274 Determination of Testosterone Thiosemicarbazone and Study of its Immunological Reactions in Urine by Adsorptive Stripping V oltammetry Jacoba Mora-Gil Castaño, Eduardo Finilla Gil, Lorenzo Calvo Blázquez, Rosa Maria García Moneó Carra, and Antonio Sánchez Misiego* Departamento de Química Analítica y Electroquímica, Universidad de Extremadura, E-06071 Badajoz, Spain Received: June 8, 1993 Final version: August 13, 1993 Abstract The electroanalytical behavior of testosterone thiosemicarbazone (TT) has been investigated by linear sweep and differential pulse voltammetry with and without adsorptive preconcentration on a hanging mercury drop electrode (HMDE). The adsorptive stripping response has been evaluated with respect to preconcentration time and potential, drop size, sean rate, and other variables. Measurement of a selected reduction wave enables the determination ofTT in the μg L- 1 range, with a detection limit of 100 ng L -I (10 min preconcentration time). The determination can also be carried out in a urine/aqueous NaCl0 4 (2/ 8) medium. The immunological interaction of TT with the testosterone specific antibody can be electrochemically monitored via the decrease in the reduction wave, as described previously. This property is discussed as a too! for the determination of testosterone and its specific antibody by competitive immunoassay with amperometric detection in real matrices such as urine. Keywords: Testosterone thiosemicarbazone, Immunology, Adsorptive stripping ·voltammetry l. Introduction Polarography has often been used in the investigation of antigen-antibody reactions. Sorne pioneering works were based on DC polarography measurements [1 - 4] and classic (non- computer-assisted) potentiometric detection [5-8]. Modern electroanalytical techniques, such as differential pulse polaro- graphy and adsorptive stripping voltammetry with improved sensitivity and selectivity, have promoted the development of a variety of electrochemical immunoassay protocols. These include methods for the determination of serum proteins [9-12] and small molecules of biological interest, like estriol [13, 14], morphine [15], and others [1 6]. These methods are usually based on monitoring the competition for antibody between an antigen and an electrochemically labeled antigen, via the polarographic response of the latter. Electroactive labels that have been employed are mercuric acetate [13], nitro groups (14], and metal ions [9]. Thiosemicarbazone derivatives belong to a wide group of substances : with pharmaceutical properties which contain carbon-sulfur bonds [17]. The polarographic behavior of these comptmnds is well documented [18] . Usually, the most analytically \ useful polarographic wave of thiosemicarbazone deriva ti ves that dueto the reduction of the C=N group, which involves 2e' and 2H+ transfer. Among the biologically significant thiosemicarbazone derivatives, testosterone 'thio- semicarbazone '- {'.:CT) (I) is of particular interest due to its steroid nature. Its speci_gc polarographic behavior, however, has not been investigated. OH (I) Testosterone and related steroids present a well-documented electroactive behavior on mercury electrodes. Smyth [19] found well-defined differential pulse waves for keto-steroids when unsaturation was present at the a. /3 position of the keto group, with reduction potentials of approximately -1.8 V vs. SCE in 0.1 M tetraethylammonium perchlorate/50% CH 3 0H. Kutner et al.[20] employed high-performance liquid chromatography (HPLC) and polarographic detection to measure a mixture of testosteroids, with a working potential of -1.7 V vs. SCE. The mobile phase employed was 1/ 15 M phosphate buffer (pH 6)- acetonitrile (1: 1). The adsorptive properties oftestosterone and related compounds on a static mercury drop electrode ha ve been described by Wang et al. [21, 22] , with detection limits in the 10 - 10 M range in an aqueous medium (0.005 M NaOH). The analytical signa! selected was a wave due to reduction of the C=C bond in the steroid ring of testosterone and its derivatives (peak potentials, Ep: -1.35 V for testosterone, -1.27 for methyltestosterone, and -1.46 for progesterone). Wang et al. postulated that the adsorptive behavior of steroids involves a planar orientation of the ring system and the electrode surface. On this basis, the thiosemicarbazone derivative studied in this work is expected to have similar adsorptive properties. In this article the results of an electroanalytical study of TT by linear sweep voltammetry with previous adsorptive preconcen- tration are described, and the interaction between testosterone, TT, and the testosterone specific antibody (Tab) is explored. The results are discussed in terms ofthe method's applicability to the electroanalytical determination of TT and Tab in urine. 2. Experimental 2.1. Apparatus For cyclic and linear sweep voltammetry, a Metrohm Herisau (Switzerland) E-612 potentiostat VA-Scanner and a Metrohm E-611 VA-Detector were used in connection with a Metrohm polarographic stand, provided with a Metrohm 'Kemula' HMDE working electrode. For differential pulse polarography, a Elec1roanaíysis 1995, 7, No. 3 © VCH Veríagsgesellschafl mbH, D-69469 Weinheim, 1995 1040-0397/95/0303-274 $ 5.00+ 25/0