On the Fluorimetric Determination of Adrenaline and Noradrenaline

Abstract

A modification of the fluorimetric method for the determination of adrenaline and noradrenaline in pharmaceutical solutions is described. In this method sulphite present is destroyed with iodine, the added excess of which is reduced with arsenous acid, a manipulation which does not affect the fluorescence obtainable.ADRENALINE and noradrenaline in low concentrations are most suitably determined by the fluorimetric method1#2. In pharmaceutical solutions, sulphite is usually present as a stabiliser. This introduces a difficulty in the determination of the catechols as these cannot be quantitatively transformed into the corresponding adrenochromes by potassium ferricyanide or by manganese dioxide if the sulphite present is not destroyed before the oxidation of the catechols.Adrenaline, however, can be determined ad modum EhrlCn3, even in the presence of sulphite by measuring the transient fluorescence obtained by oxidation with air in strongly alkaline solution. In this procedure there seems to be no interaction between the adrenochrome formed and the sulphite. A possible explanation of this phenomenon is that this reaction is performed at such a high pH that all sulphite is present as S O 3 --being non-reactive, while the oxidation with MnO, or Fe(CN), ---is performed in neutral or slightly acid solution, where the sulphite is present partly as HS0,-which is reactive forming a bisulphite compound with the adrenochromes.Noradrenaline cannot effectively be determined by the method of EhrlCn3 as the sensitivity is much lower than for adrenaline. It is, therefore, necessary first to oxidise noradrenaline with ferricyanide or MnO, to noradrenochrome and then to transform it with sodium hydroxide to the fluorescent adrenolutine. This procedure may sometimes be preferable in the determination of adrenaline as a higher fluorescent energy is obtained.As mentioned before, the sulphite present has to be destroyed in such a way that the fluorescence obtained is not affected. Several methods described are based on the separation of the catechols from the sulphite or on the destruction of the latter. The separation methods are tedious and not well suited to routine work. The destruction of the sulphite ions is usually brought about by oxidation with iodine in an acid solution such that the reaction between iodine and the catechols proceeds very slowly. The excess of iodine added has to be removed in order not to suppress the fluorescence later on. This can be done by addition of thiosulphate, an excess of which, however, also has a strong quenching influence on the fluorescence. The influence of thiosulphate is rather peculiar ; added to a solution of the adrenochrome it has no influence on 764

Fluorometric Analysis

“…Yoshinaga (416) found that the ethylenediamine method, although sensitive, lacks specificity. In the Lund procedure using Mn02 oxidation at pH 4 for adrenaline and pH 6 for noradrenaline, precise timing is necessary (79) and other improvements in technique are suggested (100); if sulfite is present, it must first be destroyed (48). Iodine also may be employed as the oxidant, at pH 4.4 and 7.2, respectively (162).…”