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A novel method was developed and validated for the quantification of the three approved CDK4/6 inhibitors (abemaciclib, palbociclib, and ribociclib) in both human and mouse plasma and mouse tissue homogenates (liver, kidney, spleen, brain, and small intestine) using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). For all matrices, pretreatment was performed using 50 μL of sample by protein precipitation with acetonitrile, followed by dilution of the supernatant. Chromatographic separation of the analytes was done on a C18 column using gradient elution. A full validation was performed for human plasma, while a partial validation was executed for mouse plasma and mouse tissue homogenates. The method was linear in the calibration range from 2 to 200 ng/mL, with a correlation coefficient (r) ≥0.996 for each analyte. For both human and mouse plasma, the accuracy and precision were within ±15% and ≤15%, respectively, for all concentrations, except for the lower limit of quantification, where they were within ±20% and ≤20%, respectively. A fit-for-purpose strategy was followed for tissue homogenates, and the accuracy and precision were within ±20% and ≤20%, respectively, for all concentrations. Stability of all analytes in all matrices at different processing and storage conditions was tested; ribociclib and palbociclib were unstable in most tissue homogenates and conditions were modified to increase the stability. The method was successfully applied for the analysis of mouse samples from preclinical studies. A new ribociclib metabolite was detected in mouse plasma samples with the same m/z transition as the parent drug.

AP5280 is an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer to which are linked tetrapeptide side chains containing bioactive platinum complexes at their C-terminal sides. We have developed and validated a rapid and sensitive analytical assay for the determination of total platinum concentrations in plasma, and free platinum of an AP5280 origin in plasma ultrafiltrate (PUF), of subjects dosed with AP5280. The total platinum levels were determined by use of graphite-furnace atomic-absorption spectrometry (GF-AAS) with Zeeman correction after appropriate dilution of the plasma sample with plasma-hydrochloric acid 0.2 mol L(-1) (1:5) as diluent. The limit of quantitation of this assay is 0.25 micromol L(-1) platinum in plasma. Linear calibration curves were obtained over the concentration range 0.25-5.0 micromol L(-1). Accuracy was between 87.7% and 104.2% and precision was 15.3% at the lowest concentration and less than 14% for all other levels tested. Accuracy and precision were thus in accordance with generally accepted criteria for analytical methods. Analysis of samples obtained from patients receiving AP5280 demonstrated the applicability of the described assay. Analysis of free platinum in PUF was performed by use of a previously validated and reported assay from our institute in which the same instrumental method is used.