pharmaceutical
Applications
note
RestekCorporation • (800)356-1688 • (814)353-1300 •
#59510
pharmaceutical
Column:
Ultra C4
Catalog #:
9102365
Dimensions: 150 x 4.6mm
Particle Size: 3µm
Pore Size:
100Å
Conditions:
Mobile Phase: 10mM ammonium
formate, pH 3.0:
acetonitrile
(60:40, v/v)
Flow:
1.2mL/min.
Temp.:
30°C
Det.:
UV @ 230nm
Sample:
Inj.:
15µL
Conc.: 333µg/mL
Solvent: acetonitrile:water
(70:30)
1
2
0 2 4 6 8 10 12 14 min.
3
LC_0085
1
2
3
LC_0118
Hydrophobic Interaction
C18 phases may provide excellent separation of steroid
components but can produce added retention for certain
hydrophobic steroids or lipophylic matrix components,
resulting in longer analysis times. The Ultra C4 stationary
phase provides excellent stability and selectivity for
steroids. Its reduced hydrophobic nature can significantly
reduce retention time while maintaining the desired selectiv-
ity. Reducing the retention time of an analyte increases its
signal-to-noise response for better detection. Retention and
resolution of two estrogens and a progestrogen using Ultra
C4 and Ultra C18 columns with the same mobile phase
conditions show that both columns offer similar resolution of
these compounds. The retention of other more hydrophobic
components should be significantly reduced on the Ultra C4
(Figure 1).
HPLC Stationary Phase Selection for the Analysis of Steroids
Steroids are an important class of pharmaceuticals. Of the top
200 prescribed drugs in 1998, 29 are classified as steroids.
1
Their uses range from contraception and hormone therapy to
treatment of bronchial asthma. Steroids of differing sub-
classes frequently are used in combinations to generate a
desired therapeutic effect.
High performance liquid chromatography (HPLC) analysis of
steroids can be performed with many types of stationary
phases. Restek’s Ultra C18, Ultra C4, Ultra Cyano, Allure
™
C18, and Allure
™
Basix columns can provide the selectivity
and retention characteristics needed to develop rapid and
robust analytical methods for a wide range of steroids.
Therefore, the choice of column phase essentially is dictated
by the sample matrix and sensitivity concerns.
1
RxList,
Figure1
The Ultra C4 column’s reduced hydrophobic tendencies provide excellent resolution and reduced analysis time for two
estrogens and a progestogen when compared to the Ultra C18 phase.
Column:
Ultra C18
Catalog #:
9174565
Dimensions: 150 x 4.6mm
Particle Size: 5µm
Pore Size:
100Å
Conditions:
Mobile Phase: 10mM ammonium
formate, pH 3.0:
acetonitrile
(60:40, v/v)
Flow:
1.0mL/min.
Temp.:
30°C
Det.:
UV @ 230nm
Sample:
Inj.:
10µL
Conc.: 333µg/mL
Solvent: acetonitrile:water
(80:20)
Peak List:
1.ß-estradiol
2.norethindrone
3.ethynyl estradiol
0 2 4 6 8 10 12 14 16 min.
