LC-MS/TOF and UHPLC-MS/MS study of in vivo fate of rifamycin isonicotinyl hydrazone formed on oral co-administration of rifampicin and isoniazid.

The formation and fate of 3-formylrifamycin isonicotinyl hydrazone (HYD) was investigated following oral co-administration of rifampicin (RIF) and isoniazid (INH) in Sprague-Dawley (SD) rats (n=5) using advanced analytical modalities. The study was carried out with 20 and 5mg/kg doses of RIF and INH, respectively. The plasma, urine and faeces samples were collected at different time points up to 48h, which were qualitatively and quantitatively evaluated for the presence of HYD after proper sample preparation. For the same, initially liquid chromatography-mass spectrometry/time-of-flight (LC-MS/TOF) method was developed in electrospray ionization (ESI) positive mode, wherein separation was achieved on a C18 column (4.6mmx250mm, 5microm), using a volatile mobile phase in a gradient mode. The presence of HYD was confirmed by accurate mass study, spiking with the standard and UV-visible spectra matching. For quantitative evaluation of HYD, a selective and sensitive ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for all the three matrices. In this case, elution of HYD was achieved on a small C18 column (4.6mmx50mm, 1.8microm) using a short gradient method. The quantitation was done by selective reaction monitoring (SRM) in ESI positive mode. The validation parameters like linearity, accuracy, precision, selectivity, matrix effect, recovery and stability were assessed as per regulatory guidelines. The calibration range was established between 1 and 200ng/ml, with r(2)>0.99 in all the cases. The back calculated values for three quality control (QC) samples, and at lower limit of quantitation (LLOQ) were within 15 and 20%, respectively, of the nominal values. Similarly, the intra- and inter-day precisions were found within 15% at the four tested levels. The HYD was found to be stable for the duration of sample preparation and analysis in the controlled experimental conditions. The analysis of in vivo samples showed a significant extent of HYD in faeces, however, the interaction product was not found in plasma and urine. To verify the results, 5mg/kg oral dose of HYD standard was given to rats separately, and its presence was studied in all the three matrices. Further, in vitro plasma stability of HYD was also carried out to explain its absence in plasma and urine, which showed approximately 55% disappearance of HYD in 2h.