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Pharmaceutical Articles

Optimizing Difficult Separations of Steroids

Using an Allure™ Biphenyl HPLC Column

By Rick Lake, Pharmaceutical Innovations Chemist
  • Increase resolution while using simple, isocratic conditions.
  • Achieve separations not possible on a C18 column.
  • Rugged and reproducible analyses.

Steroids are an unusual class of compounds, in that all structural variation is centered on a common conjugated ring system, with differences in double bonding and ring constituents producing chemical diversity. Because of the consistency in their chemical structures, it can be difficult to achieve adequate separation of steroids on an alkyl (e.g., C18) HPLC stationary phase. An optimized stationary phase can be the key to successful analyses.

When choosing a stationary phase, a separation mechanism that employs inherent differences in the chemical structures of the target analytes should be used. For analyses in which the target analytes are structurally very similar, this is especially critical. For steroids, this includes separations based on pi-pi (π-π) interactions between aromatic or unsaturated moieties: a stationary phase containing phenyl groups forms π-π bonds as the phenyl group on the stationary phase overlaps with the aromatic rings or other double bonds in the analytes.

The Allure™ Biphenyl stationary phase is a significant advancement in phenyl stationary phase chemistry, increasing retention of unsaturated compounds in reversed phase HPLC applications, while enhancing selectivity. A typical silica-based phenyl stationary phase consists of a single phenyl group bonded to a silica backbone (Figure 1). Consisting of two phenyl groups bonded end-to-end, the Allure™ Biphenyl offers a more concentrated arrangement of phenyl groups, in a sterically favorable positioning (Figure 1). This phase shows markedly better selectivity for unsaturated compounds and shows a high retention capacity, similar to that of a C18 phase.1-3

We assayed two groups of steroids, hormones and corticosteroids, on an Allure™ Biphenyl column, to determine if separation can be enhanced by exploiting differences in π-π interactions. First, we compared performances by the Allure™ Biphenyl column and a conventional C18 column of the same dimensions, using a complex mix of steroid hormones. Under identical isocratic analytical conditions, the Allure™ Biphenyl column resolved all target compounds (Figure 2), but the C18 column showed very limited resolving power (Figure 3). The Allure™ Biphenyl column also provided an overall increase in analyte retention — a very useful improvement relative to conventional phenyl phases.

The Allure™ Biphenyl column also showed enhanced selectivity in a second analysis, using corticosteroids. Under simple isocratic conditions, the Allure™ Biphenyl column provided baseline separation of hydrocortisone and prednisone and resolved isomers betamethasone and dexamethasone (Figure 4).

These analyses show that markedly better selectivity for steroids easily can be achieved, by using an Allure™ Biphenyl column under simple isocratic conditions. High retention capacity, similar to that of an octadecylsilyl phase, also is demonstrated; a useful feature unavailable from conventional phenyl phases. By increasing π-π interactions, the Allure™ Biphenyl stationary phase offers a unique and more effective alternative to hydrophobic alkyl phases for resolving chemically similar unsaturated compounds, such as steroids.


Figure 1  The unique Allure™ Biphenyl stationary phase concentrates phenyl groups in a sterically favorable positioning.



Figure 2  An Allure™ Biphenyl column resolves steroid hormones in a simple, isocratic analysis.

  1. estriol
  2. 17ß-estradiol
  3. 17α-estradiol
  4. 17δ-ethynyl estradiol

  1. testosterone
  2. estrone
  3. norethindrone

LC_BP0385

Sample:

Inj.:

10µL

Conc.:

50µg/mL each component

Sample diluent:

acetonitrile:methanol, 4:1 (v/v)

Column:

Allure™ Biphenyl (cat.# 5166365)

Dimensions:

150 x 4.6 mm

Particle size:

3µm

Pore size:

60Å

Conditions:

Mobile phase:

water:acetonitrile, 50:50 (v/v)

Flow:

1.5mL/min.

Temp.:

ambient

Det.:

UV @ 220nm


Figure 3  A C18 column shows poor resolving capability for steroids.

  1. estriol
  2. 17ß-estradiol
  3. 17α-estradiol
  4. 17δ-ethynyl estradiol

  1. testosterone
  2. estrone
  3. norethindrone

LC_BP0395

Sample:

Inj.:

10µL

Conc.:

50µg/mL each component

Sample diluent:

acetonitrile:methanol, 4:1 (v/v)

Column:

C18

Dimensions:

150 x 4.6 mm

Particle size:

3µm

Pore size:

100Å

Conditions:

Mobile phase:

water:acetonitrile, 50:50 (v/v)

Flow:

1.5mL/min.

Temp.:

ambient

Det.:

UV @ 220nm


Figure 4  An Allure™ Biphenyl column resolves all target corticosteroids, including isomers betamethasone and dexamethasone, in a simple, isocratic analysis.

Peak List

Concentration (µg/mL)
  1. hydrocortisone
  2. prednisone
  3. cortisone
  4. betamethasone
  5. dexamethasone
  • 67
  • 53
  • 53
  • 73
  • 52

LC_PH0384

Sample:

Inj.:

10µL

Sample diluent:

methanol

Column:

Allure™ Biphenyl (cat.# 5166365)

Dimensions:

150 x 4.6 mm

Particle size:

3µm

Pore size:

60Å

Conditions:

Mobile phase:

water:acetonitrile, 70:30 (v/v)

Flow:

1.5mL/min.

Temp.:

ambient

Det.:

UV @ 220nm

References

  1. Superior Separations of Unsaturated Compounds by HPLC Restek Advantage 2005v4 (lit. cat.# 580022).
  2. Improved HPLC Analysis of Steroids Restek Application Note (lit. cat.# 580020).
  3. Lake, R., and Wittrig, R., Increasing HPLC Retention and Selectivity for Unsaturated Compounds, Using π-π Interactions Pharmaceutical Canada, June 2006.
  4. References 1&2 available on request.