Development and fit-for-purpose validation of human scalp hair analysis using UHPLC-MS/MS for determining exposure to CFTR modulators

BACKGROUND: Cystic Fibrosis Transmembrane Conductance regulator (CFTR) modulators (elexacaftor (ELEXA), tezacaftor (TEZA), ivacaftor (IVA), lumacaftor (LUMA)) are CF's cornerstone pharmacologic treatment. However, a reliable noninvasive method for determining long-term CFTR modulators medication adherence is lacking. Therefore, we aimed to develop and validate a method for quantification of CFTR modulators in human scalp hair.

METHODS: LC-MS/MS was used for quantification. For method development, scalp hair from people with CF using CFTR modulators was used. Blank hair for quality control (QC) and calibration samples were donated by healthy volunteers. Solvents to remove external contamination, extraction and extraction times were tested. After method optimization, validation was performed following FDA, EMA and Society of Hair Testing guidelines.

RESULTS: Isopropanol was the best washing solvent, and with methanol the most optimal extraction solvent. Optimization of the extraction time resulted in a 90-min extraction time using ball mill. Selectivity of the developed method was confirmed for IVA, its metabolite IVA-M1, ELEXA, LUMA and TEZA. For linearity, all calibration curves met the acceptance criteria of bias less than 15%. Accuracies of the QC samples for IVA, IVA-M1 and LUMA did not comply with FDA/EMA criteria of being between 85% and 115%. ELEXA and TEZA accuracies were between 86.2% and 98.5% and complied. Precision for ELEXA and TEZA (LLOQ, LOW, MEDIUM and HIGH) ranged between 1.0 and 7.0% and 0.0-2.7% (intra CVs (%)), and 3.4-6.6% and 2.7-7.4% (inter CVs (%)).

CONCLUSION: A hair analysis for CFTR modulators was developed and concluded fit-for-purpose, demonstrating that elexacaftor and tezacaftor can be reliably quantified in hair.