax and zanubrutinib in both nanoparticle-bound and free forms. Briefly, the drugs were solubilized by diluting the sample with ethyl acetate, which extracted them from either the DcNP complex, mouse plasma, or both. Following centrifugation, the supernatants were dried with nitrogen gas and then reconstituted in acetonitrile. Extracted drug solutions were then loaded onto a Shimadzu HPLC system coupled to a 3200 QTRAP mass spectrometer (Applied Biosystems, Grand Island, NY, USA). The HPLC system consisted of two Shimadzu LC-20A pumps, a DGU-20A5 degasser, and a Shimadzu SIL-20 AC HT autosampler. A Synergi Polar-RP column (100 × 2.0 mm) with a C₈ guard column (4.0 × 2.0 mm) (Phenomenex, Torrance, CA, USA) was used for separations. Mobile phase A used water with 20 mM ammonium acetate and B used acetonitrile. The separations were done at room temperature with a flow rate of 0.55 mL/min. The mass spectrometer was equipped with an electrospray ionization (ESI) TurboIonSpray source, and the system was operated using Analyst software, version 1.5.2 (ABSciex, Framingham, MA, USA). Drug concentrations in various samples were calculated with standard curves prepared from normal mouse plasma containing known drug concentrations.