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 + 2 + 1, v/v/v) on RP-18 phase in the first direction and cyclohexane–butylacetate–methanol (8 + 6 + 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 17α-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).

Keywords:

• 17α-ethinylestradiol
• 2D-TLC
• bioassay analysis
• effect-directed analysis
• estrogenics
• ethinyl steroids
• HPTLC
• linear calibration range
• quantification
• TLC
• yeast estrogen screen assay
• YES assay

ACKNOWLEDGMENT

The authors express their appreciation to Merck-Company (Darmstadt, Germany), BioStep (Jahnsdorf, Germany), and Boistep GmbH (Jahnsdorf, Germany) for their kind support.

Notes

Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ljlc.