DEFINITIVE IDENTIFICATION OF MICROQUANTITIES OF RADIOACTIVE STEROIDS BY RECRYSTALLIZATION TO CONSTANT SPECIFIC ACTIVITY

Abstract

ABSTRACT Studies of steroid metabolism using isotopically-labeled compounds at physiological levels present unique problems in the identification of the metabolites and in the demonstration of their radiochemical purity. The submicrogram quantities of material available preclude the use of classical identification techniques. The character of the evidence obtained, the advantages and disadvantages of chromatographic and countercurrent distribution methods are discussed. Crystallization to constant specific activity is a recognized method for demonstrating that a substance is not radiochemically impure. Its parameters have never been accurately defined. Its true power is achieved only when it is preceded by extensive purification of the material to be characterized. In this way, the unknown material is first categorized by its migration rate in various solvent systems, and then by its crystalline identity with the carrier compound. The likelihood of two dissimilar steroids being both isopolar and isomorphic is held to be remote. Liquid scintillation spectrometry and gravimetry are the techniques used for the determination of constant specific activity. This method for measurement of radioactivity is extremely flexible, sensitive, and lends itself to dual-isotope experiments. Gravimetry under standardized conditions is suitably precise and much more generally applicable than spectroscopic quantitation. The parameters of the technique of rapid, forced microcrystallization are analyzed. In particular, the problem of contamination of crystals is analyzed in detail, and it is pointed out that classical concepts of purification by crystallization, developed chiefly in connection with ionic inorganic materials, must be modified when applied to nonionic steroid compounds. A mathematical analysis of the errors inherent in this technique indicates that 3 successive crystallizations of a pure radioactive compound should yield values for the specific activity which are within ± 5 % of the average of the 3 values.