Chiral separation of ketoprofen on an achiral NH2 column by HPLC using vancomycin as chiral mobile phase additive

Article abstract of DOI:10.1007/s13738-016-0951-6

A high-performance liquid chromatographic method for chiral separation of ketoprofen racemate was developed. (R)- and (S)-ketoprofen enantiomers were separated on a LiChrosorb NH₂ column (250?mm?×?4.6?mm, i.d 5?μm) at 20?°C, using 2-propanol/potassium dihydrogen phosphate buffer (pH 6.0, 0.05?M) (50:50 v/v). Containing vancomycin as the mobile phase, at a flow rate of 0.8?ml?min^?1 and detection wavelength of UV, the detector was set at 310?nm. Under these conditions, ketoprofen enantiomers could be separated with a selectivity factor (α) of 2.172 and a resolution (Rs) of 4.78 using extremely low concentrations of the vancomycin chiral additive.

Full text of DOI:10.1007/s13738-016-0951-6

J IRAN CHEM SOC
DOI 10.1007/s13738-016-0951-6
ORIGINAL PAPER
Chiral separation of ketoprofen on an achiral NH column
2
by HPLC using vancomycin as chiral mobile phase additive
Dehbiya Gherdaoui1,2 · Hafsa Bekdouche · Said Zerkout · Rachid Fegas
Michel Righezza
1
1
1,2
·
2
Received: 24 January 2016 / Accepted: 28 July 2016
©
Iranian Chemical Society 2016
Abstract A high-performance liquid chromatographic
method for chiral separation of ketoprofen racemate was
developed. (R)- and (S)-ketoprofen enantiomers were sep-
activity has been contributed mainly to the S-form, while
R-form is either inactive or reduced active form compared
to S-form [1–4].
arated on a LiChrosorb NH column (250 mm × 4.6 mm,
The literatures are rich with analytical methods for the
determination of ketoprofen enantiomers. Some of the
direct approaches for the analysis of ketoprofen enanti-
omers by HPLC and CE are either using chiral stationary
phases (CSP) or chiral mobile phase additives (CMPA)
[5–10].
Macrocyclic antibiotics among them glycol-peptides
(namely, vancomycin, teicoplanin and ristocetin A) have
been frequently employed in HPLC, capillary electrophore-
sis and capillary electrochromatography. These compounds
have a great variety of functional groups, cavities and
numerous stereogenic centers, which offer many interac-
tion possibilities including, hydrogen bonding, electrostatic
and π–π interactions, inclusion, steric interactions, dipole
stacking or combinations thereof [11–23].
2
i.d 5 µm) at 20 °C, using 2-propanol/potassium dihydro-
gen phosphate buffer (pH 6.0, 0.05 M) (50:50 v/v). Con-
taining vancomycin as the mobile phase, at a flow rate of
−1
0
.8 ml min and detection wavelength of UV, the detec-
tor was set at 310 nm. Under these conditions, ketoprofen
enantiomers could be separated with a selectivity factor (α)
of 2.172 and a resolution (Rs) of 4.78 using extremely low
concentrations of the vancomycin chiral additive.
Keywords Ketoprofen · Chiral separation · Vancomycin ·
Chiral mobile phase additive
Introduction
Vancomycin as shown in Fig. 2 has been the most com-
monly used glycopeptide in the enantioseparation. Vanco-
mycin was introduced by Armstrong et al. to resolve over
100 racemates including nonsteroidal anti-inflammatory
drugs, antineoplastic drugs, pesticides and numerous
N-derivatized amino acids [19, 24–32].
Ketoprofen, (± )-(R, S)-2-(3-benzoyl phenyl)-propionic
acid as shown in Fig. 1, is a nonsteroidal anti-inflamma-
tory drug (NSAID) that has analgesic, anti-inflammatory
and antipyretic properties. It is widely used clinically for
treatment of arthritis and rheumatic diseases. However,
(
S)-ketoprofen and (R)-ketoprofen display significantly
The low concentration of antibiotics in the run buffer is
one of the advantages of the glycopeptides as a chiral selec-
tor (CMPA). It is usually sufficient to use only 1–4 mM
solution; when they are added to the buffer fortunately they
do not absorb in UV region [9, 32–36].
different pharmacological activities. The pharmacological
*
Dehbiya Gherdaoui
ghardaouidah@yahoo.fr
The aim of this work is to present more extensive results
of the enantiomers ketoprofen separation using vancomy-
cin as CMPA, by studying the effect of its concentration on
selectivity and resolution of ketoprofen.
1
Laboratory to Research of Bioactive Products
and Enhancement of Biomass, Department of Chemistry,
Ecole Normale Superieur Vieux Kouba, 16050 Algiers,
Algeria
2
We have also compared the results obtained with those
obtained when ketoprofen was separated by C8 column,
CNRS, iSm2 UMR 7313, Aix Marseille Université,
1
3397 Marseille, France
1
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J IRAN CHEM SOC
Procedure
.05 M phosphate buffer was prepared by dissolving potas-
0
sium dihydrogen phosphate and adjusted to the desired pH
using 0.1 M potassium hydroxide. Buffer solutions were
filtered with 0.22-μm membrane filter.
Fig. 1 Chemical structure of ketoprofen
The vancomycin stock solutions were prepared by dis-
solving vancomycin in a buffer solution followed by ultra-
sonic degasification. The vancomycin solutions were stored
at +4 °C when not in use.
using acetonitrile–triethylamine acetate (TEAA) buffer (pH
5
.2, 20 mM) (35:65, v/v) containing vancomycin as the
mobile phase [9] and column based on vancomycin immo-
bilized by reductive amination to aldehyde functionalized
silica, using mobile phase composed of a mixture of 40 %
methanol in ammonium nitrate buffer (50 mM) at pH 5.0
The mobile phase was prepared by mixing the buffer
solution pH 6.0 with 2-propanol (50/50).
These conditions were obtained in previous work, when
we studied the effect of organic modifier, pH buffer and
stationary phases on the retention time of vancomycin. The
analyte ketoprofen was prepared by dissolving ketoprofen
in methanol. The flow rate was 0.8 mL/min and the injec-
tion volume was 10 μL.
[10].
Experimental
Materials and instruments
First, the ketoprofen racemate was injected by using the
mobile phase without dissolving vancomycin in it to deter-
mine the retention time of ketoprofen.
Ketoprofen racemate was injected in the second step by
using the same conditions with dissolving vancomycin in
the buffer solution.
The analysis was performed by using a chromatographic
system from Waters Alliance e2695 HPLC system with
2
998 PDA detector. The HPLC system was equipped with
Empower 3 software.
Sartorius semi-micro analytical balance bp121s, Digital
S-ketoprofen was injected in the same conditions to
determine the retention time of the enantiomer S and to
identify the two peaks.
pH meter mettler toledo and ultrasonic cleaner, NH col-
2
umn (250 mm × 4.6 mm) LiChrosorb with different diam-
eter particle manufacture by Merck KGaA.
Chemical compounds
Results and discussion
Racemate ketoprofen and S-ketoprofen from Sigma
Aldrich, all the reagents used in this work (methanol,
Effect of diameter particle of column
2
-propanol, potassium hydroxide KOH, potassium dihy-
Two chiral chromatographic columns were used with the
drogen phosphate KH PO ), were of analytical-reagent
same adsorbent material NH and the same dimensions
2
4
2
grade purchased from Sigma Aldrich.
(250 mm × 4.6 mm). These two columns have different
Fig. 2 Chemical structure of
vancomycin
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J IRAN CHEM SOC
Fig. 3 Chromatograms of
the ketoprofen in 2 mM of
vancomycin, pH 6.0 phosphate
buffer/2-propanol (50/50, v/v);
diameter column 10 μm (a) and
diameter column 5 μm (b)
Fig. 4 Chromatograms of the
ketoprofen with 5 μm diameter,
pH 6.0 phosphate buffer/2-
propanol (50/50, v/v); 1 mM of
vancomycin (c) and 1.5 mM of
vancomycin (d)
particle size: one column, with a particle size of 5 μm, was
used for analytical purposes (measurement of additive con-
centrations) and the other, with a particle size of 10 μm,
was used to study the effect of particle to the separation
Fig. 3.
concentration on factor retention, selectivity and resolu-
tion of ketoprofen enantiomers. The chiral resolution and
selectivity increased with the increasing vancomycin con-
centration in the mobile phase, and ketoprofen was sepa-
rated with a good selectivity of 2.17 at low concentration of
For the same mobile phase and vancomycin concentra-
tion, Fig. 3 shows a better separation when we use a col-
umn with particle size of 5 μm then using another with
particle size of 10 μm (that is to say with much lower peak
widths). Using columns with small particle size allows to
achieve good separation with increased efficiency and thus
increased resolution.
2 mM and short time retention on achiral NH column and
mobile phase consisting 2-propanol/buffer pH 6.0 (50/50).
2
Comparative study
Among the most mentioned studies in the chiral separa-
tion of ketoprofen with the present work the study of Guo
[
9], and Bouchair [10]. Table 1 shows a comparison of the
Effect of chiral selector concentration
enantioselectivity and resolution in this work with values
reported in the literature. Good resolution was obtained
The concentration of the chiral selector is an important var-
iable controlling the chiral recognition.
The influence of vancomycin concentration content in
the mobile phase on the chiral separation was investigated
by varying the vancomycin concentration in the range of
in the use of column NH with vancomycin as CMPA
and the chiral Column vancomycin immobilized on diol
with selectivity of 2.17 and 2.02, respectively. The use of
2
the NH column enables to obtain better resolution than
2
the use of C8 column by low concentration of the chiral
selector.
0
–2 mM Fig. 4. Figure 5 shows the effect of vancomycin
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J IRAN CHEM SOC
1
.2
1
6
5
4
3
2
1
0
0
0
.8
.6
S
selectivity
Resolution
2.5
R
0
.5
1
1.5
2
2.5
0
0.5
1
1.5
2
concentration (mM)
concentration (mM)
(
a)
(b)
Fig. 5 Vancomycin concentration effect on retention factor (a), selectivity and resolution of the ketoprofen enantiomers (b)
Table 1 Comparison of the enantioselectivity with resolution of ketoprofen enantiomers
Chromatography condition
Selectivity
1.20
Resolution
1.95
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Column, Hypersil BDS C8 column
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Column vancomycin immobilized on diol
Mobile phase: 50 mM ammonium nitrate buffer:
Methanol (60:40, v/v), pH 5.0
2.02
2.17
3.62
4.78
Bouchair [10]
Column LiChrosorb NH2
Mobile phase: 2-propanol/potassium dihydrogen
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