A high‐throughput LC–MS/MS method for simultaneous determination of isoniazid, ethambutol and pyrazinamide in human plasma

RATIONALE: Tuberculosis (TB) remains a challenging global infectious disease, mainly affecting the lungs. First‐line anti‐TB drugs play a crucial role in slowing down the rapid spread of TB. In addition, the patient might benefit from therapeutic drug monitoring since it has become an accepted clinical tool for optimizing TB treatment. METHODS: A simple and sensitive liquid chromatography/tandem mass spectrometry method was developed to monitor the plasma level of isoniazid, ethambutol and pyrazinamide in plasma samples. A one‐step extraction procedure using an Ostro™ plate was applied, and extracts were analyzed by gradient elution followed by detection on a mass spectrometer by multiple reaction monitoring mode. RESULTS: The analytes were separated within 4.2 min and over the concentration range of 0.2–10 μg/ml for isoniazid and ethambutol and 1–65 μg/ml for pyrazinamide. The method was successfully validated according to the European Medicine Agency guideline for the selectivity, linearity and lower limit of detection, precision and accuracy, matrix effect, extraction recovery, carryover, dilution integrity and stability, and applied for quantification of analytes in clinical samples from TB patients. CONCLUSIONS: The presented method allows sensitive and reproducible determination of selected anti‐TB drugs with advantages such as low sample volume requirement, short run time of analysis, one‐step sample preparation procedure with capabilities for phospholipids removal, and a low quantification limit as well as a high degree of selectivity.