Abstract
The development of a quantitative paper chromatographic method for the routine determination of reducing C 21O 5 and C 21O 6 corticosteroids from guinea pig urine extracts by means of a single chromatographic step and direct densitometry following reaction with blue tetrazolium has been described. Among a number of paper chromatographic systems tried, the system 1,2-dichloroethane-methanol-water was found to give the most suitable results. The phase diagram of this ternary system was constructed and a number of compositions and other variables affecting the resolution have been studied. An empirical system with a mobile phase consisting of 1–2% methanol in ethylene chloride saturated with H 2O was found to give a good resolution and reproducibility. Treating the papers with 1,2-dichlorobenzene prior to reaction with blue tetrazolium caused the formation of a much bluer formazan with an absorbancy of roughly three times that obtained without treatment. The all-over precision achieved following cortisol (20–120 μg) deposition on paper, chromatography, and densitometry was on the average 10% (coefficient of variation).
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