Significant matrix effect in fludarabine quantification in plasma: implications for CAR-T cell therapy monitoring.
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BACKGROUND: Event-free survival (EFS) in patients with B-cell acute lymphoblastic leukemia (B-ALL) significantly improves with adequate exposure (as Area Under the Curve: AUC0-inf or AUC) to fludarabine (FLU) during lymphodepletion prior to chimeric antigen receptor (CAR) T-cell infusion. A cumulative AUC of FLU exceeding 14 mgxh/L is considered optimal, making therapeutic drug monitoring (TDM) crucial for personalized dosing and prolonged CAR T-cell persistence. RESEARCH DESIGN AND METHODS: We developed and validated an European Medicines Agency (EMA)-compliant high-performance liquid chromatography-tandem mass spectrometry method (HPLC-MS/MS) for FLU TDM, based on similar and published methods, to support clinicians during the patient conditioning phase. All EMA acceptance criteria were met. RESULTS: Analysis of real plasma samples revealed unexpectedly high FLU concentrations and, consequently, an excessively high AUC. Analysis of plasma samples from a child receiving FLU therapy highlighted the necessity for accurate drug response verification in real matrices. Preparation of quality controls in FLU-free plasma consistently failed without preliminary dilution. Analysis of internal standard range variation enabled more precise and accurate analyses. CONCLUSIONS: This experience highlights the importance of complementing standard validation tests with analyses in each patient's plasma, collected immediately before FLU infusion, to ensure reliable TDM to support CAR T-cell therapy.