RESUMO
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
RESUMO
BACKGROUND: The novel cystic fibrosis transmembrane conductance regulator (CFTR) modulators, ivacaftor, lumacaftor, and tezacaftor, are the first drugs directly targeting the underlying pathophysiological mechanism in cystic fibrosis (CF); however, independent studies describing their pharmacokinetics are lacking. The aim of this study was to develop a quantification method for ivacaftor and its 2 main metabolites, lumacaftor and tezacaftor, in plasma and sputum using liquid chromatography with tandem mass spectrometry. METHODS: The developed method used a small sample volume (20 µL) and simple pretreatment method; protein precipitation solution and internal standard were added in one step to each sample. Liquid chromatography with tandem mass spectrometry was performed for a total run time of 6 minutes. The method was validated by assessing selectivity, carryover, linearity, accuracy and precision, dilution, matrix effects, and stability. RESULTS: The selectivity was good as no interference from matrices was observed. In the concentration range from 0.01 to 10.0 mg/L, calibration curves were linear with a correlation coefficient >0.9997 for all compounds. The within-run and between-run accuracy were between 99.7% and 116% at the lower limit of quantitation (LLOQ) and between 95.8% and 112.9% for all concentrations above LLOQ for all analytes in plasma and sputum. Within-run and between-run precisions were <12.7% for LLOQ and <6.7% for the higher limit of quantitation. Samples were stable, with no significant degradation at examined temperatures and time points. Clinical applicability was revealed by analyzing samples from 2 patients with CF. CONCLUSIONS: The presented method enables simultaneous quantification of ivacaftor, lumacaftor, and tezacaftor in plasma and sputum and is an improvement over previous methods because it uses smaller sample volumes, a simple pretreatment protocol, and includes tezacaftor. In future studies, it can be applied for examining pharmacokinetics characteristics of new CF transmembrane conductance regulator modulators.