Determination of Isoniazid, Acetylisoniazid and Isonicotinic Acid in Human Urine by High Performance Liquid Chromatography Coupled with Postcolumn Photochemical Reaction and Fluorescence Detection

Abstract

A high performance liquid chromatographic method involving postcolumn photochemical reaction and fluorometric detection has been developed for the determination of Isonicotinic acid, acetylisoniazid and Isoniazid in human urine. These compounds are separated by reversed phase chromatography using 0.07M phosphate buffer (pH 6.8) containing 100mM hydrogen peroxide as a mobile phase. The compounds in the column effluent are irradiated with ultraviolet
light to give fluorescence. The fluorescence is monitored with excitation at 316nm and emission at 418nm. The calibration curves for Isonicotinic acid, acetylisoniazid and isoniazid are linear over the ranges of 0.1-120, 1.0-180 and 0.5-200ng, respectively. The mean recoveries of isonicotinic acid, acetylisoniazid and isoniazid from urine are more than 92%. This method can be applied for acetylator phenotyping.