Determination of rifampicin and its main metabolite in plasma and urine in presence of pyrazinamide and isoniazid by HPLC method

A reversed phase HPLC method is described for the simultaneous estimation of rifampicin and its major metabolite desacetyl rifampicin, in the presence of isoniazid and pyrazinamide, in human plasma and urine. The assay involves simple liquid extraction of drug, metabolite and internal standard (rifapentine) from biological specimens and their subsequent separation on a C₁₈ reversed phase column and single wavelength UV detection. In plasma as well as in urine samples, all the three compounds of interest eluted within 17 min. Using methanol–sodium phosphate buffer (pH 5.2; 0.01 M) (65:35, v/v) as mobile phase under isocratic conditions, it was established that isoniazid, pyrazinamide and ascorbic acid (added to prevent oxidative degradation of analytes) did not interfere with the analyte peaks. Recoveries (extraction efficiency) for drug were greater than 90% in both plasma and urine, whereas for metabolite the values were found to be 79 and 86% in plasma and urine, respectively. The plasma and urine methods were precise (total coefficient of variation ranged from 5 to 23%) and accurate (−7 to 5% of the nominal values) for both the analytes. Individual variance components, their estimates and their contribution to the total variance were also determined. Using the same method, unknown samples supplied by WHO were assayed and good correlations were obtained between the found and intended values. The method developed proved to be suitable for simultaneous estimation of rifampicin and desacetyl rifampicin in plasma and urine samples.