1. Yvonne Anna Minar, Barbara Milz and Bernd Spangenberg*
2D-Thin Layer Chromatography of 17α-Ethinylestradiol on RP-18 W plate, detected by YES-Test
Yvonne Anna Minar, Barbara Milz and Bernd Spangenberg*
University of Offenburg, Department of Process Engineering, Badstrasse 24, D Offenburg, Germany

2. First of all I want to explain, how the YES-test work.
The Yeast Estrogen Screen Test (YES-Test according to Routledge and Sumpter) is used for detecting estrogenic active compounds.
First of all I want to explain, how the YES-test work.

3. Yeast Estrogen Screen (YES) test according to Routledge and Sumpter 1
MUG MU
MUG MU
X-ß-GAL
Indigo
4-Methylumbelliferyl ß-D-Galaktopyranosid
5-bromo-4-chloro-3-indoxyl-β-D-galactopyranosid
5,5'-dibromo-4,4'-dichloro-indigo
4-Methylumbelliferon

4. Planar Yeast Estrogen Screen Test (p-YES)
Plate immersion into alkaline buffer (pH 12)
(LacZ-buffer)
Plate after separation (RP-18 W plate)
Plate immersion into yeast suspension
4 h
1-12 h
Physical detection
(UV-366 nm)
Detection of effect
B. Spangenberg, C.F. Poole, C. Weins, Quantitative Thin Layer Chromatography. A Practical Survey, Springer, Berlin, 2010

5. Detection reactions for p-YES test
1 h
Detection of effect
12 h

6. Formulas of estrogenic acting compounds
Estrone (E1)
Estriol (E3)
17ß-Estradiol (E2)
Resveratrol
Zearalenone (ZEA)
17α-Ethinyl estradiol (EE2)
Bisphenol A (BPA)
Diethylstilbestrol (DES)

7. 2D p-YES, separation of 17α-ethinylestradiol (EE2) on RP-18 W plate
1. Direction: Hexane, ethyl acetate, acetone ( , v/v)
H2SO4 staining
YES-test (MUG)
2. Direction: Acetone and water ( , v/v)
Y. A. Witos, B. Milz, B. Spangenberg, “Two-Dimensional Thin-Layer Chromatography of 17α-Ethinylestradiol on RP-18 W Plate, Detected by Effect-Directed Analysis Using the YES Test”, J. Planar Chromatogr.-mod. TLC 30 (2017) 136–141.

8. The method can be used for phytoestrogenic investigations.
The detection limit for 17α-ethinylestradiol (EE2) is 30 pg per spot. That is bad in comparison to the detection limit for EE2 on silica plates with 50 fg per spot.
The method can be used for phytoestrogenic investigations.
Y. A. Witos, B. Milz, B. Spangenberg, “Two-Dimensional Thin-Layer Chromatography of 17α-Ethinylestradiol on RP-18 W Plate, Detected by Effect-Directed Analysis Using the YES Test”, J. Planar Chromatogr.-mod. TLC 30 (2017) 136–141.

9. 2D separation of a red clover extract (Trifolium pratense) on RP-18 W plate
1. Direction: Hexane, ethyl acetate, acetone ( , v/v)
H2SO4 staining
native fluorescence
YES-test
2. Direction: Acetone and water ( , v/v)

10. 2D separation of a red clover extract (Trifolium pratense) on RP-18 W plate
YES-test + MUG
2D-plate of red clover after YES-Test and dipping in LacZ-buffer.
Do we really have eight estrogenic active compounds in the red clover extract? 6 3 7 5 1 2 4 8

11. dipped in alkaline LacZ-buffer (without YES-test)
1D separation of a red clover extract (Trifolium pratense) on RP-18 W plate
Red clover tracks
dipped in alkaline LacZ-buffer (without YES-test)
native fluorescent

12. dipped in alkaline LacZ-buffer (without YES-test)
1D separation of a red clover extract (Trifolium pratense) on RP-18 W plate
Red clover tracks
dipped in alkaline LacZ-buffer (without YES-test)
native fluorescent
Fluorescence enhancement !

13. After YES-test we observe a single new band (compound 1)!
1D separation of a red clover extract (Trifolium pratense) on RP-18 W plate
After YES-test we observe a single new band (compound 1)!
dipped in yeast suspension and LacZ-buffer (YES-test, MUG)
dipped in alkaline LacZ-buffer (without YES-test) ? ? ? ? 1 ? ?
Red clover tracks

14. 1D separation of a red clover extract (Trifolium pratense) on RP-18 W plate
Red clover tracks
dipped in LacZ-buffer (YES-test X-ß-GAL)
YES-test using X-ß-GAL (red channel) 3 3 ? ? 1 1 1 3

15. YES-test + X-ß-GAL (red channel)
2D separation of a red clover extract (Trifolium pratense) on RP-18 W plate
YES-test + X-ß-GAL (red channel)
YES-test + MUG 3 1 2 6 3 7 5 1 2
We have just three estrogenic active compounds in the red clover extract! 4 8

16. YES-test + X-ß-GAL (red channel)
2D separation of a red clover extract (Trifolium pratense) on RP-18 W plate
YES-test + X-ß-GAL (red channel)
1: Formononetin (an isoflavone) 3 1
2: Coumestrol 2 6 3 7 5 1 2
3: Daidzein (degradation product of formononetin) 4
(Trifolium pratense)

17. Summary:
The method is well suited to detect phytoestrogenic active compounds. The very alkaline LacZ-buffer (pH = 12) enhances native fluorescing bands! Only a X-ß-Gal detection in combination with a 2D-separation on RP-18 W plates can verify phytoestrogens for sure!

18. The HPTLC-Team Offenburg
Thank you very much for your attention!