A method for microdetermination of cortisol and corticosterone in blood by the isotope derivative dilution analysis was established by the use of thiosemicarbazide-³⁵S as the labeled reagent and ³H-labeled corticosteroid for correcting the loss during extraction and separation procedures.
Cortisol and corticosterone are reacted with thiosemicarbazide-³⁵S in acetic acidmethanol at 65° for 90min, and the thiosemicarbazones-³⁵S formed are extracted with dichloroethane. After addition of the carrier to the extract, it is submitted to repeated thin-layer chromatography at a low temperature of 10°, using solvent systems of chloroform-ethanol (9:1, v/v) and benzene-acetone (1:1), by which corticosteroids are separated from each other and from substances present in blood. The amount of isolated cortisol and corticosterone thiosemicarbazones are estimated from the radioactivity of ³H and ³⁵S.
According to this method, the values of cortisol and corticosterone are in the range of 0.001-10.0μg, recovery rate is ca.100%, and relative standard deviation is 1.0-6.7%. The determination can be made with good accuracy and precision. Comparative examination of the same sample by this method and by fluorometry gave a slightly higher values from this method, and the relative standard deviation showed a large dispersion of 6.0-18.0%. However, the analytical precision and sensitivity of this method are better than fluorometry. Determination of 0.1-5.0ml of sample plasma by this method showed. relative standard deviation of 0.8-3.4%, with small dispersion, and accuracy of determination was found to be satisfactory.