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RP-HPLC Analysis of Seledive Serotonin Reuptake Inhibitors
Using Allure- Basix and Ultra PFP Polar Stationary Phases
By Rick Lake, Pharmaceutical Innovations Chemist
• Good retention and selectivity without ion-pairing chromatography.
• Practical at acidic pH (Ultra PFP phase) or neutral pH (Allure" Basix phase).
Improved peak shape for basic compounds, compared to alkyl phases.
Selective seroton in reuptake inhibitors (SSRIs) are
a novel class of antidepressants that have gained
mu ch acceptance in th e medical community.
Although they have been found to be no more
effective than the "older" tr icyclic antidepressants,
they produce fewer side effects. Historically, SSRIs
have been analyzed using ion-pairing chromatog­
raphy (IPC) on alkyl stationary phases (e.g., CI8).
IPC is a good alterna tive when reversed phase
chro matograp hy (RPC) on hydrophobic alkyl
phases cannot provide adequa te separat ion. IPC
has disadvantages, however, including artifactual
peaks, slow column equilibrium, poo r peak shape,
and incompatibility with MS detection. Because of
these downsides to IPC, we evaluated the use of
polar stationary phases, including the Allure"
Basix and Ultra PFP phases, for analysis of SSRIs.
The chemical structures of SSRIs (Figure 1) reveals
that these compounds are polar bases capable of
ionic separations. To ensure complete ionization, a
pH value approximately 2 units from an analyte's
pKa should be used. SSRIs have high pKa values
(fluvoxamine maleate: 8.7, fluoxetine: 9.1, sertra­
line HCl: 9.5), however, and two pH units above
these analytes' pKa values will be outside the acidic
to neut ral opera ting range for silica-based
columns. Because SSRIs are basic, th eir retention
can be increased by increasing the mobile phase
pH. According to acid-base equilibria, as pH
decreases, bases gain a proton (ionize), making
them more hydrophili c and less retained by RPc.
Thus, the greatest retention of SSRIs would occur
at neutral pH, rath er than at an acidic pH.
At neutral pH, an Allure" Basix column shows
good retention, selectivity, and peak shape for
SSRIs (Figure 2). This stationary phase and pH are
a good choice if optimum retention and selectivi­
ty are desired. Adding an amine modifier can alter '
selectivity and improve peak shape (Figure 3). As
the concentration of amine modifier is increased,
the retent ion of basic analytes decreases, and the
peaks shar pen. This could be an effective way to
produ ce alternate selectivity and enhance peak
2006 vol. 1
Figure 1 Selective serotonin reuptake inhibitors (SSRls) are a
chromatographic challenge.
Fluoxetine Fluvoxamine Maleate Sertraline Hel
r NH
,
~r"o 0"'-F
A'
co n 0 NH -y--<J- \
co
F , ONHO o=<Jo
Figure 2 At neutral pH, an Allure' Basixcolumn provides good
retention, selectivity, and peak shape for SSRls.
sample:
Inj.:
Cone.:
Sample diluent:
Column:
Dimensions:
Particle size:
Pore size:
Conditions:
Mobile phase:
Flow:
Temp.:
Del.:
Ret Time
Peak List: (min.) Asymmetry selectivity (a)
1. uracil
2. impurity
3. f1uvoxaminemaleate 7.001 1.13
4. sertraline HCI 11.596 1.33 1.82
5. fluoxetine 12.639 1.15 1.10
,
I
10 2QMin.
lCPH0353
lOliL
100f.1g/mL each component
water:acetonitnle,50:50
Allure" Basix (cal.# 9161565)
150x 4.6mm
5f.1m
60.'1
20mMpotassium phosphate dibasic in water (pH7):acetonitrile, 40:60
l mL/min.
30°C
UV@ 230 nm
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