By means of paper chromatographic separation and colorimetric determination,it is possible to develop a method for the determination of .5α-dihydrocortisone acetate(allo-pregnane-17α,21-diol-3,11,20-trione-2l-acetate).The proposed procedure is given below.Prepare a chloroform solution of the sample (ca.3 mg/ml).Impregnate the chromatographic paper (Whatman No.1 or Chinese Snow White No.1) with a mixture of formamide and methanol (6:4),and spot in duplicate a suitable amount of the sample solution (30-100 μg).Develop the chromatogram with cyclohexane-benzene-chloro-form (1:1:1) for about five hours,using descending technique.Remove the paper,cut off one strip and reveal the spots by immersing first in Tollen's reagent,then in 5% sodium thiosulphate solution and wash thoroughly with water.The steroid appears as a dark brown spot.Cut out the corresponding part on the remaining strip,elute with chloroform,collect about 4 ml of the liquid in a small beaker.Evaporate off,dissolve the residue in 5 ml 95% ethanol,add 0.5 ml tetramethylammonium hydroxide solution (10 ml of 10% aqueous solution and enough 95% ethanol to 100 ml) and 0.5 ml triphenyltetrazolium chloride solution (500 mg in 100 ml 95% ethanol).Mix well and determine the optical density at 480 mμ after 15 minutes,using a blank paper strip similarly treated as the reference.Calculate the content from a calibration curve.