New approach to rifampicin stability and first-line anti-tubercular drug pharmacokinetics by UPLC-MS/MS

Successful tuberculosis (TB) therapy requires achieving sufficient exposure to multiple drugs. Limited stability of
several first-line anti-TB drugs might compromise reliable therapeutic drug monitoring (TDM). We developed
and validated a sensitive and selective UPLC-MS/MS method for simultaneous quantification of isoniazid (INH),
pyrazinamide (PZA), rifampicin (RIF), its metabolite 25-desacetylrifampicin and degradation products: rifampicin
quinone and 3-formyl-rifampicin. Analysis was completed from a very small plasma volume (20 μL) using
only protein precipitation with methanol. Chromatographic separation was achieved on a Kinetex Polar C18
column (2.6 μm; 150 × 3 mm) with a mobile phase consisting of 5 mM ammonium acetate and acetonitrile, both
containing 0.1 % formic acid, in gradient elution. The analytes were detected using a positive ionization mode by
multiple reaction monitoring. The LLOQ for RIF and its degradation products was 0.1 μg/mL, 0.05 μg/mL for
INH, and 0.2 μg/mL for PZA. The method was validated based on the FDA guidance. The application of the
method was confirmed in the analysis of RIF, INH, and PZA, as well as RIF metabolism/degradation products in
plasma samples of patients with TB. Based on the detailed stability study of the analyzed compounds at various
storage conditions, we proposed recommendations for handling the plasma and serum samples in TDM and other
pharmacokinetic studies.