A highly sensitive and specific LC-MS/MS technique was developed. It is validated for quantifying rifapentine and 25-desacetyl rifapentine in K2EDTA human plasma. The concentration ranges are 60.061 ng/ml to 8008.134 ng/ml for Rifapentine and 30.000 ng/ml to 4000.015 ng/ml for 25-desacetyl rifapentine. The separation of analytes were achieved using reverse phase chromatography employing a Supelco discovery C18 column (10 cm × 4.6 mm, 5 μm particle size). Detection was performed via electro spray ionization in positive ion mode [M+H]+. A triple quadrupole mass spectrometer, with multiple reaction monitoring of specific ions for each analyte and their respective deuterated internal standards (IS) were used. Retention times for rifapentine and 25-desacetyl rifapentine were 2.45 minutes and 1.77 minutes, respectively. The retention time for the IS rifapentine D9 and 25-desacetyl rifapentine D8 were 2.30 minutes and 1.68 minutes, respectively. The total run time of the method was 8.00 minutes. No interfering peaks or matrix effects were noticed during validation, confirming the reliability of the results. The validation report encompasses standard curves, quality control sample recovery, stability of experiments, and meeting stringent criteria for sensitivity, reproducibility, and accuracy. This method has been effectively used in a rifapentine bioequivalency study with healthy adult Asian male volunteers, highlighting its suitability for pharmacokinetic investigations.
Key words: Rifapentine , LC-MS/MS, Method Validation, Human plasma, Internal Standard, Pharmacokinetics.
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