Quantification of Letrozole, Palbociclib, Ribociclib, Abemaciclib, and Metabolites in Volumetric Dried Blood Spots: Development and Validation of an LC-MS/MS Method for Therapeutic Drug Monitoring

Abstract

Therapeutic drug monitoring (TDM) may be beneficial for cyclin-dependent kinase 4/6 inhibitors (CDK4/6is), such as palbociclib, ribociclib, and abemaciclib, due to established exposure-toxicity relationships and the potential for monitoring treatment adherence. Developing a method for quantifying CDK4/6is, abemaciclib metabolites (M2, M20), and letrozole in dried blood spots (DBS) could be useful to enhance the feasibility of TDM. Thus, an optimized LC-MS/MS method was developed using the HemaXis DB10 device for volumetric (10 µL) DBS collection. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column. Detection was performed with a triple quadrupole mass spectrometer, utilizing ESI source switching between negative and positive ionization modes and multiple reaction monitoring acquisition. Analytical validation followed FDA, EMA, and IATDMCT guidelines, demonstrating high selectivity, adequate sensitivity (LLOQ S/N ≥ 30), and linearity (r ≥ 0.997). Accuracy and precision met acceptance criteria (between-run: accuracy 95-106%, CV ≤ 10.6%). Haematocrit independence was confirmed (22-55%),with high recovery rates (81-93%) and minimal matrix effects (ME 0.9-1.1%). The stability of analytes under home-sampling conditions was also verified. Clinical validation supports DBS-based TDM as feasible, with conversion models developed for estimating plasma concentrations (the reference for TDM target values) of letrozole, abemaciclib, and its metabolites. Preliminary data for palbociclib and ribociclib are also presented.

Keywords: Ctrough; abemaciclib; dried blood spot; letrozole; mass spectrometry; palbociclib; plasma exposure; ribociclib; therapeutic drug monitoring.

Figure 1

Representative SRM chromatograms of a blank DBS sample (a), a blank DBS sample containing the ISs (b), an LLOQ sample (c), and DBS samples from patients treated with abemaciclib (d), palbociclib (e), and ribociclib (f). The measured concentrations were 110 ng/mL for abemaciclib, 86 ng/mL for M20, 104 ng/mL for M2, and 84 ng/mL for letrozole, 58 ng/mL for palbociclib and 50 ng/mL for letrozole, 653 ng/mL for ribociclib, and 114 ng/mL for letrozole.

Figure 2

Percent difference (%Difference) of ECpla relative to actual Cpla obtained for each analyte. The accepted range (±20%) of %Difference is represented by dashed lines.

Figure 3

Relationship between actual and estimated plasma values of abemaciclib (a), M2 (b), M20 (c), and letrozole (d) based on Bland–Altman plot (left) and Passing–Bablok regression (right). The lower and upper limits of agreement (±1.96 SD of the bias) are reported as dashed lines in Bland–Altman plots and expressed as ng/mL, while the bias is reported as a solid line. In the Passing–Bablok regression, the 95% confidence interval is reported as dashed lines.

Figure 4

Incurred sample reanalysis. Dashed lines represent the accepted range (±20%) of percent difference (ISR %Difference).

Figure 5

DBS sample process from blood collection by finger prick with the HemaXis device to extraction of the analytes.

Figure 6

MS/MS mass spectra of abemaciclib (a), M2 (b), M20 (c), palbociclib (d), ribociclib (e), and letrozole (f) with chemical structures and identification of the main fragment ions.