GRAFTED 2d-Thin LAYER CHROMATOGRAPHY (2d-Tlc) OF 17α-Ethinylestradiol DETECTED BY BIO-DIRECTED ANALYSIS

Abstract

& We present a two dimensional (2D) planar chromatographic separation of estrogenic active compounds on RP-18 (Merck, 1.05559) and silica gel (Merck, 1.05721) phase. A mixture of 13 substances was separated using a solvent mix consisting of methanol-acetonitrile-water (2 þ 2 þ 1, v=v=v) on RP-18 phase in the first direction and cyclohexane-butylacetate-methanol (8 þ 6 þ 1, v=v=v) in the second direction on silica gel plate. Both developments were carried out over a distance of 70 mm. We used the grafted method to combine both plates in a 2D-separation. This 2D-separation method can be used to quantify 17a-ethinylestradiol (EE2) in an effect-directed analysis using the yeast strain Saccharomyces cerevisiae BJ3505. The test strain (according to McDonnell) contains the estrogen receptor. Its activation by estrogen active compounds is measured by inducting the reporter gene lacZ that encodes the enzyme ß-galactosidase. This enzyme activity is determined on plate by using the fluorescent substrate MUG (4-methylumbelliferyl ß-D-galactopyranoside).