Quantification of clofazimine, linezolid, levofloxacin, and moxifloxacin in breast milk by online solid phase extraction coupled to LC-MS/MS

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.