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Enhanced Selectivity for Steroids: HPLC Stationary Phase for Unsaturated Compounds

by Richard Lake, Randall Romesberg and Michael Wittrig, Restek Corp.

Click here to enlage Figure 1. Baseline resolution of endogenous (beta-estradiol) and contraceptive steroids, through π-π interactions with the Allure™ Biphenyl stationary phase. 1. beta-estradiol 2. ethynyl estradiol 3. norethindrone 4. norgestrel

In developing and validating pharmaceutical methods, maximum resolution among analytes, or among an analyte, its degradation products, synthesis intermediates, and process interferences, is critical for compound specificity and system suitability. Resolution is especially important for stability-indicating methods that also are used to investigate the abundance of degradation products.

Many active pharmaceutical compounds have unsaturated frameworks, or contain unsaturated functional groups. We have evaluated some commonly used HPLC stationary phases to determine the best choice for maximizing selectivity for unsaturated compounds. We used analyses of steroids, a diverse class of pharmaceuticals that share a common fused hydrocarbon ring structure, and simple isocratic methods, to compare stationary phases. Differences in steroid ring unsaturation and ring substituents create the chemical diversity of these compounds and, for our purposes, provide a traceable, systematic model for determining the effects of ring unsaturation on chromatographic selectivity (1,2).

Steroids are hydrophobic compounds and, as such, commonly are separated through hydrophobic interactions, using reversed phase HPLC columns with simple alkyl chain stationary phases (e.g., C18, C8). For alternate selectivity, or for normal phase separations, a cyano stationary phase is used. A third separation mechanism that can be employed when unsaturation is present relies on pi-pi (π-π) interactions. In steroids, π-π interactions can occur when the fused ring moieties containing double bonds overlap. The Allure™ Biphenyl stationary phase employs this mechanism in retaining and resolving unsaturated compounds.

Overall, our analyses yielded conclusive and reproducible evidence that the Allure™ Biphenyl stationary phase provides the greatest selectivity for unsaturated compounds. Through π-π interactions, this phase is capable of greater retention of unsaturated compounds and, most importantly, can resolve compounds on the basis of small differences in ring unsaturation. The contraceptive hormones in Figure 1 illustrate this resolving power. The C18 phase resolves beta-estradiol and ethynyl estradiol, which have differing functional groups, but cannot resolve ethynyl estradiol and norethindrone, which differ in fused ring structure. In contrast, the Allure™ Biphenyl column resolves all compounds to baseline. Most important, the Allure™ Biphenyl column exhibits a resolution specific to differences in hydrocarbon ring structure, as noted by the superior resolution between ethynyl estradiol and norethindrone. We have obtained comparable results from analyses of corticosteroids and endogenous steroids—for additional details and chromatography, request References 1 and 2.

References

1. Restek Advantage, 2005v4, 2005. 2. Improved HPLC Analysis of Steroids: Introducing the Allure™ Biphenyl Column for Unsaturated Pharmaceuticals Application Note 580020, Restek Corporation, 2005. References available on request from Restek.

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