We developed and validated a method to quantify clofazimine, linezolid, levofloxacin, and moxifloxacin in breast milk. Inconsistencies in analyte behavior necessitated the development of separate sample preparation methodologies for the less polar analyte, clofazimine, and the more polar compounds linezolid, levofloxacin, and moxifloxacin. Clofazimine was extracted via online solid-phase extraction using a Synergi Hydro-RP column with a total run time of 7 min, while the polar compounds were extracted using a Restek Viva BiPh column for a total run time of 6.50 min. Both groups were separated by liquid chromatography on an Agilent Poroshell 120 EC-C18 column. Detection was performed using tandem mass spectrometry with electrospray ionization and multiple reaction monitoring. Calibration curves fitted weighted quadratic regression equations over the ranges of 0.0477–6.00 μg/mL for clofazimine, 0.191–24.0 μg/mL for linezolid, and 0.240–30.2 μg/mL for levofloxacin and moxifloxacin. The method demonstrated precision and inter/intra-day accuracies that readily met acceptance criteria according to the FDA and EMA guidelines. The average recoveries for clofazimine, linezolid, levofloxacin, and moxifloxacin were 68.0%, 94.1%, 104.5%, and 106.0%, respectively; mean precision ranged from 0.8% to 4.7%. The robust methodology, proven to be selective and reliable when tested in different lots of breast milk, was applied to the analysis of clinical samples collected from women on treatment for drug-resistant tuberculosis. Comparison of the observed drug breast milk concentrations with maternal plasma samples taken at the same timepoints provide much-needed data regarding the exposure of second-line TB drugs in pregnant and breastfeeding women and their infants.
Clobazam (CLB) and its active metabolite N-desmethylclobazam (N-CLB) exhibit substantial interindividual variability in paediatric patients with refractory epilepsy, highlighting the value of therapeutic drug monitoring (TDM). This study aimed to develop and validate a rapid and reliable ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the simultaneous quantification of CLB and N-CLB and to investigate clinical factors associated with their concentrations in plasma. The method required 50 μL of plasma and achieved chromatographic separation within 3 min. The linear ranges were 25–800 ng/mL for CLB and 237.5–7600 ng/mL for N-CLB, and the method was validated according to the core elements of the ICH M10 guidelines. The validated method was applied to 61 paediatric patients with refractory epilepsy, whose concomitant antiseizure medications and CYP3A4 and CYP2C19 genotypes were evaluated. Multivariate analysis revealed that the daily CLB dose was positively associated with CLB concentration (β = 5.75; 95% CI: 3.01–8.49), whereas the use of VPA was associated with lower CLB concentration (β = −88.37; 95% CI: −134.43 to −42.31) and the use of STP was associated with higher CLB concentrations (β = 206.29; 95% CI: 137.11–275.47). For N-CLB, poor CYP2C19 metabolism (β = 4354.34, 95% CI: 3259.61–5449.07) and STP use (β = 3005.10, 95% CI: 1740.35–4269.84) were associated with higher concentrations. The method is rapid, requires a small plasma volume, and is suitable for routine simultaneous TDM of CLB and N-CLB. Integrating TDM with CYP2C19 genotyping may support individualized CLB therapy and safety monitoring in paediatric patients with refractory epilepsy.
