Appendix VIII J. Tetrazolium Assay of Steroids

The coloured reaction products tend to adsorb onto the surface of the glassware. To avoid low results, the glassware should be treated with the coloured reaction products before use. The treated glassware should be reserved for this assay and should be washed only with water between assays.

Carry out the following procedure protected from light. Dissolve a quantity of the substance being examined in sufficient aldehyde-free absolute ethanol to produce a solution containing 300 to 350 µg in 10 mL unless otherwise specified in the monograph. Transfer 10 mL to a 25-mL graduated flask, add 2 mL of triphenyltetrazolium chloride solution, displace the air in the flask with oxygen-free nitrogen, immediately add 2 mL of dilute tetramethylammonium hydroxide solution and again displace the air with oxygen-free nitrogen. Stopper the flask, mix the contents by gently swirling and allow to stand in a water bath at 30° for 1 hour unless otherwise specified in the monograph. Cool rapidly, add sufficient aldehyde-free absolute ethanol to produce 25 mL, mix and immediately determine the absorbance of the resulting solution in a stoppered cell at the maximum at 485 nm, Appendix II B, using in the reference cell a solution prepared at the same time and in the same manner using 10 mL of aldehyde-free absolute ethanol. Repeat the operation using the specified BPCRS or EPCRS in place of the substance being examined ensuring that the period of time that elapses between the addition of the tetramethylammonium hydroxide solution and the measurement of the absorbance is the same as for the test solution.