Development and Validation of LC-MS/MS Method for Nintedanib and BIBF 1202 Monitoring in Plasma of Patients with Progressive Pulmonary Fibrosis Associated with Systemic Sclerosis

Background: Nintedanib (NIN), an intracellular inhibitor of tyrosine kinases that inhibits processes fundamental to the progression of pulmonary fibrosis (PPF), is used in the treatment of patients with PPF associated with systemic sclerosis. During NIN therapy, adverse events lead to a permanent dose reduction and treatment discontinuation. Therapeutic drug monitoring (TDM) can be used to manage and optimize drug administration based on the measurement of drug concentrations. Therefore, TDM can be helpful in minimizing the impact of adverse events and help patients remain in therapy. The aim of this study was to develop and validate a new bioanalytical UPLC-MS/MS method enabling the determination of NIN and its active metabolite in the plasma of patients with PPF associated with systemic sclerosis. Methods: Sample preparation was carried out using protein precipitation with an extraction mixture: acetonitrile neutralized with 2 M sodium carbonate. Analytes and the internal standard (intedanib-d3) were monitored using mass spectrometry (MS) and positive-ion-mode electrospray ionization by MRM. Chromatographic analysis was performed on a Zorbax SB-C18 column kept at 40 °C using isocratic elution. The mobile phase contained 0.1% formic acid in water; acetonitrile (35:65 v/v) was pumped at a flow rate of 0.3 mL/min. The analysis time was 5 min. Results: The method was verified according to the EMA guidelines over a concentration range of 2.00-200.00 ng/mL. The correlation coefficients for the calibration curves were found to be 0.9991 and 0.9957 for NIN and its metabolite BIBF 1202, respectively. The within- and between-run precision and accuracy of LLOQ were evaluated for NIN and BIBF 1202 to be within RSD 2.96%, 4.53%, 5.51%, and 6.72% and in the ranges of 102.2-107.3%, 98.0-101.8%, 104.3-114.2%, and 99.1-104.9, respectively. The stability of the analytes in plasma after 4 h at 30 °C was found to be satisfactory, meeting the assumed bias criteria below 15%. Conclusions: The proposed method was successfully applied to analyze two active compounds-NIN and BIBF 1202-in plasma samples at two time points: trough (pre-dose concentration) and 2-3 h (maximum concentration) after the administration of NIN.

BIBF 1202; LC-MS/MS; nintedanib; progressive pulmonary fibrosis; systemic sclerosis; therapeutic drug monitoring.