The selectivity of an HPLC column is an important aspect to consider when developing and validating pharmaceutical assays, especially stability-indicating methods. These methods require the analyte to be identified specifically, and quantified, from among degradation products, synthesis intermediates, and process interferences. Specificity, an important validation characteristic, is the ability to unequivocally assess the analyte in the presence of other expected components. By increasing selectivity in the HPLC column, a mechanism can be achieved that more easily produces specificity, by better resolving the analyte from possible interferences. Considering that many pharmaceutical compounds are unsaturated (1) or contain unsaturated functional groups, we compared commonly used silica-based stationary phases to find the one that offers the best selectivity when analyzing unsaturated compounds.

Figure 1 Common steroid structure

We chose the analysis of steroids to investigate the selectivity of commonly used stationary phases for differences in an analyte’s degrees of unsaturation. Steroids are a unique class of pharmaceuticals in that they share a common fused hydrocarbon ring system. The hydrocarbon skeleton of a fully saturated steroid consists of a phenanthrene (2) linked to a cyclopentane (3) (Figure 1). Differences in ring unsaturation and ring substituents (4) 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. Certain carbon positions contribute more often than others to structural variation. For instance, carbons in position 1 through 5 often participate in double bonding, and carbon 17 often contains one of a variety of functional groups (sometimes multiple linked). By concentrating on these dynamic positions in the steroid structure, the effect of differences in analyte ring unsaturation and in analyte functional groups can be used to determine and predict chromatographic behavior of HPLC stationary phases. In an attempt to limit operational bias, and to limit the effect of eluent on separation mechanisms, all analyses were performed with the same instrumentation and under the same isocratic conditions, without the use of buffers.

Figure 2 Biphenyl phase composition

Steroids are hydrophobic molecules (5) that typically are analyzed using hydrophobic reversed-phase packings, like the C8 and C18 alkyl chain stationary phases. In this separation mechanism, hydrophobic compounds are attracted to the hydrophobic bonded stationary phase, and separation is created as the more hydrophobic molecules stay associated with the hydrophobic phase for longer periods of time. For alternate retention, by normal phase chromatography, a cyano-embedded stationary phase, using polar interaction, is used. Polar interaction involves dipole-dipole or dipole-induced dipole (6) interactions. These occur when a permanent dipole in the stationary phase interacts with polar molecules or repels negatively charged electron clouds in nonpolar molecules. Another, and more selective, mechanism for separating steroids is ?-? interactions (7). ?-? interactions are noncovalent bonds between organic compounds containing aromatic rings or unsaturated bonds. These interactions are used by stationary phases containing phenyl groups. With steroids, ?-? interactions can occur when the ?-electron clouds in their fused ring moieties (8) overlap with phenyl groups on the stationary phase. The Allure™ Biphenyl stationary phase offers a unique composition that stearically enhances the chances of ?-? interactions—end to end bonded phenyl groups (Figure 2)

In our first analysis, we investigated a group of seven corticosteroids (10) (Figure 3) to determine which stationary phases provided the best retention and selectivity. ( Corticosteroids on Allure™ Biphenyl, Corticosteroids on Ultra C18, Corticosteroids on Ultra Cyano and Corticosteroids on Pinnacle™ II Phenyl) From this analysis, we observed that the Allure™ Biphenyl and the C18 stationary phases performed the best, as they provided the fewest coelutions, as seen in Table 1, and the greatest retention. Only the Allure™ Biphenyl column was capable of resolving all analytes. This stationary phase also most closely duplicated the chromatography of the C18 phase, showing capability and mechanisms similar to the C18 column, but with enhanced selectivity. More important, when the structures of hydrocortisone, cortisone, and prednisone are compared to the chromatography, a more significant result is revealed. Hydrocortisone and prednisone have identical functionality at position 17, but differ in ring saturation, whereas hydrocortisone and cortisone have nearly identical ring saturation, but differ in position 17 functional group orientations. Cortisone is resolved to baseline by the C18 stationary phase, but hydrocortisone and prednisone co-elute. Thus, the C18 phase is selective for large differences in functional group orientation, but not for subtle differences in hydrocarbon ring unsaturation. The Allure™ Biphenyl stationary phase resolves hydrocortisone and prednisone almost to baseline, indicating that this phase can better resolve compounds with subtle differences in ring unsaturation.

Next, we analyzed contraceptive hormones, to confirm the enhanced selectivity of the Allure™ Biphenyl stationary phase for degrees of analyte unsaturation, as seen in the corticosteroid analysis. ( Contraceptive Hormones on Allure™ Biphenyl and Contraceptive Hormones on Ultra C18) The chemical structures of ß-estradiol, ethynyl estradiol, and norethindrone show differences and similarities in functional group and ring structures comparable to those of the corticosteroids (Figure 3). ß-estradiol and ethynyl estradiol differ in the orientation and structure of the position 17 ethynyl functional group, but have similar hydrocarbon ring unsaturation. Ethynyl estradiol and norethindrone have identical position 17 functional groups, but have small differences in hydrocarbon ring saturation. As expected, the C18 phase completely resolves ß-estradiol and ethynyl estradiol, but cannot resolve ethynyl estradiol and norethindrone. Overall, again, retention and selectivity are markedly better for the Allure™ Biphenyl phase, which completely resolves all three compounds. Most important, the Allure™ Biphenyl phase shows greater resolving capability for unsaturation differences in the hydrocarbon ring structure, as noted by the superior resolution of ethynyl estradiol and norethindrone.

Finally, we directly compared the Allure™ Biphenyl and C18 stationary phases for selectivity and efficiency, using the analysis of endogenous hormones ß-estradiol, testosterone, and progesterone (Endogenous Steroids on Allure™ Biphenyl and Endogenous Steroids on Ultra C18) under the same isocratic conditions. ß-estradiol and testosterone contain significant differences in ring double bonding, but have very similar functional groups (Figure 3). Therefore, by comparing resolution of these two compounds, we can make a direct correlation between hydrocarbon ring variations and resolution. The C18 column yielded a resolution of 3.42 between the two compounds, and the Allure™ Biphenyl column yielded a resolution of 5.94 — a 43% increase — which shows the Allure™ Biphenyl phase exhibits better specificity for ring variation. To compare column efficiencies, we calculated the USP tailing factors for the eluting peaks. The C18 column provided tailing factors of 1.31 and 1.25, and the Allure™ Biphenyl column provided tailing factors of 1.14 and 1.10, respectively. This indicates that the Allure™ Biphenyl stationary phase exhibits better efficiency, as well as better selectivity.

Figure 3 Steroid Structures

The best choice in stationary phase is always determined by the specific application. If increased selectivity and efficiency are desired, these analyses of steroids demonstrate that the Allure™ Biphenyl stationary phase, through ?-? interactions, offers a unique alternative to hydrophobic or polar interactions when analyzing compounds with saturation differences in the hydrocarbon ring structure. ?-? interactions offer better retention, selectivity and efficiency for unsaturated compounds or unsaturated functional groups. Moreover, the structure of the Allure™ Biphenyl stationary phase offers many of the same retention characteristics of hydrophobic reversed phase packings (C18). The increased selectivity and efficiency exhibited by the Allure™ Biphenyl stationary phase should be well suited for the development and validation of stability-indicating methods and potency assays. Increased selectivity can provide better resolution among an analyte and its degradation products, and increased efficiency will provide the capability to achieve tighter system suitability parameters when analyzing an analyte at high concentration.