A UPLC-MS-MS method for the simultaneous quantification of first-line antituberculars in plasma and in PBMCs

Objectives: TB is currently the second cause of death among patients affected with infectious diseases. Quantification of drug levels in plasma and in cells where Mycobacterium tuberculosis persists and grows may be useful in understanding the appropriateness of dosage regimens. We report a new and fully validated chromatographic method to quantify first-line antituberculars in plasma and PBMCs. The method was used for plasma and cell quantification of antituberculars in patients undergoing treatment with standard oral therapy. Methods: Ethambutol, isoniazid, pyrazinamide and rifampicin were extracted from plasma and PBMCs using two separate and optimized procedures; analysis was performed using UPLC coupled with a mass-mass detector system (UPLC-MS-MS). Antitubercular levels in patients were assayed at the end of the dosing interval (C-trough) and 2 h post-dose (C-max). Results: The method was accurate and precise. Recovery and the matrix effect were reproducible. While rifampicin intracellular concentrations were similar to plasma values (median intra-PBMC C-max = 7503 ng/mL versus median plasma C-max = 6505 ng/mL), isoniazid and pyrazinamide intracellular concentrations were lower than plasma values (median intra-PBMC C-max = 12 ng/mL versus median plasma C-max = 3258 ng/mL for isoniazid and median intra-PBMC C-max = 2364 ng/mL versus median plasma C-max = 26988 ng/mL for pyrazinamide) and ethambutol intracellular concentrations were significantly higher than plasma values (median intra-PBMC C-max = 73334 ng/mL versus median plasma C-max = 2244 ng/mL). Conclusions: The method was suitable for both therapeutic drug monitoring and for pharmacokinetic analysis. Should the clinical usefulness of measuring antitubercular drug intracellular concentrations be confirmed, this method could be useful to enhance the clinical application of intra-PBMC evaluation.