Development and Clinical Evaluation of an LC-MS/MS Method for the Determination of Isoniazid in Cerebrospinal Fluid from Tuberculous Meningitis

Purpose: Isoniazid (INH), a first-line anti-tuberculosis agent, exhibits variable cerebrospinal fluid (CSF) penetration owing to NAT2 genetic polymorphisms. This study developed an LC–MS/MS method to quantify INH in the human CSF for therapeutic drug monitoring in patients with tuberculous meningitis. Methods: The LC-MS/MS method developed and validated in this study includes linearity, sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability evaluations, all of which meet the standards for bioanalytical method validation. This method was applied to a real-world prospective observational study to compare CSF trough concentrations of INH between patients receiving conventional anti-tuberculosis therapy and those receiving conventional therapy combined with intrathecal INH injection, and a pharmacokinetic study was conducted in one TBM patient. Results: The validated LC‑MS/MS method achieved a total run time of 3.5 min, demonstrated linearity over the range of 5– 4000 ng/mL, and had an LLOQ of 5 ng/mL, with all validation parameters meeting acceptance criteria. In the clinical cohort, the median CSF trough concentration of INH was significantly higher in the intrathecal therapy group (1130.00 ng/mL, N=117) than in the conventional therapy group (155.00 ng/mL, N=45, P< 0.001). Target attainment rates at ≥ 120 ng/mL and ≥ 250 ng/mL were also significantly greater in the intrathecal group (84.62% and 77.78%, respectively) versus the conventional group (57.78% and 35.56%, both P< 0.001). Pharmacokinetic analysis in a TBM patient after intrathecal INH administration revealed a T m ₐₓ of 6 h, C m ₐₓ of 2810 ng/mL, and an elimination half‑life of approximately 14 h. Conclusion: A sensitive and reliable LC-MS/MS method was successfully developed and validated for quantification of INH in human CSF. This study confirms that intrathecal administration of INH can significantly increase drug concentrations in the CSF of TBM patients, providing methodological support and preliminary clinical evidence for optimizing treatment strategies in TBM. Keywords: LC-MS/MS, isoniazid, cerebrospinal fluid, tuberculous meningitis