In vitro and in vivo investigation of metabolic fate of rifampicin using an optimized sample preparation approach and modern tools of liquid chromatography–mass spectrometry

Biotransformation products of rifampicin formed in vitro and in vivo were identified using modern tools of liquid chromatography–mass spectrometry (LC–MS). For the same, first the mass fragmentation pattern of rifampicin was delineated by conducting multiple-stage MS (MSn), high resolution MS (HR-MS) and hydrogen/deuterium exchange MS (HDE-MS) experiments in an electro-spray ionization (ESI) mode. Then in vitro metabolism was investigated by incubating the drug with rat liver S9 fraction and microsomes at pH 7.4 in the presence of NADPH for 2 h at 37 °C. In vivo metabolites were generated by administering the drug to Sprague–Dawley (SD) rats (n = 5) at a dose of 50 mg kg−1 and collecting blood, urine and feaces at different time points up to 72 h. The samples were processed using an optimized sample preparation approach, involving protein-precipitation using acetonitrile; followed by liquid-freeze separation at −20 °C; and solid-phase extraction of aqueous layer. HPLC conditions were optimized using known standards of rifampicin metabolites or degradants. The extracted and concentrated samples were then subjected to LC-DAD–MS/time-of-flight (LC-DAD–MS/TOF) analyses in both ESI positive and negative modes. The structures of metabolites were elucidated by comparing their accurate mass values and fragmentation patterns with those of rifampicin, use of in silico identification tools and relative retention time matching with the standards. The nature of the metabolites and their relative amounts varied. In total, 21 metabolites were identified, of which many structures were new, not reported till date.