Validation of a Method for Quantifying Elexacaftor, Elexacaftor-M23, Tezacaftor, Tezacaftor-M1, and Ivacaftor by HPLC-PDA

Abstract: Cystic fibrosis is a genetic disorder primarily managed with pharmacological modulators of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, including Elexacaftor, Tezacaftor, and Ivacaftor. This study aimed to develop and validate an Ultra-Fast Liquid Chromatography with Photodiode Array Detection (UFLC/PAD) method for the simultaneous quantification of these three drugs and two of their major pharmacologically active metabolites (Elexacaftor-M23 [Ele-M23] and Tezacaftor-M1 [Tez-M1]) in plasma. Methods: A Shimadzu UFLC/PAD system equipped with a Shim-pack VP-ODS column was employed, using an isocratic mobile phase composed of water and acetonitrile (35:65). Itraconazole was used as the internal standard for plasma calibration curves ranging from 0.1 to 10 μg/mL. Plasma samples were prepared via protein precipitation using methanol, followed by centrifugation. The method was validated for linearity, limit of quantification (LOQ), precision, and accuracy. Results: The LOQ for each analyte was 0.1 μg/mL. Intra-day precision (coefficient of variation, CV%) ranged from 3.8% to 7.2%, while inter-day precision ranged from 8.6% to 16.3%, all within acceptable criteria. Retention times were 2.83 min for Ele-M23, 3.2 min for Ele, 5.6 min for Tez, 7.9 min for Tez-M1, 12.8 min for Iva, and 16.6 min for Itraconazole. Conclusion: The method is rapid, reproducible, and suitable for the simultaneous quantification of CFTR modulators and their primary metabolites in plasma. It is well-suited for clinical laboratory use in therapeutic drug monitoring and the optimization of cystic fibrosis treatment.