Simultaneous Microdetermination of Rifampin, Deacetylrifampin, Isoniazid, and Acetylisoniazid in Plasma by Liquid Chromatography with Dual Electrochemical and Spectrophotometric Detection

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Abstract
A rapid liquid chromatographic method for simultaneous determination of isoniazid (INH), acetylisoniazid (AINH), rifampin (RIF), and deacetylrifampin (DARIF) in microsamples of deproteinized plasma is described. The compounds and internal standard (IS) (diphenylcarbazide) were separated on a 10-µm, 8 mm × 10-cm phenyl Radial Pak cartridge in conjunction with a binary linear gradient system at a flow rate of 3 ml/min. A dual electrochemical (+ 800 mV) and Spectrophotometric (334 nm) detection system with a computerized data station was employed to measure the above compounds in the effluent. Prior to injection, the plasma sample was diluted (2:1) with a pH 3, 0.075 M phosphate buffer after adding the internal standard (6.67 µg/ml of plasma) and passed through an Amicon Centrifree-MS filter at 2000g. Under these conditions, no interference in the analysis was observed, and the retention times of AINH, INH, IS, DARIF, and RIF were 3.95, 4.89, 15.82, 17.25, and 19.34 min, respectively. The linearity of the assay for all four compounds was excellent (r > 0.9925), and the between- and within-day CV was not >8% at any concentration. This method is currently being used for therapeutic monitoring and pharmacokinetic studies of INH, RIF, and their major metabolites in patients with tuberculosis.

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