Development and Validation of a UPLC-MS/MS Method for Therapeutic Drug Monitoring, Pharmacokinetic and Stability Studies of First-Line Antituberculosis Drugs in Urine

Abstract

Tuberculosis (TB) remains one of the leading global causes of mortality. Several methods have been established to detect anti-TB agents in human plasma and serum. However, there is a notable absence of studies analyzing TB drugs in urine. Thus, our objective was to validate a method for quantifying first-line anti-TB agents: isoniazid (INH), pyrazinamide (PZA), ethambutol (ETH), and rifampicin (RIF), along with its metabolite 25-desacetylrifampicin, and degradation products: rifampicin quinone and 3-formyl-rifampicin in 10 µL of urine. Chromatographic separation was achieved using a Kinetex Polar C18 analytical column with gradient elution (5 mM ammonium acetate and acetonitrile with 0.1% formic acid). Mass spectrometry detection was carried out using a triple-quadrupole tandem mass spectrometer operating in positive ion mode. The lower limit of quantification (LLOQ) was 0.5 µg/mL for INH, PZA, ETH, and RIF, and 0.1 µg/mL for RIF's metabolites and degradation products. The method was validated following FDA guidance criteria and successfully applied to the analysis of the studied compounds in urine of TB patients. Additionally, we conducted a stability study of the anti-TB agents under various pH and temperature conditions to mimic the urine collection process in different settings (peripheral clinics or central laboratories).

Keywords: ethambutol; isoniazid; pyrazinamide; rifampicin; therapeutic drug monitoring.

Figure 1

MRM chromatograms of anti-TB drugs in human urine: (A) unspiked blank human urine; (B) blank human urine spiked at the LLOQ level (0.1 mg/L for RIF, RIF-Q, 3-F-RIF, and 25-D-RIF; 0.5 mg/L for INH, PZA, RIF, and ETH), internal standards at levels 0.05 mg/L for RIF-IS, 0.5 mg/L for PZA-IS and ETH-IS; (C) human urine from the patient undergoing treatment for active TB disease with first-line RIF-containing regimen (measured concentrations: 18.5 mg/L for RIF, 2.4 mg/mL for RIF-Q, 0.5 mg/mL for 3-F-RIF, 3.1 mg/L for 25-D-RIF, 36.5 mg/L for INH, 79.5 mg/L for PZA, and 235.6 mg/L for ETH). Abbreviations: 25-D-RIF—25-desacetylrifampicin; 3-F-RIF—3-formylrifampicin; ETH—ethambutol; ETH-IS—ethambutol-D₄; INH—isoniazid; PZA—pyrazinamide; PZA-IS—pyrazinamide-15N,D₃; RIF-Q—rifampicin quinone; RIF—rifampicin; RIF-IS—rifampicin-D₃.

Figure 2

Changes in RIF and its metabolites (RIF-Q, 3-F-RIF, and 25-D-RIF) areas after 1 h, 8 h, and 24 h at 37.5 °C in pH 4 to pH 8. Abbreviations: 25-D-RIF—25-desacetylrifampicin; 3-F-RIF—3-formylrifampicin; RIF-Q—rifampicin quinone; RIF—rifampicin.

Figure 3

Changes in RIF and its metabolites (RIF-Q, 3-F-RIF, and 25-D-RIF) areas after 1 h, 8 h, and 24 h at 20.5 °C in pH 4 to pH 8. Abbreviations: 25-D-RIF—25-desacetylrifampicin; 3-F-RIF—3-formylrifampicin; RIF-Q—rifampicin quinone; RIF—rifampicin.