Postcolumn Iodine–Azide Reaction as a Detection System in HPLC for the Determination of Methylthiouracil in Urine

Abstract

This article describes a system for determining methylthiouracil in urine based on the sensitizing induction of methylthiouracil on the reaction between iodine and azide ions and combined techniques of high-performance liquid chromatography and simple postcolumn detection. The optimal conditions for iodine–azide reaction and high-performance liquid chromatographic separation were determined. The reproducibility, linearity, and recovery were evaluated under the optimal conditions. The methylthiouracil standards added to normal urine show that the response of the detector, set at 350 nm (corresponding to unreacted iodine in the postcolumn iodine–azide reaction), is linear within the concentration range of 0.4–5.0 nmol mL⁻¹ urine. The relative standard deviation values for precision and recovery within the calibration range varied from 0.9 to 4.8% and from 94 to 105%, respectively. Limits of detection (LOD) and quantitation (LOQ) were 0.3 and 0.4 nmol mL⁻¹ urine, respectively.

Keywords:

• Column liquid chromatography
• iodine–azide reaction
• methylthiouracil
• postcolumn reaction
• urine

Acknowledgments

The presented experiment was conducted with the support of Grant No. N204 14732/3728 from the Ministry of Science and Higher Education, Poland. The author thanks Prof. W. Ciesielski from the Department of Instrumental Analysis, University of Łódź, for his contribution in discussions.