2d-Thin LAYER CHROMATOGRAPHY (2d-Tlc) FLASH TEST OF 17α-Ethinylestradiol AND RELATED STEROIDS DETECTED BY FLUORESCENCE DENSITOMETRY

Abstract

& We present a two dimensional (2D) planar chromatographic separation of ethinyl steroids on cyanopropyl phase. A mixture of nine steroids was separated by using a cyanopropyl-coated silica gel plate (Merck, 1.16464) with the solvent mix of dichloromethane, methanol, cyclohexane (95 þ 5 þ 60, v=v) in the first direction and with a mixture of water, acetonitrile, ethanol, dioxane, and NH 3 (25%) (8 þ 2 þ 1 þ 1 þ 0.05, v=v) in the second direction. Both developments were carried out over a distance of 70 mm. The steroids were catalytically transformed into fluorescent compounds that can be sensitively detected. The fluorescence was measured by a simple 8-bit CCD-camera. The range of linearity covers two magnitudes of power using the extended Kubelka-Munk expression for data transformation. This separation method is inexpensive, fast, and reliable and can be used to quantify 17a-ethinylestradiol (EE2) in the effluents of waste water treatment plants at an LOD of 2 ng per spot. Using a solid phase extraction pretreatment procedure achieves an LOD of 2 ng L À1 water.

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

“…[5] The method allows applying large amounts of sample (up to 50 mL) without losing separation efficiency. The key-step in this analysis is the use of cyanopropyl-plates for an orthogonal separation, using normal phase conditions in the first and RP-conditions in the second run.…”

“…Figure 4 shows the separation of large amounts of 13 estrogenic-acting compounds (1 mg each) after staining with sulfuric acid according to the literature. [5] The various Rf-values are summarized in Table 1. Especially the separation of E2 (Rf 1 ¼ 0.49, Rf 2 ¼ 0.49) and EE2 (Rf 1 ¼ 0.53, Rf 2 ¼ 0.62) is good and provides robust separation conditions even with large application volumes of more than 100 mL.…”

“…EE2 and all other steroids were thus converted into coloured and strongly fluorescent compounds. [5] As a biological method, a yeast estrogen screening assay (YES-assay) was utilized. The test strain was generated by McDonnell et al [8,9] It is derived from the yeast strain Saccharomyces cerevisiae BJ3505 (protease deficient, MATa, PEP4::HIS3, prb-1-delta1.6R, HIS3-delta200, lys2-801, trp1-delta101, ura3-52gal2can1).…”

“…[4] Recently, a method was published that separated different estrogenic acting compounds by 2D-TLC separated on cyano-propyl phase and using fluorescing detection. [5] This method is suitable for monitoring 17a-ethinylestradiol contents in water samples down to a quantification level of 2 ng per liter. For this a time-consuming pre-treatment procedure was used.…”