Dried Blood Spot Method Development and Clinical Validation for the Analysis of Elexacaftor, Elexacaftor-M23, Tezacaftor, Tezacaftor-M1, Ivacaftor, Ivacaftor Carboxylate, and Hydroxymethyl Ivacaftor Using LC-MS/MS.

Abstract

The highly effective Cystic Fibrosis Transmembrane conductance Regulator (CFTR) modulator, elexacaftor-tezacaftor-ivacaftor, is now widely being used by people with cystic fibrosis. However, few independent studies have detailed the pharmacokinetics (PK) of CFTR modulators. Blood collection by venipuncture is the gold standard for PK measurements, but it is invasive. The aim of this study was to develop and clinically validate a quantification method for elexacaftor, tezacaftor, ivacaftor, and their main metabolites in dried blood spots (DBSs) using liquid chromatography with tandem mass spectrometry. Linearity, accuracy, precision, stability, hematocrit (Hct), spot-to-spot carryover, spot volume, and extraction efficiency were validated in DBS for all analytes. The clinical validation of elexacaftor-tezacaftor-ivacaftor in patients was performed by comparing 21 DBS samples with matched plasma samples. The preset requirements for linearity, within-run and between-run accuracy, precision, Hct, spot volume, and extraction efficiency were met. Puncher carryover was observed and resolved by punching 3 blanks after each sample. The samples remained stable and showed no notable degradation across the tested temperatures and time intervals. Corrected DBS values with the Passing-Bablok regression equation showed good agreement in Bland-Altman plots, and acceptance values were within 20% of the mean for a minimum of 67% of the repeats, according to the EMA guidelines. A quantification method for the analysis of elexacaftor, tezacaftor, ivacaftor, and their main metabolites was developed and clinically validated in DBS. This method could be valuable in both clinical care and research to address unanswered PK questions regarding CFTR modulators.