Development and standardization of levothyroxine analysis by high-performance capillary electrophoresis

This study demonstrates the development of a stability indicating capillary electrophoretic assay of levothyroxine. The optimum separation environment of the assay was determined by examining the effect of pH, buffer concentration, and sample additives on the levothyroxine peak resolution. Phosphate buffer (100 mM), pH 2.5, in a 27-cm capillary produced a satisfactory levothyroxine peak at approximately 10 min. An increase in the concentration of separation buffer increased the migration time and peak area of the levothyroxine peak. The peak symmetry was improved with the addition of methanol and acetonitrile in the sample matrix. However, acetonitrile and methanol concentrations above 25% produced current leakage, probably caused by the formation of microbubbles within the capillary. Standard plot of levothyroxine was established in the range of 3.75 to 60.00 micrograms/ml; with a sample size of 20 nl, this corresponds to 75-2000 micrograms of levothyroxine detected per run. Intra- and interday variability remained < 5% for the standard samples. Separation of levothyroxine from its possible deiodinated degradation products, triiodo- and diiodothyronine T3 and T2, was also obtained under these operating conditions.