NEW COLORIMETRIC APPLICATIONS OF THE COLORED FERRICYANIDE-ORTHO-DIANI SIDINE SYSTEM. INDIRECTCOLORIMETRIC ESTIMATION OF CYSTEINE By J. AP, T•C•AS, I"'. BUSCAP, ONS and C. RODRIGUEz-RoDA* Presented September 6,/960, Seminar international Federation of Societies of Cosmetic Chemists, Barcelona, Spain THE OUTSTANDING importance of cysteine is well-known, as its presence is fundamental in various biochemical processes. It has been proved that among other actions, cysteine has a beneficial effect on the body as it minimizes the injurious effects produced by radio- activity, x-rays and by the intoxications due to heavy metals. It has also a powerful antihistaminic action. Moreover, the fact that cystine con- tained in wool, hair and horn materials of human or animal origin yields cysteine when its sulfur bonds are broken, gives interest to the study of the amounts of cystine produced in these breakings, thus allowing a better under- standing of these reactions. All these reasons led us to the study of the possible application to the quantitative estimation of cysteine by a new colorimetric method by means of a reaction we previously developed for the determination of various mercapto derivatives (1). First of all we undertook a wide bibliographic study of the different methods in existence for its determination. Their number and their diversity is extraordinary this shows clearly the interest shown in its estimation. The main methods used for cysteine determination can be classified as: Volumetric methods (iodometric, generally) Colorimetric methods (based on oxidation, formation of azo-derivatives, complexes, etc.) Electrometric methods (potentiometric, amperometric, polarographic) Colorimetric methods are among the most frequently used. Among them some may be mentioned: those which are based on the reaction between cysteine and ferric ion in presence of different aromatic amines (2, 3) or * Analytical Chemistry Dept., Faculty of Science, Univ. of Barcelona, Barcelona, Spain. 307

3O8 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS between cysteine itself and sodium nitroprussiate (4). Hazeloop based his method on the colored reaction between cysteine and o-benzoquinone in chloroform solution (5) Sch6berl based his on the reduction of phos- photungstic acid followed by the colorimetric determination of the blue compound obtained (6). The Hellerman (7) method must be plxced among the iodometric cysteine is treated with an excess of o-iodosobenzoic acid, yielding cystine and iodobenzoic acid the excess of the former being titrated iodometrically. Barnstein's method can be listed among the gasometric procedures: cysteine is oxidized to cystine by means of a solution of I• in KI. The excess of I• is determined by measuring the released Ne after I2 has reacted with hydrazine (8). The amperometric methods using Hg +•, Cu • and Ag 4 salts were studied by Kolthoff (9•11) and are among the newest electrical developments. It appeared to us, having carried out the study of these existing methods, that if our fbrmerly reported method for the determination of mercapto derivatives was applicable to the estimation of cysteine it could be of a greater simplicity and sensitivity. TH• Basins OF TH• M•'rHOD In previous papers (12, 13) we reported that it is possible to determine colorimetrically some oxidizing agents taking advantage of the fact that they transform o-dianisidine into quinonic compounds which possess a strong bluish-green color, this color turning red upon acidification. Later on we made use of this ferricyanide o-dianisidine system for the indirect colorimetric estimation of organic and inorganic reducing agents. We have thus studied the determination of Sn +=, As +a, S=Oa -2, S=O• -• and H=O= (14, 15) and of many mercapto derivatives (1). o 250• O / N o,o0 X EO0 300 400 500 •00700 8 0 900 I000 Figure 1.•Absorption spectrum in presence of cystine at pH 1.5 and at pH 3.