Analytical Method Development and Validation for Clofazimine in Human Plasma and Application of LC-MS/MS Based Methods for Clofazimine and Bedaquiline in Indian MDR-TB Patients Stratified by HIV Coinfection.

Multidrug-resistant tuberculosis (MDR-TB) remains a major cause of morbidity and mortality worldwide, with people living with HIV experiencing persistently poor treatment outcomes. Clofazimine and bedaquiline are cornerstone drugs in contemporary all-oral MDR-TB regimens, yet their complex pharmacokinetics and substantial interindividual variability complicate regimen optimisation, particularly in vulnerable populations. Robust plasma-based drug quantification is essential to characterise exposure-response relationships and inform individualised therapy. We developed and validated a rapid, sensitive and specific liquid chromatography-tandem mass spectrometry assay for quantifying these drugs in human plasma. The methods were successfully applied on plasma samples from study participants with MDR-TB with or without HIV coinfection who received clofazimine and bedaquiline as part of MDR-TB therapy assessing the Cmax, Tmax and AUC in both groups. The method addresses key analytical challenges posed by clofazimine's extreme lipophilicity and reliably quantifies concentrations across a broad, clinically relevant range, including potentially toxic exposures. Linearity was obtained between 0.0313 to 4.0 mg/L, and the method met bioanalytical method validation criteria along with interlab comparison with a reference laboratory, all within acceptable limits. We observed significantly lower Cmax concentrations of both drugs among HIV-infected participants compared to HIV-uninfected participants (p = 0.011 for clofazimine and p = 0.02 for bedaquiline), highlighting the need for therapeutic drug monitoring in this vulnerable group. This work provides a robust analytical foundation for pharmacokinetic, therapeutic drug monitoring and exposure-response studies of key MDR-TB drugs and supports efforts to optimise treatment outcomes, especially among people living with HIV.