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Microdetermination of adrenocortical steroids by double isotope method. I. Determination of cortisol and corticosterone in blood as thiosemicarbazone-35S.

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.