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Published on: September 26, 2025
A Validated LC-MS/MS Method for Unbound Rifapentine Quantitation Reveals Nonlinear Plasma Protein Binding and
Wenmei Qiao1,2, Mutong Fang1,3, Miaona Liu1,2
1The Third People's Hospital of Shenzhen, The Second Affiliated Hospital of Southern University of Science and Technology, National Clinical Research Center for Infectious Diseases, Shenzhen, Guangdong, 518112, People's Republic of China.
Background And Objective:
Therapeutic drug monitoring (TDM) typically uses total drug concentration (Ct), but pharmacological effects depend on free concentration (Cf), especially for highly protein-bound drugs like rifapentine (RFPT) (96-99% bound). Monitoring Cf 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 Cf, Ct, 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 (R2 = 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 Cf detection. Cf correlated nonlinearly with Ct. Ct was significantly higher in adults than children (20.91 ± 14.08 versus 14.64 ± 8.47 μg/mL, P = 0.03), but Cf (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 Ct-Cf relationships highlight the necessity of direct Cf monitoring, particularly with altered protein binding. Similar free fractions in adults and children despite dose-related Ct differences suggest tailored dosing may mitigate toxicity. Adjustments based solely on Ct may not be universally applicable.
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