The removal of non-steroidal pigments of urinary extracts by adsorption on charcoal.

The extraction of steroids from urine with the usual organic solvents is accompanied by the extraction of undesirable pigments which complicate the isolation of urinary steroids by paper chromatography. Even after preliminary fractionations by paper chromatographic techniques, Burton et al. (1) found that the residues were highly pigmented and bulky. This made the task of detecting minute amounts of urinary steroidal metabolites very difficult. Interfering urinary pigments were also encountered by Romanoff et al. (2) in the most polar fraction, despite preliminary fractionations on silica gel. Preliminary studies in our laboratories have shown that these pigments are adsorbed on charcoal. Although no data were presented, Zaffaroni has reported that charcoal adsorption was capable of producing artifacts and was not especially suitable for the quantitative recovery of steroids (3). The opinion that artifacts may be produced by adsorption of steroids on charcoal has also been expressed by others (4, 5). Finally, Meyer (6) has shown an approximately 14 per cent conversion of cortisone to adrenosterone after extraction from charcoal in a Soxhlet apparatus by methylene chloride, benzene, and ethyl acetate. He found only a 44 per cent recovery of cortisone after its adsorption from aqueous media by charcoal and subsequent elution in a Soxhlet apparatus with the above solvents. The elution of steroids from charcoal at room temperature with a number of solvent systems was investigated. A solution of 40 per cent benzene in absolute ethyl alcohol (volume per volume) proved to be the most satisfactory for the quantitative elution of steroidal substances (both 17ketosteroids and corticosteroids) from charcoal. This could be accomplished at room temperature with a minimal elution of the pigments associated with urinary extracts.