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Sub-Department ofEndocrine Pathology, University of Liverpool, The Liverpool Clinic, Liverpool, L7 7DE

(Received 17 October 1974)

Although mild methods of hydrolysis are available, hot acid hydrolysis is frequently used to hydrolyse oestriol conjugates before estimation of the free steroid. Assessment of losses arising from hydrolysis, extraction and other manipulations is possible, using radioactive oestriol. Tritiated oestriol is preferable to ¹⁴C-labelled oestriol because of the high specific activity of tritium-labelled compounds. For radioimmunoassay, compounds of very high specific activity have been prepared, labelled in the 2,4 positions, with or without additional radioactivity at positions 6,7 or 6,9.

We have found that oestriol labelled in the 2,4 positions is unsuitable for following procedural losses if hot acid hydrolysis is used.

Tritiated steroids (Radiochemical Centre, Amersham) were checked for radiochemical purity by thin-layer chromatography, followed by scanning in a radiochromatogram scanner. No evidence of impurity was found. The steroid (either 20 000