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• 16 •
2008 vol. 2
Pharmaceutical
Separating NSAIDs through Aromatic Selectivity
Improve Retention by Using An Allure® Biphenyl HPLC Column
By Rick Lake, Pharmaceutical Innovations Chemist, and Benjamin Smith, Applications Technician
Figure 1
Aromatic rings make NSAIDs candidates for separation
through
π
-
π
interactions.
Figure 2
The retention capacity of the Allure® Biphenyl phase
far exceeds that of conventional phenyl phases.
• Optimize retention and selectivity of non-steroidal anti-inflammatory drugs, for better separations.
• Orthogonal separations with simple mobile phase changes
• Increased retention requires higher organic content, increasing desolvation efficiency in LC/MS.
Non-steroidal anti-inflammatory drugs (NSAIDs),
in either prescribed or over-the-counter formula-
tions, are widely used to treat pain, fever, and
inflammation. While steroidal anti-inflammatory
drugs all share a similar, four-ring chemical struc-
ture, NSAIDs have more diverse chemical struc-
tures, complicating their analysis. The work we
report here is based on three common classes of
NSAIDs: arylalkanoic acids, 2-arylpropionic acids
(profens), and oxicams.
NSAIDs have a high carbon to heteroatom ratio
and, therefore, historically have been separated
through reversed phase HPLC on C18 columns. A
conventional C18 stationary phase separates com-
pounds based mainly on their overall hydropho-
bicity. Considering the carbon to heteroatom ratio,
this is an effective separation mechanism for
NSAIDs. Newer stationary phases are available,
however, and we set out to determine if other
phases, using other separation mechanisms, such
as
π
-
π
interactions, could be more effective for
assaying NSAIDs.
When selecting a stationary phase, it is advanta-
geous to exploit inherent differences in the target
analytes’ chemical structures. Among these three
classes of NSAIDS, there are some common func-
tional groups, like halogens, amines, and car-
boxylic acids, but no one group is shared across the
entire list of analytes (Figure 1). However, all of the
target analytes do share one basic structural com-
ponent – the six-carbon aromatic ring. Aromatic
rings are common components of drug molecules,
and they can be targeted using a phenyl-based sta-
tionary phase.
As a retention mechanism, phenyl stationary phas-
es employ
π
-
π
interactions between the phenyl
groups in the stationary phase and any unsaturat-
ed bonds in the analyte. The use of conventional
phenyl phases has been somewhat limited due to
their moderate retention capacity, relative to that
of a C18 phase. Figure 2 illustrates the relative
retention capacities of NSAID test probes on an
Allure® Biphenyl column, a conventional phenyl
column and a C18 column. Note that, in all cases,
as commonly seen in practice, the conventional
phenyl phase yields only moderate retention com-
pared to that of a C18 column. However, the
Allure® Biphenyl phase, which is a stationary
Arylalkanoic acids
Oxicams
Diclofenac
Sulindac
Piroxicam
Naproxen
16.000
14.000
12.000
10.000
8.000
6.000
4.000
2.000
0.000
Retention Capacity (k')
naproxen
sulindac
piroxicam
diclofenac
naproxen
sulindac
piroxicam
diclofenac
▼
▼ ▼ ▼ ▼ ▼
▼ ▼
◆
▲
◆
▲
◆
▲
◆
◆
▲
◆
▲
◆
▲
◆
▲
Acetonitrile
comparable to C18
Methanol
retention increases,
exceeding that of C18
▲
Biphenyl
◆
C18
▼
Phenyl
For each analyte all columns were assayed under identical isocratic conditions. The equivalent elutropic
strength between acetonitrile and methanol was determined by the relative retention capacities of the C18
phase.
Columns:
5µm, 4.6mm x 150mm
Mobile Phase: 10mM potassium phosphate (pH 2.5): acetonitrile or methanol
Det.:
UV @ 254nm
Flow:
1.0 mL/min.
2-Arylpropionic acids
Ketoprofen
▲