Development, validation and clinical use of a LC-MS/MS method for the simultaneous determination of the nine main antituberculosis drugs in human plasma

The treatment of tuberculosis, in particular the multi-drug resistant tuberculosis, remains a challenge mainly because of the therapy duration and the pharmacokinetic variability of the drugs included in the regimen. The monitoring of antituberculosis drugs is a recent tool that could improve the outcome of patients. We developed a LC-MS/MS method allowing the simultaneous quantification of the four first-line drugs (isoniazid, rifampicin, pyrazinamide and ethambutol), a metabolite of isoniazid (acetylisoniazid) and the five main second-line drugs (rifabutin, levofloxacin, moxifloxacin, linezolid and bedaquiline). An isotopologue standard was used for each drug. The protein precipitation was performed with acetonitrile and the separation was carried out using an EC-18 column and a gradient elution. The validated ranges for each drug were adapted to monitor the plasma concentration at 2 h (peak) and 6 h to evaluate their enteric absorption. The intermediate precision (CV) and the trueness at the limit of quantification were ≤ 10.1% and ≤ 10.7%, respectively. Preliminary data were obtained for 12 patients. The results showed that 38% of the patients had infra-therapeutic levels for both rifampicin and isoniazid, that the leading cause of an impaired oral absorption seemed to be malabsorption and that the effective concentrations for rifampicin were in the lower range of the therapeutic interval.