(115g) Enantioseparation of Mitotane On Kromasil CHI-TBB Using CO2 at High Pressure and Methanol, Ethanol and 2-Propanol as a Modifier

Introduction: Mitotane (o,p'-DDD) is a synthetic derivative of the insecticide dichlorodiphenyltrichloroethane (DDT) and have potent adrenotoxic effects(1). It is a chiral drug marketed in the form of racemic mixture and has been extensively used in the treatment of adrenocortical carcinoma (ACC), tumor of the outer layer of the adrenal gland. In the USA, about 25 new cases occur each year but in Southern Brazil, approximately 10 times that many cases are diagnosed each year, mainly in the states of São Paulo and Paraná(2). Recent pre-clinical research on the power of antineoplasic mitotane enantiomers revealed the S-enantiomer is four times more potent in comparison with the R-enantiomer(3) and for these studies to continue, it is essential to obtain the enantiomers in pure form and in significant quantity. The preparative chromatography with chiral stationary phase has been a well established tool for the fast separation of enantiomers, especially when combined with the use of a supercritical eluent leading to experimental system with high efficiency and high separation factor. The use of supercritical fluid chromatography may result in higher production rates as the resolution per time in general is better in this process type(4). The aim of this work was to separate the mitotane enantiomers on a chiral stationary phase and CO2 with modifier, methanol, ethanol or 2-propanol, as mobile phase in semi-preparative scale and to study the effect of modifier concentration on the separation behavior at constant temperature and pressure. Experimental: The racemic mitotane was separated using the column Kromasil® CHI-TBB as chiral stationary phase (CSP) and carbon dioxide at high pressure as mobile phase, varying the composition of solvents modifiers, methanol, ethanol or 2-propanol. Two levels of percentage were defined, 2.6 and 4.8% and the conditions of temperature, 35°C, and pressure, 160 bar, were kept constant. The separation unit is capable of processing semipreparative scale and includes a cooler of CO2, a bath for cooling, a pump piston high-precision (Gilson 805 Manometric Module, USA) and a preparative HPLC pump (Shimadzu LC 6AD Liquid Chromatography, Japan). The system also includes a cell mixture, five valves block, four valves micrometers, a sample injection system (Rheodyne 712i, USA) through a valve the of six doors, a chiral chromatographic column and a heating system. The detection system consists of a UV spectrophotometer (SPD 10A Shimadzu, Japan) and a control module (Shimadzu SCL 10A, Japan) coupled, as shown in Figure 1. Results: In scientific literature, there is no description of the enantioseparation of mitotane racemic using supercritical fluid chromatography method with the chiral column Kromasil® CHI-TBB, as described in this work. The separation factor (α) more promising with value of 1.47, was achieved for the addition of 4.8% methanol as the best resolution (Rs) for the enantiomer 1 with value of 1.06. The high separation factor obtained with the addition of methanol may be related to its dielectric constant, higher than that of other solvents, which favors the solubility of the both enantiomers of mitotane. High dielectric constant means less force of attraction between enantiomer and the activities sites of CSP due the increase in the capacity of solvatation of enantiomers. However, using 4.8% methanol the nonspecific forces were minimized, while the specific forces were little influenced, revealing consequently larger separation factor in this condition. The highest retention factors (k), with values of 2.54 and 2.70, for the enantiomers 1 and 2, respectively, were obtained adding up 2.6% ethanol as the highest resolution value (Rs) of 1.00 for the enantiomer 2. The values for the theoretical plates (N) were higher than those obtained in the separation of mitotane by liquid chromatography with the same CSP and the same temperature and flowrate, 35°C and 3 mL/min(5) reaching 7727 and 5670 with the addition of 4.8% ethanol for enantiomer 1 and 2.6% ethanol for enantiomer 2, respectively. The increase in the percentage of co-solvents caused an increase in separation factor when the methanol was added and in the retention factor when 2-propanol was added. References: (1) Terzolo, M., Daffara, F., Conton, P. A., Sperone, P., Bollito, E., Hahner, S., Ambrosi, B., Mannelli, M., Allolio, B. ?Adjuvant mitotane treatment fot adrenocortical carcinoma?. The New England Journal of Medicine, v. 356, pp. 2372-2380, 2007. (2) Ribeiro, R. C., Michalkiewicz, E. L., Figueiredo, B. C., DaLacerda, L., Sandrini, F., Pianovsky, M. D., Sampaio, G., Sandrini, R. ?Adrenocortical tumors in children?. Brazilian Journal of Medical and Biological Research, v. 33, pp. 1225-1234, 2000. (3) Zancanella, P., Santana, C. C., Figueiredo, B. C. and Oliveira, B. H. ?Enantioselective metabolismo of mitotane?. Submitted to Journal of Pharmaceutical Sciences, 2008. (4) Johanssen, M. ?Separation of enantiomers of ibuprofen on chiral stationary phases by packed column supercritical fluid chromatography?. Journal of Chromatography A, v. 937, pp. 135, 2001. (5) Dias, R. M. Msc. Thesis, State University of Campinas, 2007.