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  • Title: A sensitive method for quantitation of rifabutin and its desacetyl metabolite in human biological fluids by high-performance liquid chromatography (HPLC).
    Author: Lewis RC, Hatfield NZ, Narang PK.
    Journal: Pharm Res; 1991 Nov; 8(11):1434-40. PubMed ID: 1665904.
    Abstract:
    Sensitive HPLC-UV methodology has been developed and validated for quantitating rifabutin, an antimycobacterial, and its 25-desacetyl metabolite, LM-565, in human plasma and urine. The HPLC separation for both plasma and urine samples was performed on an ODS, 5-microns, reverse-phase column (25 cm x 4.6-cm ID) using a mobile phase of acetonitrile/0.05 M potassium phosphate, pH 4.2, with triethylamine, (38:61.5:0.5, v/v), at a flow rate of 1.0 ml/min. The separation eluate was monitored by absorbance at 275 nm. Plasma samples (1 ml) were spiked with an internal standard (medazepam), buffered at pH 7.4 and extracted with 80:20 (v/v) hexane:ethyl acetate, and then back extracted with acidified water (0.05 M H3PO4). Linearity was established between 5.0-800 and 2.5-400 ng/ml for rifabutin and LM-565, respectively. Intraday imprecision for rifabutin and LM-565 plasma quality controls prepared at 7.3 and 3.2 ng/ml, respectively, was less than 15% relative standard deviation (RSD). Absolute recovery for parent drug and metabolite, from plasma, was greater than 90% throughout the respective dynamic ranges and greater than 70% for medazepam. Urine samples (1 ml) were acidified with 50 microliters of 3.6 M H2SO4 and diluted with 0.1 M ammonium acetate. Linearity was established between 100 and 5000 ng/ml for both rifabutin and LM-565. Intraday imprecision for a urine control at 200 ng/ml was less than or equal to 12% RSD for either component. The method is currently being used to support Phase I kinetics program for rifabutin in prophylaxis of MAC infection of AIDS patients. Application of this method to a bioavailability assessment is presented.
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