Development and Validation of a Quantitative LC-MS/MS Method for CDK4/6 Inhibitors Palbociclib, Ribociclib, Abemaciclib, and Abemaciclib-M2 in Human Plasma

Background:The cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, palbociclib, ribociclib, and abemaciclib, are standard-of-care agents for patients with hormone receptor-positive human epidermal growth factor receptor 2-negative metastatic breast cancer. In support of therapeutic drug monitoring and clinical pharmacokinetic studies, a liquid chromatography coupled with tandem mass spectrometry assay for the simultaneous quantitation of CDK4/6 inhibitors and the major active metabolite M2 of abemaciclib in human plasma has been developed.Methods:Analytes were extracted from 50 & mu;L of human plasma by precipitating proteins with methanol and then collecting the supernatant. Reversed-phase high-performance liquid chromatography was performed for analyte separation using a biphasic gradient at a flow rate of 0.25-0.5 mL/min. The total run time was 9.5 minutes. The analytes were detected using MS/MS with electrospray ionization operating in positive ion mode.Results:Validation according to the US Food and Drug Administration's guidance showed that the new assay produced accurate (94.7%-107%) and precise (within-run: 1.2%-8.2%; between-run: 0.6%-7.5%) measurements of all analytes over a concentration range of 5-2000 ng/mL. Overall, analyte recoveries were consistent (mean values: 110%-129%). The analytes were also stable in human plasma and the final extract under various storage conditions. Finally, the clinical applicability of the assay was confirmed by quantitation of all analytes in plasma samples obtained from patients treated with CDK4/6 inhibitors. Reproducibility of the measured analyte concentrations in study samples was confirmed successfully by incurred sample reanalysis.Conclusions:A sensitive liquid chromatography coupled with tandem mass spectrometry method to measure CDK4/6 inhibitors was developed and validated according to the Food and Drug Administration criteria. Quantitation of all analytes in clinical plasma samples confirmed that the assay is suitable for therapeutic drug monitoring and clinical pharmacokinetic studies of CDK4/6 inhibitors.

Automated summary

A sensitive liquid chromatography coupled with tandem mass spectrometry method was developed to measure the concentration of CDK4/6 inhibitors and the major active metabolite M2 of abemaciclib in human plasma. The method was validated according to the criteria of the Food and Drug Administration, and can accurately and precisely measure the concentrations of all analytes, making it suitable for therapeutic drug monitoring and clinical pharmacokinetic studies.