Development and Validation of a UPLC-MS/MS Method for Therapeutic Drug Monitoring, Pharmacokinetic and Stability Studies of First-Line Antituberculosis Drugs in Urine
Mohamed Abouzid,
Katarzyna Kosicka-Noworzyń,
Marta Karaźniewicz-Łada,
Prakruti Rao,
Nisha Modi,
Yingda L. Xie,
Scott K. Heysell,
Anna Główka,
Leonid Kagan
Affiliations
Mohamed Abouzid
Department of Physical Pharmacy and Pharmacokinetics, Poznan University of Medical Sciences, 3 Rokietnicka Street, 60-806 Poznań, Poland
Katarzyna Kosicka-Noworzyń
Department of Physical Pharmacy and Pharmacokinetics, Poznan University of Medical Sciences, 3 Rokietnicka Street, 60-806 Poznań, Poland
Marta Karaźniewicz-Łada
Department of Physical Pharmacy and Pharmacokinetics, Poznan University of Medical Sciences, 3 Rokietnicka Street, 60-806 Poznań, Poland
Prakruti Rao
Division of Infectious Diseases and International Health, University of Virginia, 345 Crispell Drive, Charlottesville, VA 22903, USA
Nisha Modi
Public Health Research Institute, Department of Medicine, Rutgers New Jersey Medical School, Newark, NJ 07013, USA
Yingda L. Xie
Public Health Research Institute, Department of Medicine, Rutgers New Jersey Medical School, Newark, NJ 07013, USA
Scott K. Heysell
Division of Infectious Diseases and International Health, University of Virginia, 345 Crispell Drive, Charlottesville, VA 22903, USA
Anna Główka
Department of Bromatology, Poznan University of Medical Sciences, 3 Rokietnicka Street, 60-806 Poznań, Poland
Leonid Kagan
Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA
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).
