What is the Use of Tetramethylammonium Hydroxide in the Zimmermann Reaction?

The reaction described by Zimmermann (1935) is now widely used for the estimation of 17-keto-steroids, particularly in crude urine extracts, and depends upon the violet colour developed by mixing solutions of potassium hydroxide, dinitro-benzene, and ketosteroid. A careful study of the reaction was made by Callow, Callow, and Emmens (1938), and subsequently many modifications have been proposed with the object of simplifying the procedure or rendering it more specific. The main disadvantages of its use are that different steroids have different colour equivalents, and when used for crude biological extracts, materials other than steroids react to produce a non-specific interference. Wilson (1954) has suggested carrying out the reaction at a lower temperature to give more nearly equal colour equivalents, and various formulae have been proposed to correct for the interfering chromogens by the use of extinctions at two or three wavelengths.

Alternatively, the chromogen may be extracted with organic solvent, which leaves a considerable proportion of the interfering material in the residual solution. Most of these modifications have retained the use of potassium hydroxide in the reaction, but more recently, Bongiovanni, Eberlein, and Thomas (1957) and Glenn (1959) have proposed the use of an organic base instead, and it was of interest to know if this offered any advantage in the estimation of ketosteroids. 

The use of tetramethylammonium hydroxide (T.M.A.H.) in place of potassium hydroxide in the Zimmermann reaction has been investigated. Although various pure steroids have different colour equivalents, a comparison of the results of 17-ketosteroid and 17-hydroxycorticosteroid estimations on a series of urines showed that the differences for the two reagents were only small and may be ignored. Tetramethylammonium hydroxide has the considerable advantage over potassium hydroxide of stability and need not be prepared freshly. The correction procedures available are discussed.

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