A Liquid Chromatography-Tandem Mass Spectrometry Method for the Quantification of Cystic Fibrosis Drugs (Caftors) in Plasma and Its Application for Therapeutic Monitoring

Abstract

Cystic fibrosis (CF) is a life-threatening disorder caused by mutations in the CFTR gene, leading to defective chloride ion transport and thickened mucus in the respiratory and gastrointestinal systems. CFTR modulators, including ivacaftor, lumacaftor, tezacaftor, and elexacaftor, have improved patient outcomes, but interindividual pharmacokinetic variability and potential drug-drug interactions require therapeutic drug monitoring (TDM) for optimal efficacy and safety. In this context, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous quantification of CFTR modulators and their major active metabolites in human plasma to support pharmacokinetic studies and routine TDM. The multiplex LC-MS/MS assay was established using plasma protein precipitation, followed by chromatographic separation on an Xselect HSS T3 (Waters^®) column and positive electrospray ionization mode detection. The method was validated based on FDA and EMA guidelines for specificity, linearity, accuracy (89.8-107.8%), repeatability (1.1-8.1%), intermediate fidelity (1.3-10.9%), matrix effects, and stability, demonstrating a robust performance with excellent precision and accuracy. International interlaboratory comparisons confirmed the reliability of the assay. The developed method can be applied for the clinical monitoring of caftors' plasma concentrations and preliminary data suggest that it can also be applied to alternative matrices, such as breast milk. This method will serve to characterize caftors' pharmacokinetic variability and monitor drug-drug interactions to further refine personalized dosing strategies and enhance precision medicine treatments for patients with CF.

Keywords: CFTR modulators; LC-MS/MS; cystic fibrosis; method development; pharmacokinetics; precision medicine; quantification; therapeutic drug monitoring; validation.

Figure 1

Multiplex plasma analysis using UHPLC-MS/MS of the four caftors and active metabolites. For the sake of readability, the corresponding internal standards are not shown. Chromatographic profile of a CAL6 plasma quality control sample (IVA 5 µg/mL, LUM 40 µg/mL, TEZ 10 µg/mL, ELX 15 µg/mL, IVA-M1 5 µg/mL, TEZ-M1 15 µg/mL, and ELX-M23 15 µg/mL).

Figure 2

Accuracy profiles of the analytes and their metabolites on three sets of analyses with a tolerance interval of 30% and a risk α at 5%. Mean bias obtained over 3 days for each concentration of QC (five levels: L1, L2, M1, M2, H) are reported in red. Upper and lower β-expectation tolerance intervals (β = 95%, black lines) and acceptance limits (±30%, green dotted lines) are also presented.

Figure 3

Qualitative evaluation of the matrix effect. Chromatograms of six different blank plasma extracts with post-column infusion of a CAL6 sample of each analyte (IVA 5 µg/mL, LUM 40 µg/mL, TEZ 10 µg/mL, ELX 15 µg/mL, IVA-M1 5 µg/mL, TEZ-M1 15 µg/mL, and ELX-M23 15 µg/mL).

Figure 4

Short-term stability testing of all the analytes in plasma and whole blood. Quantification performed on three QC samples (low, medium, and high concentrations) over time (at 0, 2, 4, 6, 24, 48, and 72 h) at room temperature (rt) and at +4 °C. The mean deviation of the measured values from the nominal concentration at T0 for the three QC samples is shown. Upper and lower acceptance limits (±15%, red dotted lines) are also presented.

Figure 5

Freeze-thaw stability testing of all the analytes in plasma. Quantification was performed on three QC samples (low, medium, and high concentrations) over three freezing/thawing cycles. The mean deviation of the measured values from the nominal concentration at T0 for the three QC samples is shown. Upper and lower acceptance limits (±15%, red dotted lines) are also presented.

Figure 6

Inter-laboratory comparison. Results of the plasma concentrations obtained from samples analyzed simultaneously in our laboratory (Laboratory A) and two European laboratories (namely, Laboratory B or C).

Figure 7

Chromatogram of a patient receiving IVA + LUM dual therapy, collected 3 h after dosing. The plasma concentrations of IVA, LUM, and IVA-M1 were 0.7, 17.8, and 1.9 µg/mL, respectively. The measured concentrations in this patient are in the range of expected concentrations based on available pharmacokinetic data [2]. Corresponding internal standards are not shown.

Figure 8

Chromatogram of a patient receiving IVA + TEZ + ELX triple therapy, collected 20 h after dosing. The plasma concentrations of IVA, IVA-M1, TEZ, TEZ-M1, ELX, and ELX-M23 were 1.76, 2.80, 3.39, 3.26, 6.92, and 1.88 µg/mL, respectively. The measured concentrations in this patient are higher than what has been previously reported [2]. Corresponding internal standards are not shown.