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Background and Objective <p>Therapeutic drug monitoring (TDM) typically uses total drug concentration (<i>C</i><sub>t</sub>), but pharmacological effects depend on free concentration (<i>C</i><sub>f</sub>), especially for highly protein-bound drugs like rifapentine (RFPT) (96–99% bound). Monitoring <i>C</i><sub>f</sub> 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 <i>C</i><sub>f</sub>, <i>C</i><sub>t</sub>, and clinical factor correlations in adults and children.</p> Methods <p>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.</p> Results <p>The method showed excellent linearity (<i>R</i><sup>2</sup> = 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 <i>C</i><sub>f</sub> detection. <i>C</i><sub>f</sub> correlated nonlinearly with <i>C</i><sub>t</sub>. <i>C</i><sub>t</sub> was significantly higher in adults than children (20.91 ± 14.08 versus 14.64 ± 8.47 μg/mL, <i>P</i> = 0.03), but <i>C</i><sub>f</sub> (0.085 ± 0.09 versus 0.054 ± 0.05 μg/mL, <i>P</i> = 0.06) and free fractions (0.38 ± 0.29% versus 0.35 ± 0.20%, <i>P</i> = 0.65) showed no significant difference.</p> Conclusions <p>The LC–MS/MS method is rapid, sensitive, and suitable for routine free RFPT TDM. Nonlinear <i>C</i><sub>t</sub>–<i>C</i><sub>f</sub> relationships highlight the necessity of direct <i>C</i><sub>f</sub> monitoring, particularly with altered protein binding. Similar free fractions in adults and children despite dose-related <i>C</i><sub>t</sub> differences suggest tailored dosing may mitigate toxicity. Adjustments based solely on <i>C</i><sub>t</sub> may not be universally applicable.</p>

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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

  • 期刊论文

  • 发表日期：

    2026年1月29日

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  • Wenmei Qiao,
  • Mutong Fang,
  • Miaona Liu,
  • Tian He,
  • Peize Zhang,
  • Wei Li

摘要

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.