Steroids separation

S. Habibi-Goudarzi, K. J. Ruterbories, J. E. Steinbrunner, and D. Nurok, A computer-aided survey of systems for separating steroids by two-dimensional thin-layer chromatography, J PC, 7 161 (1988). 

Other workers have successfully applied these principles to the optimization of their separations, and their papers attest to the value of this method Antle67 has separated steroids by normal phase on an amine bonded phase, and Lehrer68 has separated phenols and cresols by reverse phase on a C8 bonded phase. Both provide interesting chromatograms. 

Halomethyldimethylsilyl ethers of steroids possess longer retention times than TMS derivatives, which makes it possible to separate steroids with different numbers of hydroxyl groups. Thomas and co-workers [332,333] exploited chloromethyldimethylsilyl derivatives of steroids, the retention times of which are up to three times longer... 

Other sorbents that have also been used for separating steroids in TLC are cellulose, kieselguhr, alumina, polyamide, magnesium oxide, and celite. An-drostanes can be analyzed using cellulose layers impregnated with 1,2 propanediol. Layers consisting of alumina and magnesium oxide can be used to separate some sterols and sterol acetates. 

For further identification of the separated steroid alkaloids, Bushway et al. used UV absorption ratios at 215, 225, 235 and 245 nm. For the detection, a wavelength of 215 nm was preferred to 208 nm because of a more stable baseline, and with sufficient sensitivity. The sensitivity could be increased by using acetonitrile - water as mobile phase. Such a solvent system allowed detection at 200 nm. 

Solanum and veratrum alkaloids Separation steroidal alka-loids(Fig.10.1 and 10.2) Zorbax Sil 6 Mm 250x4.6 n-Hexane-Me0H-Me,C0(18 1 1) n-Hexane-EtOH-Me CO(18 1 1) 4... 

Determination of lipophilicity. Reversed phase TLC has been used to predict the lipophilicity of certain drugs. Silica plates impregnated with methyl-silicone-diethyl ether, or Cl8 plates with octadecylsilane groups on the silica surface have been used for these applications. Biagi et al. (1994) have separated steroids and their acetates on reversed phase TLC based on the solvent strength and lipophilicity of the substance. It is possible that this technique may also be used to determine the relative lipophilicity of different vegetable and egg lecithins. 

Eye drops containing fluprednisolone acetate have been tested [80] in a way similar to that previously described for hydrocortisone acetate preparations [63], After submitting to appropriate test conditions, the steroid extracts were chromatographed on fluorescent silica gel layers, the separated steroid ester and alcohol spots localised in UV light, eluted and determined quantitatively by UV spectrophotometry or colorimetrically with tetrazolium compounds. The stability of tablets containing oestr-4-en-17 jS-ols and the influence of the quality of the auxiliary material... 

An alternative approach based on visual inspection of simulated chromatograms was used by Johnson and Nurok et al. [39,40] for selecting a two-dimensional system for separating steroids by continuous-development TLC. It was found that the best separation was for normal-phase/reversed-phase systems, which is not surprising due to the large differences in selectivity between these constituent systems. It should be noted that dual-phase TLC plates for developing in the latter mode are commercially available and are coated with a strip of silica gel layer contiguous with a bonded C]8 layer or vice versa. 

Separation should be performed according to the chemical nature of steroids (consideration of the separable steroids) or ... 

The spot elution technique is a simple procedure in the quantitative TLC of steroids. After elution from the plate, the separated steroids can be quantified by colorimetric, fluorimetric, gas chromatographic, mass spectrometric or radiochemical methods. By adding the high efficiency and selectivity of TLC to the sensitivity of the method chosen for quantitation, many difficult analytical problems can be solved in small laboratories equipped with basic TLC equipment and a spectrophotometer or fluorimeter. Besides its advantages, the use of the spot elution technique also has some problems. These are as follows ... 

The effluent in the individual tubes was pooled into five bands, as indicated by the peaks on the chart. These bands, which contained cyclohexane, methylene chloride, a small amount of ethylene glycol stripped from the colunm, and a separated steroid, were first subjected to vacuum distillation to remove the volatile solvents. The remaining glycol-steroid residue was then mixed with about ten volumes of water. The glycol was trapped in the aqueous solution, and the steroid was removed by extracting three times with an equal volume of ethylene chloride. When this solvent was removed by distillation, the steroid remained as a crystalline residue and was dissolved in 10 ml. of acetone for further identification by narrow-strip paper chromatograms. Evaporation of the acetone and recrystallization from cold ethanol or anhydrous ethyl ether produced the individual steroids, of anal3rtical purity, in recovery yields that exceeded 85%. 

The potential of GC-MS in determining a multitude of steroid metabolites simultaneously in a single steroid profile is still unsurpassed. In particular, GC bears the greatest potential for separating steroids, and MS allows for the highest specificity in determining steroid metabolites. The benchtop GC-MS systems currently available are easy to operate, can be tuned automatically, and present the most robust developments in the MS instrumental field, currently and in the near future. Furthermore, reasonable prices and computerized data management make benchtop instruments suited for routine clinical use. 

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