A Validated LC-MS/MS Method for Unbound Rifapentine Quantitation Reveals Nonlinear Plasma Protein Binding and Comparable Free Fractions Between Adults and Children with Tuberculosis

Background and objective Therapeutic drug monitoring (TDM) typically uses total drug concentration (C t ), but pharmacological effects depend on free concentration (C f ), especially for highly protein-bound drugs like rifapentine (RFPT) (96-99% bound). Monitoring C f is critical for optimizing efficacy and minimizing hepatotoxicity in patients with tuberculosis (TB) with individual variability. Addressing limitations of existing assays, this study developed a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying free RFPT and evaluated C f , C t , and clinical factor correlations in adults and children. Methods A validated LC-MS/MS method used Centrifree ultrafiltration (CF-UF) for free RFPT isolation and isotope internal standard (rifapentine-d8) for quantification. Validation included specificity, linearity (3.00-299.60 ng/mL), accuracy, precision, matrix effects, and stability. Clinical samples from 58 patients with TB (adults and children) receiving RFPT were analyzed. Total and free RFPT, albumin, and biochemical parameters were compared. Results The method showed excellent linearity (R 2 = 0.9999), accuracy (93.11-102.67%), and precision (intra-/inter-day RSD ≤ 7.40%). The lower limit of quantification (LLOQ) was 3.00 ng/mL, suitable for clinical C f detection. C f correlated nonlinearly with C t . C t was significantly higher in adults than children (20.91 ± 14.08 versus 14.64 ± 8.47 μg/mL, P = 0.03), but C f (0.085 ± 0.09 versus 0.054 ± 0.05 μg/mL, P = 0.06) and free fractions (0.38 ± 0.29% versus 0.35 ± 0.20%, P = 0.65) showed no significant difference. Conclusions The LC-MS/MS method is rapid, sensitive, and suitable for routine free RFPT TDM. Nonlinear C t -C f relationships highlight the necessity of direct C f monitoring, particularly with altered protein binding. Similar free fractions in adults and children despite dose-related C t differences suggest tailored dosing may mitigate toxicity. Adjustments based solely on C t may not be universally applicable.