Development and validation of a bioanalytical method for the quantification of the CDK4/6 inhibitors abemaciclib, palbociclib, and ribociclib in human and mouse matrices using liquid chromatography-tandem mass spectrometry

Abstract

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.

Keywords: Abemaciclib; LC-MS/MS; Palbociclib; Plasma; Ribociclib; Tissue homogenates.

Fig. 1

Mass spectra with the molecular structure and proposed MS fragmentation pattern of abemaciclib (a), palbociclib (c), ribociclib (e), and their stable isotopically labeled internal standards (b, d, f, respectively). The figure was created using ChemDraw Professional 15.0 and MATLAB R2017a software using the output from Sciex Analyst software

Fig. 2

Representative chromatograms of control human plasma spiked with internal standards (a), spiked at the LLOQ (b), and spiked at the ULOQ (c) at the mass transitions of ribociclib (m/z435→322), ribociclib-IS (m/z441→322), palbociclib (m/z448→380), palbociclib-IS (m/z456→388), abemaciclib (m/z507→393), and abemaciclib-IS (m/z515→393). Y-axis in chromatograms of a and b series for the analyte mass transitions (m/z435→322, m/z448→380, and m/z507→393) have the same scale, but Y-axis scale in chromatograms from c are adjusted to the highest response. The figure was created using the output form Sciex Analyst software along with GraphPad Prism 7 software

Fig. 3

Chromatograms of blank mouse plasma at the mass transitions of ribociclib (a), palbociclib (b), and abemaciclib (c), and representative chromatograms of samples from preclinical studies of ribociclib (d, 20 mg/kg orally administered, t = 8 h, 625 ng/mL), palbociclib (e, 10 mg/kg orally administered, t = 2 h, 311 ng/mL), and abemaciclib (f, 10 mg/kg orally administered, t = 24 h, 74.3 ng/mL). The additional figure included in d shows the same acquired with different gradient to increase the resolution between the two peaks. The figure was created using the output form Sciex Analyst software along with GraphPad Prism 7 software

Fig. 4

Plasma concentration-time curve (a) and tissue concentration (b) of ribociclib over 24 h in female FVB mice after oral administration at 20 mg/kg. Data are presented as mean ± SD (n = 6). The figure was created using the GraphPad Prism 7 software