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This article compares different methods for determination of iodine in biological samples. Most frequently, indirect methods are applied in clinical practice. Sandell-Kolthoff spectrophotometric method of iodine determination [1] ranks among the most widely used ones; researchers also developed other modifications of the above method. Regarding direct determination, the following methods are considered the most suitable ones: emission spectrometry with inductively coupled plasma (ICP-OES), electrochemical methods, mass spectrometry with inductively coupled plasma (ICP-MS), and nuclear activation analysis (NAA) [2]. The ICP-MS and NAA are costly, but iodine can be analyzed directly regardless of its form, oxidation state, and isotopic distribution of iodine [3]. On the contrary, electrochemical, and ICP-OES determinations can be complicated with remarkable spectral interferences and also with dependence on possible presence of various forms and oxidation states of iodine in samples [4]. We compared three most frequently used methods for determination of iodine in selected biological materials: Sandell-Kolthoff spectrophotometric determination, ICP-OES and ICP-MS techniques [5]. The samples of biological materials such as foods and food supplements corresponding to content of iodine in urine were selected (Jooste and Strydom 2010). A NIST-SRM-1549 Milk powder certified reference material (CRM) with certified iodine concentration (3.38+0.6 mg/kg) was used to control the accuracy of the total iodine determination. The least accurate results were obtained by ICP-OES in analyses of given CRM (5.64+0.39 mg/kg), better results were acquired by spectrophotometric method (4.53+0.23 mg/kg) and the most accurate results were provided by ICP-MS method (3.36+0.12 mg/kg of iodine).