Validation of a universal and highly sensitive two-dimensional liquid chromatography–tandem mass spectrometry methodology for the quantification of pyrazinamide, ethambutol, protionamide, and clofazimine in different biological matrices.

• A global method determines four anti-TB drugs in various biological milieus. • Two-dimensional column-switch arrangement improves the method performance. • The extraction methods of tissues and feces are optimized. • Extra-liver tissue samples are calculated by liver curves. • Co-administration of anti-TB drugs enlarges the plasma exposures of protionamide. A novel and potent anti-tuberculosis drug combination pyrazinamide (PZA), ethambutol (EMB), protionamide (PTO), and clofazimine (CFZ) that rapidly kills Mycobacterium tuberculosis (Mtb) in the lungs has been identified using the artificial-intelligence-enabled parabolic response surface approach. A universal and highly sensitive two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) method for the simultaneous determination of PZA, EMB, PTO, and CFZ in various biological samples in different states (liquid samples: plasma, bile, and urine; solid samples: tissue and feces) using simple pretreatment was established and validated. For the first dimension of this column-switching arrangement, the automated purification and enrichment of the drugs were achieved on a Polar-RP column. The subsequent analytical separation was performed on an Agilent Zorbax SB-Aq column, and the total loop time was 7.5 min. The positive-ionization mode with multiple reaction monitoring was used for detection. The sensitivity was good with no carry-over detected, and the lower limit of quantification ranged from 100 to 500 pg/mL. This quantification method was fully validated and proved to be robust in accordance with US Food and Drug Administration guidelines. High recoveries (85.3–111.4%) and accuracies (92.1–109.3%), together with high precision values (0.5–13.8%), were verified in all matrices. All standard curves showed favorable linearities with r 2 > 0.995. This validated method was applied to study plasma pharmacokinetics, tissue distribution, and excretion in Sprague–Dawley rats after oral administration of the drug combination. [ABSTRACT FROM AUTHOR]