Development and application of a multimatrix LC-MS/MS method for quantifying elexacaftor-tezacaftor-ivacaftor: Expanding therapeutic drug monitoring in cystic fibrosis from systemic circulation to airways and sweat

Background: Therapeutic drug monitoring (TDM) is becoming increasingly essential in cystic fibrosis (CF), as a growing number of children and adults are now eligible for elexacaftor/tezacaftor/ivacaftor (ETI) CF Transmembrane conductance Regulator (CFTR) modulator therapies. Although plasma remains the benchmark, its invasive nature limits practical use. Analytical approaches based on dried matrices currently available still rely on venipuncture and intensive workflows. To overcome these limitations, we developed an advanced, multidimensional TDM framework for ETI quantification that incorporates truly venipuncture-free, self-collected, and quantitative dried blood spot (DBS) sampling, along with non-invasive nasal airway swabs (NAS) and sweat. We also evaluated ETI in airway and sweat fluids, offering insight into drug distribution and activity at key target tissues. Methods: Selectivity, specificity, linearity, accuracy, precision, and inter/intraday stability of the methods applied to plasma, NAS, and sweat specimens were investigated according to ICH M10 guidelines and IATDMCT for DBS. Results: Analytical performance of the methods in all tested matrices were demonstrated and met the acceptance criteria of ICH M10 guidelines for bioanalytical-method validation, with DBS additionally fulfilling the IATDMCT DBS-specific recommendations. DBS measurements were statistically equivalent to plasma concentrations, supporting their utility as a minimally invasive surrogate for TDM. ETI levels in NAS were significantly higher than in plasma or sweat, indicating localized accumulation of the drug on the airway surface, a key site of CFTR activity. Sweat samples, while showing lower analyte amounts, contained detectable levels in all analytes within their biologically active concentrations. Conclusions: This integrated analytical approach provides a holistic view of systemic and local ETI distribution, increasing the potential for personalized TDM, pharmacokinetics/pharmacodynamics study, and optimization of CFTR modulator therapy.