Quantification of Brigatinib in Plasma

Free brigatinib in plasma samples was separated from protein-bound brigatinib by dialyzing the samples across a semipermeable dialysis membrane (8000-Da mass cutoff) using Thermo Scientific™ Single-Use RED (rapid equilibrium dialysis) Plates. For each RED dialysis unit, an aliquot (200 µL) of plasma was transferred into the donor chamber and an aliquot (350 µL) of warm (37 °C) phosphate buffered saline (PBS) was transferred into the receiver chamber of the dialysis unit. The RED devices were incubated at 37 °C with gentle shaking (250 rpm on a rotator shaker) for six hours. A 125-µL aliquot of plasma and 150-µL aliquot of PBS were removed from the dialysis unit and matrix-matched to a final composition of 50:50 (v:v) plasma:PBS, in Rain-X® treated 96-well plates. The matrix-matched samples were immediately frozen at − 80 °C. The protein binding assay and sample analysis were conducted at Charles River Laboratories, Inc. (Worcester, MA, USA). Samples were analyzed for brigatinib concentrations using liquid chromatography with tandem mass spectrometry (LC-MS/MS) methods that have been previously reported [11 (link)] using a dual-range assay with a lower limit of quantitation of 0.100 ng/mL and an upper limit of quantitation of 500 ng/mL.