Acetylation phenotyping of isoniazid using a simple and accurate high-performance liquid chromatography

A simple, specific, accurate and reproducible method for the analysis of isoniazid and its major metabolite, N-acetylisoniazid in urine using high-performance liquid chromatography (HPLC) is described. The assay is performed after extraction of isoniazid, N-acetylisoniazid and 5-(4-methylphenyl)-5-phenylhydantoin (internal standard) from urine using a mixture of chloroform:isopropanol (70:30, v/v) and eluted from a 5 microns C-18 reversed phase column at ambient temperature with a mobile phase consisting of 10 mM sodium acetate:methanol:acetonitrile (40:40:20, v/v) containing 10 mM dioctylsulphosuccinate sodium and adjusted to pH 2.9 with sulphuric acid (less than 1 ml), at a flow rate of 1 ml/min with u.v. detection at 266 nm. Quantification was achieved by the measurement of the peak height ratio, and the absolute recoveries ranged from 94 to 99%. Within-day coefficients of variation ranged from 2.81 to 4.54% for isoniazid and from 2.37 to 3.75% for N-acetylisoniazid. Between-day CVs varied from 3.27 to 5.62% and from 2.5 to 4.91% for isoniazid and N-acetylisoniazid, respectively. Preliminary stability tests using a urine sample from a subject showed an increase in mean isoniazid concentration of about 25% after 1 month storage at -20 degrees C. The method was used for acetylation phenotyping of five individuals.