Reversed-phase high-performance liquid chromatographic separation of some 2-arylpropionic acids using vancomycin as chiral stationary phase

Article abstract of DOI:10.1007/s13738-015-0635-7

Abstract A rapid, sensitive and reproducible HPLC method has been developed for enantioseparation of six non-steroidal anti-inflammatory drugs, which are acidic compounds: carprofen, fenoprofen, flurbiprofen, ibuprofen, indoprofen and ketoprofen. The effects of the mobile phase composition on retention times and resolutions of the analytes were studied. A column based on vancomycin immobilized by reductive amination to aldehyde functionalised silica was prepared in house and used. The prepared sorbent shows a great stability and selectivity over a range of pH (4-6), and the separation was carried out using the mobile phase composed of a mixture of 40% of methanol in ammonium nitrate buffer (50 mM) at pH 5.0. Another mobile phase consisted of 50% of methanol in phosphate buffer (5A mM) at pH 5.0 was also prepared and tested. The two mobile phases are the optimum conditions obtained. All experiments were conducted at flow rate 0.6 ml/min, using a UV detector wavelength at λ = 254 nm.

Full text of DOI:10.1007/s13738-015-0635-7

J IRAN CHEM SOC
DOI 10.1007/s13738-015-0635-7
ORIGINAL PAPER
Reversed‑phase high‑performance liquid chromatographic
separation of some 2‑arylpropionic acids using vancomycin
as chiral stationary phase
Nabila Bouchair1,2 · Michel Righezza · Abderrezak Hamdi1
2
Received: 14 August 2013 / Accepted: 18 February 2014
©
Iranian Chemical Society 2015
Abstract A rapid, sensitive and reproducible HPLC
method has been developed for enantioseparation of six
non-steroidal anti-inflammatory drugs, which are acidic
compounds: carprofen, fenoprofen, flurbiprofen, ibupro-
fen, indoprofen and ketoprofen. The effects of the mobile
phase composition on retention times and resolutions of
the analytes were studied. A column based on vancomycin
immobilized by reductive amination to aldehyde function-
alised silica was prepared in house and used. The prepared
sorbent shows a great stability and selectivity over a range
of pH (4–6), and the separation was carried out using the
mobile phase composed of a mixture of 40 % of methanol
in ammonium nitrate buffer (50 mM) at pH 5.0. Another
mobile phase consisted of 50 % of methanol in phosphate
buffer (5 mM) at pH 5.0 was also prepared and tested. The
two mobile phases are the optimum conditions obtained.
All experiments were conducted at flow rate 0.6 ml/min,
using a UV detector wavelength at λ = 254 nm.
Introduction
Usually, the enantiomers of racemic drugs show different
pharmacological effects. Thus, one enantiomer may be the
effective agent while the other isomer is probably inactive
or even toxic. The consequences of chirality may extend
to drug metabolism and enantiomeric interconversion,
where one enantiomer is converted into another in vivo.
Thus, 2-arylpropionic acids which are widely used as anti-
inflammatory agents provide an example of stereoselective
activation. When the drug R-ibuprofen was administered in
man, there was a rapid appearance of the S-ibuprofen in the
blood [1–4].
These consequences of chirality have increased attention
paid by researchers to the studies in the field of stereose-
lective synthesis and catalysis, as well as many techniques
for chiral separations. On the other hand, in regard to the
manufacture of chiral drugs, it is very important to examine
the chiral purity when a single enantiomer is to be used.
Thus, it is important during initial testing of a drug, to be
able to isolate and separate the enantiomers to assess which
is responsible for the potency, the toxicity and/or unwanted
side effects.
Keywords Chiral selector · Enantioseparation · Profens ·
Reversed-phase high-performance liquid chromatography ·
Vancomycin
Chromatographic methods have become very popular
in HPLC where most of these methods involve the use of
either a chiral selector chemically bonded to the stationary
phase, or added to the mobile phase. In both cases there is
formation of reversible diastereoisomeric complexes with
the solute enantiomers. The separation results from the dif-
ferences in stability between the complexes and therefore
leads to a difference in retention times.
*
Nabila Bouchair
nabila_bouchair@yahoo.fr
1
2
Currently, a number of chiral stationary phases have
been described in the literature for HPLC, which have been
shown to be efficient for the separation of enantiomeric
drugs and other compounds [5–22]. However, there is at
University of Jijel, City Ouled Aissa, B.P. 98, 18000 Jijel,
Algeria
Aix Marseille Université, CNRS, iSm2 UMR 7313,
1
3397 Marseille, France
1
3

J IRAN CHEM SOC
Table 1 Effect of buffer pH on
retention and enantioseparation
pH
Ketoprofen
Indoprofen
Ibuprofen
Flurbiprofen
Fenoprofen
Carprofen
4
k1
α
3.06
1.66
2.13
3.02
1.33
1.01
1.71
1.27
0.57
4.67
1.80
2.45
3.26
1.28
1.08
12.54
1.24
1.13
Rs
5
k1
α
3.79
2.02
3.62
3.75
1.62
2.19
2.38
1.74
1.97
6.88
2.54
4.1
4.43
1.59
2.04
14.36
1.57
1.81
Rs
6
k1
α
2.63
1.62
1.73
3.47
1.46
1.71
2.21
1.60
1.42
6.45
2.46
2.78
4.00
1.47
1.79
16.12
1.60
1.84
Rs
Mobile phase: 50 mM ammonium nitrate buffer:MeOH (60:40, v/v %), pH 5.0, flow rate: 0.6 ml/min,
detection wavelength 254 nm
k retention factor of the first-eluted enantiomer, α selectivity, R resolution
1
s
Fig. 1 Effect of buffer molarity
on: a retention; b resolution
1.40
a
1
1
0
0
0
0
0
.20
.00
.80
.60
.40
.20
.00
Ketoprofen
indoprofen
ibuprofen
flurbiprofen
fenoprofen
carprofen
0
20
40
60
80
100
120
Molarity
5
4
3
2
1
0
b
Ketoprofen
indoprofen
ibuprofen
flurbiprofen
fenoprofen
carprofen
0
20
40
60
80
100
120
Molarity
present no chiral stationary phase in HPLC that is capable
of separating all classes of drugs, so that analysts have to
select a convenient phase for a particular separation from a
large selection of chiral stationary phases.
Since the introduction of macrocyclic antibiotics as chi-
ral selectors by Armstrong et al. [15], these compounds,
especially vancomycin, teicoplanin and ristocetin A, have
served as chiral stationary phases for a wide range of drugs.
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J IRAN CHEM SOC
Table 2 Variation of retention
Percentage of OM Ketoprofen Indoprofen Ibuprofen Flurbiprofen Fenoprofen Carprofen
20 %
factor (k ), selectivity (α)
1
and resolution (R ) of the
s
THF
enantiomers with percentages
of different organic modifiers
in ammonium nitrate buffer
k1
α
4.76
1.96
2.42
4.18
1.73
2.35
3.51
1.50
1.25
7.87
2.61
3.37
5.52
1.47
1.71
13.12
1.50
1.71
(50 mM, pH 5.0)
Rs
0 %a
k1
2
2
3
4.58
1.98
2.92
4.25
2.06
3.18
4.16
1.94
1.94
8.79
3.09
4.46
6.34
1.66
1.99
14.78
1.77
2.17
α
Rs
5 %
k1
3.13
2.07
2.19
3.05
2.31
2.83
2.62
2.09
1.87
4.51
3.50
3.01
3.77
1.82
1.81
6.90
1.97
1.90
α
Rs
0 %
k1
1.78
1.96
2.42
1.76
2.26
2.71
1.54
2.03
2.24
2.41
3.37
4.15
2.03
1.68
1.68
2.84
1.90
2.19
α
Rs
ACN
20 %
k1
4.04
1.50
2.16
4.18
1.32
1.51
4.15
1.35
1.22
9.56
1.73
2.39
5.34
1.30
1.35
15.71
1.31
1.37
α
Rs
2
3
5 %
k1
2.35
1.43
1.30
2.28
1.31
1.21
1.96
1.30
1.00
3.60
1.64
2.17
2.93
1.27
1.33
6.93
1.28
1.36
α
Rs
0 %
k1
1.49
1.40
1.27
1.55
1.28
0.98
1.19
1.28
0.88
2.09
1.57
1.70
1.75
1.24
0.89
3.58
1.25
0.97
α
Rs
MeOH 40 %
k1
α
2.98
2.06
3.34
3.79
1.64
2.05
2.29
1.68
1.84
5.82
2.42
3.54
3.65
1.57
2.22
13.82
1.50
1.81
Rs
5
0 %
k1
1.68
1.95
2.50
1.74
1.61
1.50
1.02
1.77
1.31
2.40
2.35
1.50
1.81
1.56
1.43
4.69
1.49
1.48
α
Rs
OM organic modifier, k retention factor of the first-eluted enantiomer
1
a
0
.1 % TEA
The high enantioselectivity of these compounds may be
attributed to their abilities to form various interactions,
for example. electrostatic, hydrogen bonding, π–π inter-
actions, etc. Drugs which have been extensively studied
are the 2-arylpropionic acids. A common pharmacologi-
cal aspect of this group of non-steroidal anti-inflammatory
compounds is the enantiomeric interconversion due to
metabolism.
separation of seven racemic non-steroidal anti-inflamma-
tory drugs (NSAIDs) and most of the drugs were success-
fully enantioseparated [23]. However, it proved difficult
to separate using the vancomycin-bonded chiral station-
ary phase, the majority of profen derivatives using a single
mobile phase [16, 23].
In this work, we have sought to explore the capac-
ity of the sorbent vancomycin, as prepared by Svensson
et al. [24, 25], to separate six profen drugs. Vancomy-
cin is a tricyclic glycopeptide antibiotic used to treat
Vancomycin has been used as a mobile phase addi-
tive in capillary liquid chromatography for stereoselective
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J IRAN CHEM SOC
Fig. 2 Chromatograms of the profens in 50 mM, pH 5.0 ammonium nitrate buffer/MeOH (60/40, v/v %); a carprofen, b fenoprofen, c flurbipro-
fen, d ibuprofen, e indoprofen, f ketoprofen
Table 3 Variation of retention
MeOH %
Ketoprofen
Indoprofen
Ibuprofen
Flurbiprofen
Fenoprofen
Carprofen
factor (k ), selectivity (α)
1
and resolution (R ) of the
s
40
k1
α
enantiomers with percentages of
methanol in sodium dihydrogen
phosphate buffer (50 mM, pH
5.52
2.00
2.75
6.13
1.64
2.1
3.52
1.72
1.43
9.26
2.41
3.08
7.20
1.63
2.07
23.47
1.65
2.02
5
.0)
Rs
5
0
k1
α
2.70
1.93
2.36
3.05
1.66
1.72
1.84
1.74
1.28
3.86
2.46
2.40
3.56
1.57
1.91
8.79
1.64
2.07
Rs
k1 retention factor of the first-eluted enantiomer
gram-positive infections by inhibiting bacteria muco-
peptides biosynthesis. It is produced by the growth of
some strains of Streptomyces orientalis bacteria and is
used for treatment of serious staphylococcal infections
in humans.
The six profens examined are: ketoprofen, indopro-
fen, ibuprofen, fenoprofen, flurbiprofen and carprofen.
The mobile phase constituents: organic modifiers, pH and
buffer concentration were all investigated.
Vancomycin is reported to have 6 pK values: 2.18, 7.75,
a
8
.89, 9.59, 10.4 and 12.0 [26, 27] with 18 chiral centres,
Experimental
three cavities and five aromatic rings in the aglycon part
that bears two sugar units. The molecule has a molecular
weight of 1449 and contains one carboxylic group, nine
hydroxyl groups, seven amido groups and two amino
groups [28].
Instrumentation and chromatographic conditions
HPLC experiments were performed using an isocratic
HPLC pump (L-6200, VWR International Darmstadt,
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J IRAN CHEM SOC
Table 4 Influence of molar
concentration of phosphate
buffer on retention factor (k1),
selectivity (α) and resolution
Phosphate buffer (mM) Ketoprofen Indoprofen Ibuprofen Flurbiprofen Fenoprofen Carprofen
50a
k1
α
1.53
1.63
1.63
1.85
1.44
1.38
1.11
1.55
1.30
2.31
1.96
2.35
1.91
1.37
1.20
5.08
1.36
1.41
(Rs) of the enantiomers of
profens
Rs
5a
k1
α
2
2
1
1.90
1.64
2.07
2.21
1.45
1.20
1.32
1.58
1.30
2.84
2.02
2.73
2.34
1.38
1.34
6.13
1.39
1.56
Rs
5b
k1
α
1.44
1.59
1.44
1.71
1.40
1.16
1.02
1.52
1.14
2.14
1.91
2.75
1.78
1.34
1.29
4.73
1.33
1.35
Rs
0b
k1
α
2.00
1.65
2.20
2.35
1.45
1.29
1.41
1.58
1.40
2.96
2.02
1.43
2.49
1.39
1.64
6.49
1.41
1.74
Rs
5b
k1
2.54
1.58
1.83
2.92
1.40
1.37
1.66
1.51
1.46
3.65
1.89
2.63
3.02
1.35
1.47
7.72
1.36
1.63
α
Rs
k1 retention factor of the first-eluted enantiomer
a
Disodium hydrogen phosphate buffer (Na HPO ):MeOH (50:50, v/v %), pH 5.0
2
4
b
Trisodium phosphate buffer (Na PO ):MeOH (50:50, v/v %), pH 5.0
3
4
Germany), a UV–VIS detector (L-4250, VWR Interna-
tional Darmstadt, Germany), EZchrom software (VWR
International Darmstadt, Germany) and an autosampler
All experiments were performed at room temperature,
detected by UV absorbance at 254 nm. The flow rate was
0.6 ml/min.
(AS-2000A, VWR International Darmstadt, Germany).
Preparation of the column
Solvents and reagents
The column was synthesized using Lichrospher 100 DIOL
according to the procedure described previously by Sven-
sson et al. [24, 25]. Silica diol stationary phase has a sur-
Methanol (MeOH) was purchased from Sigma-Aldrich
(France), acetonitrile (ACN) from Merck KGaA (Ger-
2
many), tetrahydrofuran (THF) from Prolabo (France), tri-
ethylamine (TEA) from Fluka Analytical (Belgium), water
face coverage of 3.87 µmol/m and a specific surface area
2
of 350 m /g. The stationary phase was modified with a
for HPLC (H O) from SDS (France) and acetic acid glacial
70-mM solution of sodium periodate in water/methanol
(80/20 v/v) cooled to 0 °C at 0.5 ml/min for 4 h followed
by flushing with water. Immobilization of the chiral selec-
tor was carried out by a reductive amination with a 10-mg/
ml solution of vancomycin hydrochloride and sodium
cyanoborohydride in 50 mM NaH PO at pH 7 for 12 h.
2
(HOAc) from Carlo Erba Reagents (France).
Ammonium nitrate (NH NO , ≥99.5 %) was obtained
4
3
from Sigma-Aldrich (France), sodium dihydrogen phosphate
monohydrate (NaH PO ·H O), disodium hydrogen phosphate
2
4
2
anhydrous (Na HPO ) and trisodium phosphate 12-hydrate
2
4
2
4
(
Na PO ·12H O) from VWR international (Germany). All
Finally CSP was flushed by a solution 50 mM ammonium
nitrate in water/methanol (60/40, v/v) for 1 h.
3
4
2
profens were purchased from Sigma-Aldrich (France).
Lichrospher 100 DIOL (150 × 3 mm I.D.), 5 µm, was
purchased from VWR international (France).
Vancomycin hydrochloride from Streptomyces orientalis
was purchased from Sigma-Aldrich (France).
Results and discussion
All buffer mobile phases were prepared with specified con-
centrations of salt and TEA. Solution pH was controlled with
glacial acetic acid before the addition of organic modifier.
In our experiments, to optimize the enantioseparation, we
first evaluated the chromatographic parameters k , α and
1
Rs as a function of pH and molarity of ammonium nitrate
1
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J IRAN CHEM SOC
Fig. 3 Chromatograms of the profens in 5 mM, pH 5.0 phosphate buffer/MeOH (50/50, v/v %); a carprofen, b fenoprofen, c flurbiprofen, d ibu-
profen, e indoprofen, f ketoprofen
buffer in a mixture with methanol. Using different organic
modifiers and optimized pH and molarity, all chromato-
graphic parameters were calculated.
values of profens are between 4 and 4.4 leading to nega-
tively charged species at pH 5. At this pH value, the chiral
selector still possesses positive charges, allowing it to inter-
act electrostatically with the analytes.
Effect of buffer pH on retention and enantioseparation
Effect of buffer molarity on retention
and enantioseparation
The effect of the pH of the aqueous mobile phase on ana-
lyte retention and enantioseparation is critical since the
ionization of acidic analytes and chiral selector can be con-
trolled according to this.
The commercially available column Chirobiotic V con-
taining vancomycin is described as being stable between
pH 3.5 and 7. From the values listed in Table 1, it appears
that chiral recognition operates in the tested pH range.
Most compounds tested exhibit good enantioseparation
between pH 4 and 6 with a maximum at pH 5. The pKa
The concentration of the buffer is a very effective experi-
mental parameter for controlling analyte retention and res-
olution. A range of ammonium nitrate buffer concentrations
between 30 and 100 mM was investigated. Separation of
all profens was observed in this range of concentrations.
In our studies, an increase of the molarity gave a decrease
of analyte retention (Fig. 1a), but the selectivity values
remained quite constant. Most of the profens exhibited
1
3

J IRAN CHEM SOC
maximum values of resolution at the concentration of
with the mobile phases consisting of: 50 mM sodium dihy-
drogen phosphate buffer, pH 5: MeOH (50/50, v/v %),
5
0 mM (Fig. 1b).
2
5 mM disodium hydrogen phosphate buffer, pH 5: MeOH
Effect of organic modifier on retention
and enantioselectivity
(50/50, v/v %) and 5 mM phosphate buffer, pH 5: MeOH
(50/50, v/v %).
Figure 3 shows the chromatograms of the six profens
using the mobile phase consisting of 5 mM phosphate
buffer and MeOH (50/50, v/v %) at pH 5.
In the chromatographic conditions, 50 mM ammo-
nium nitrate buffer and pH 5, it was found that the elu-
tion strength of methanol is lower than the other organic
modifiers, tetrahydrofuran and acetonitrile. In the case of
tetrahydrofuran and acetonitrile both solvents were tested
at percentages 20, 25 and 30 % v/v, while methanol was
investigated at 40 and 50 % v/v. For all solvents tested, the
retention of the profens decreased with increasing content
of organic modifier. With respect to tetrahydrofuran, all
the analytes were completely resolved for all solvent com-
positions, however, relatively short retention times were
observed for 30 % v/v. In the case of 20 and 25 % v/v, the
retention times were acceptable (<18 min for both enanti-
omers), except for carprofen and flurbiprofen which had a
relatively long retention times, as can be seen in Table 2.
Adding TEA to the mobile phase of tetrahydrofuran
caused a decrease in the retention time of ketoprofen and
an increase in retention times for the other profens.
For acetonitrile as mobile phase (25 and 30 % v/v), the
resolution of some analytes was not complete. At 20 % v/v
acetonitrile, the six profens enantiomers were completely
resolved with acceptable retention times (Table 2).
With methanol, analyte retention and resolution values
represented in Table 2 show that the optimum conditions
were achieved using a mobile phase consisting of 40 % v/v
methanol.
Conclusion
In this work, a column based on vancomycin immobi-
lized by reductive amination to aldehyde functionalized
silica was prepared in house and investigated for the chi-
ral liquid chromatographic analyses of six NSAIDs. The
effects of key experimental parameters on the enanti-
oseparation were investigated. The selectivity, resolution
and retention factors were studied for variation in buffer
pH, solvent composition and nature of organic modifier.
Good enantioseparations were obtained at pH 5 for all
profens investigated with tetrahydrofuran, acetonitrile
and methanol and 50 mM of ammonium nitrate buffer.
Replacement of the ammonium nitrate buffer by a phos-
phate buffer gave good separations with shorter retention
times. From the results obtained, the vancomycin sta-
tionary phase showed high enantioselectivity and stabil-
ity over the pH range investigated (pH 4–6). From these
results it is suggested that the chiral recognition mecha-
nism for vancomycin is due to electrostatic interactions
(charge–charge) between the negatively charged acidic
compounds and the positively charged chiral selector.
In conclusion, the results obtained for all profens show
good separatory performance for the prepared column
without deterioration of the vancomycin stationary phase
incorporated into the silica. The results were reproduc-
ible and therefore the column can be considered robust
and suitable for routine analysis.
The chromatograms of the six profens obtained using
optimum conditions of pH and buffer concentration are
shown in Fig. 2 with the mobile phase consisting of 40 %
v/v methanol.
Effect of phosphate buffer
Although the six profens were successfully separated
using the optimum conditions discussed above, the reten-
tion times of carprofen and flurbiprofen were relatively
long, therefore, phosphate buffers were investigated.
Sodium dihydrogen phosphate buffer, disodium hydro-
gen phosphate buffer and phosphate buffer were tested
at different concentrations with a fixed amount of metha-
nol, 50 % v/v. By changing the ammonium nitrate buffer
to sodium dihydrogen phosphate buffer, it was found that
the elution strength of methanol become weaker. The best
retentions and resolutions were obtained with 50 % v/v
methanol at pH 5 (Table 3). Almost all analytes showed
good separation with the mobile phases tested (Tables 3,
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