Detection of Thyroxine in Dietary Supplements Using an Enzyme-Linked Immunosorbent Assay
Journal of health science
3,5-diiodo-L-tyrosine, C 15 H 11 I 4 NO 4 , mol.wt. 776.87] is one of the thyroid hormones prescribed for the symptoms of hypothyroidism. It is also possible that mild thyroid hormones excess over many years may increase the risk for a serious heart rhythm problem and fractures caused by excessive calcium loss from bones. In general, LC/MS is used for the assay of T4 in supplements with enzymatic hydrolysis and extraction procedure. 5) This assay method is timeconsuming and requires proficient
... equires proficient techniques. Enzyme-linked immunosorbent assay (ELISA) has become an increasingly important alternative detection method for the determination of pesticides, 6) toxins 7) and forensic drugs 8) as a screening tool. ELISA for the detection of T4 in serum have been developed as a specific and rapid method, and a commercial test kit based on ELISA for clinical diagnosis of various thyroid disorders is available on the market. The present study examined an ELISA procedure for the detection of T4 in supplements made from Chinese herbal preparations advertised as weight reducers. MATERIALS AND METHODS Materials ---Five samples were offered by consumers with thyroid gland disease and liver trouble. Eight samples were collected from Aichi prefecture (Japan) by drug inspectors in 2002. All samples were claimed to be extracts of animal organs and traditional Chinese herbs, for use as weight reducers. Reagents and Instrumentation ---Dried thyroid of Japanese Pharmacopoeia quality was purchased from Teikoku Hormone Mfg. (Tokyo, Japan) and 0.01 mol/l phosphate buffered saline (PBS, for tissue washing) and protease (for biochemistry) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Reducing buffer solution was prepared as a solution in 0.11 mol/l sodium chloride Thyroxine (T4), one of the thyroid hormones, in adulterated dietary supplements was analyzed using two different methods; enzyme-linked immunosorbent assay (ELISA) and LC/MS. To release T4 from thyroglobulin, samples were first hydrolyzed with proteolytic enzyme, and then the supernatant was diluted and directly analyzed using a commercial free T4 ELISA kit for diagnostic discrimination. In contrast, T4 was extracted with ethyl acetate from the supernatant, and then ethyl acetate layer was evaporated. The residue was dissolved in the mobile phase and analyzed by LC/MS with electrospray ionization (ESI) interface under positive ion mode. These methods were applied to the analyses of 13 dietary supplements advertised as weight reducers. T4 was detected in four of the samples and the analytical results by ELISA agreed well with those obtained by LC/MS. The ELISA technology described here is available for the screening of T4 in adulterated supplements.