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), but pharmacological effects depend on free concentration (C), especially for highly protein-bound drugs like rifapentine (RFPT) (96-99% bound). Monitoring Cis 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, C, 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= 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 Cdetection. Ccorrelated nonlinearly with C. Cwas significantly higher in adults than children (20.91 ± 14.08 versus 14.64 ± 8.47 μg/mL, P = 0.03), but C(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-Crelationships highlight the necessity of direct Cmonitoring, particularly with altered protein binding. Similar free fractions in adults and children despite dose-related Cdifferences suggest tailored dosing may mitigate toxicity. Adjustments based solely on Cmay not be universally applicable.