Chiral separation and modeling of baclofen, bupropion, and etodolac profens on amylose reversed phase chiral column

Article abstract of DOI:10.1002/chir.22717

Chiral resolution of baclofen, bupropion, and etodolac profens was obtained with amylose derivatized chiral reversed stationary phase (carbamate groups). The eluent used for bupropion and etodolac was MeOH–water (20:80, v/v) and for baclofen was water–methanol (95:5, v/v). The eluent run rates, finding wavelength and temperature, were 1.0?mL/min, 220?nm and 27?±?1?°C for all the eluents. The magnitude of the retardation factors for S- and R-enantiomers of baclofen, bupropion, and etodolac were 1.37, 2.62, 2.25, 3.25, 1.8, and 3.0. The magnitudes of separation and resolution factors were 1.90, 1.44, and 1.67 and 2.77, 2.35, and 2.04. Limits of detection and quantitation were 1.0–2.0 and 5.1–10.0?μg/mL. Chiral recognition mechanisms were recognized by simulation and high-performance liquid chromatography (HPLC) experiments. It was seen that hydrogen interactions, hydrophobic interactions, and π–π exchanges were the chief interactions for chiral recognition mechanisms. The described methods may be exploited for the chiral separation of baclofen, bupropion, and etodolac profens in any unknown sample.

Full text of DOI:10.1002/chir.22717

Received: 23 March 2017
DOI: 10.1002/chir.22717
Revised: 29 April 2017
Accepted: 5 May 2017
R E G U L A R A R T I C L E
Chiral separation and modeling of baclofen, bupropion, and
etodolac profens on amylose reversed phase chiral column
Imran Ali1
| Mohd. Suhail1 | Zeid A. Alothman2 | Abdulrahman Alwarthan2
1 Department of Chemistry, Jamia Millia
Islamia, (Central University), New Delhi,
India
2 Department of Chemistry, College of
Science, King Saud University, Riyadh,
Kingdom of Saudi Arabia
Abstract
Chiral resolution of baclofen, bupropion, and etodolac profens was obtained with
amylose derivatized chiral reversed stationary phase (carbamate groups). The eluent
used for bupropion and etodolac was MeOH–water (20:80, v/v) and for baclofen was
water–methanol (95:5, v/v). The eluent run rates, finding wavelength and tempera-
ture, were 1.0 mL/min, 220 nm and 27 1 °C for all the eluents. The magnitude
of the retardation factors for S‐ and R‐enantiomers of baclofen, bupropion, and
etodolac were 1.37, 2.62, 2.25, 3.25, 1.8, and 3.0. The magnitudes of separation
and resolution factors were 1.90, 1.44, and 1.67 and 2.77, 2.35, and 2.04. Limits
of detection and quantitation were 1.0–2.0 and 5.1–10.0 μg/mL. Chiral recognition
mechanisms were recognized by simulation and high‐performance liquid chroma-
tography (HPLC) experiments. It was seen that hydrogen interactions, hydrophobic
interactions, and π–π exchanges were the chief interactions for chiral recognition
mechanisms. The described methods may be exploited for the chiral separation of
baclofen, bupropion, and etodolac profens in any unknown sample.
Correspondence
Imran Ali, Department of Chemistry, Jamia
Millia Islamia (Central University)
New Delhi, India.
Email: drimran_ali@yahoo.com, drimran.
chiral@gmail.com
KEYWORDS
amylose chiral column, baclofen, bupropion, chiral HPLC‐separation, docking studies, etodolac
1 | INTRODUCTION
are guidelines for selling racemic drugs all over the world.
The most crucial agencies issuing these guidelines are the
The chiral separation of racemic drugs became important
since the guidelines of the US Food and Drug Administration
(FDA) for marketing of optically active drugs were pub-
lished.1 The FDA formulated certain guidelines due to the
dissimilar pharmaceutical properties of the enantiomers. In
several research papers, it was mentioned that one enantiomer
is pharmaceutically active while the other is dormant or lethal
or ballast. A mostly inactive enantiomer creates a range of
side effects and harm in the human body.2 This is due to cer-
tain features, such as a dissimilar stereoselective metabolism,
allocation rate, excretion, and clearance in the human body.
Consequently, medical practitioners, researchers, academi-
cians, industry, and government are looking for a practical
approach to the chiral separation of racemates. Now there
FDA, the European Medicines Agency, and Canadian and
Japanese health agencies.3-6
Aryl propionic acids, so‐called profens, are nonsteroidal
antiinflammatory (NSAID) medicines. These are sold for
curing puffiness and arthritis.7,8 The most regularly used
profens are baclofen, bupropion, and etodolac (Figure 1).9
The WHO in 2001 reported profens as the most usually pre-
scribed medicines in about 100 countries and, hence, incor-
porated in the list of indispensable medicines.10
Approximately 30 million people use NSAIDs every day
for many diseases.11. The yearly world sale market of these
drugs is $12 billion US, and is assumed to increase to $30
billion US by the end 2017.12 Profens show serious problems
despite their wide applications.13-17 The serious undesirable
386
© 2017 Wiley Periodicals, Inc.
wileyonlinelibrary.com/journal/chir
Chirality. 2017;29:386–397.

ALI ET AL.
387
2 | MATERIALS AND METHODS
2.1 | Chemicals and reagents
The ( )‐mixtures of baclofen, bupropion, and etodolac
were extracted from the commercial drugs available in
the market. Methanol of high‐performance liquid
chromatography (HPLC) grade was provided by Merck
(Bombay, India). Ionized water was obtained through a
Millipore Milli‐Q (Bedford, MA) water production
machine. Standard solutions of 1.0 mg/L concentration
of bupropion and etodolac were made in MeOH, while
of baclofen was made in methanol–water (5:5, v/v),
methanol, respectively.
2.2 | Instrumentation
FIGURE 1 Enantiomeric structures of baclofen, bupropion and
etodolac
HPLC runs were made on a machine manufactured by
Shimadzu (Japan). The HPLC machine comprised software
Class VP, pump (LC‐10 AT VP), and ultraviolet range
detector (SPD‐10A). The stationary phase used was tris‐
(3,5‐dimethylphenyl carbamate (carbamate groups), packed
in a steel column (15.0 x 0.46 cm), with trade name
AmyCoat‐RP (Kromasil, Bohus, Sweden).
18-20
effects are ulceration, gastric bleeding, vomiting,
dys-
pepsia,21 bowel swelling,22 and mucus injury in the small
intestine.13 Profens also create risk to the kidneys concerning
fluid and salt preservation. Profens are also accountable for
heart‐related issues, producing ten and two times an addi-
tional probability of a heart assault.14,23 As per a research
described in the American Journal of Medicine, ~107,000
patients are admitted to hospitals yearly due to gastric
problems after consumption of profens.24 Out of these,
~16,500 persons died yearly.25 Profens also show risk effects
on the central nervous system after interaction with
fluoroquinolones in combination therapy.26 Every so often,
people suffered coma, resulting in death.16 The 50% side
effects and toxicities may be reduced by describing optically
active profens. Besides, it is not known which enantiomer is
creating these problems. There are chances that all these side
effects and problems may be controlled by prescribing the
optically pure profens.
2.3 | HPLC conditions
All the experiments were made on an HPLC machine as
depicted with 20.0 μL of profens solution loaded onto the
HPLC machine. The eluent used for bupropion and etodolac
was methanol–water (20:80, v/v) and for baclofen was
methanol–water water–methanol (59:5, v/v). The eluent run
rate, finding wavelength, and temperature were 1.0 mL/min,
220 nm, and 27 1 °C for all the eluents. The eluents were
cleaned and degassed every day before use. The eluents were
cleaned through a nylon membrane (25 mm diameter with
0.45 μm pore size). The retention (k), separation (α), and
resolution (Rs) factors were ascertained.
A thorough look for scientific articles in the literature27-33
confirm that only some research articles described chiral res-
olution of baclofen, bupropion, and etodolac. Most of the
articles described the enantiomeric resolution of a single
drug, while some used costly and toxic solvents along with
a high resolution time. Therefore, there is an immense need
to produce fast, economic, and efficient enantiomeric resolu-
tion of the separation of these molecules. Additionally, there
is also a requirement to describe the chiral recognition mech-
anism. As a result, chiral separation methods were produced
for baclofen, bupropion, and etodolac on an amylose
derivatized chiral reversed stationary phase. In addition, a
chiral recognition mechanism was evaluated by simulation
of the column with simulation studies to describe the enantio-
meric mechanism. These results of these studies are pre-
sented in this article (Figure 1).
2.4 | Simulation studies
The exchanges of enantiomers of the reported profens with
amylose derivatized chiral selector (Figure 2).were
ascertained by docking studies. The results achieved were
utilized to fix the chiral recognition mechanisms.
2.5 | Procedure
The docking study of the compounds baclofen,
bupropion, and etodolac were done with an Intel dual
CPU (1.86 GHz) with Windows XP operating software.
Marwin Sketch software was exploited to sketch the
structures of different antipodes. The structures were

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ALI ET AL.
FIGURE 2 2D (a) and 3D (b) structures of tris‐(3,5‐ dimethylphenyl carbamate) amylose chiral selector
sparked to 3D and saved in a PDB file.34 After that, the
structure of amylose derivatized chiral stationary phase
was sketched using Marwin Sketch software. The struc-
ture of amylose derivatized chiral stationary phase was
docked using AutoDock Tools (ADT) 4.2 by handing
over Gastegier charges, integrating nonpolar hydrogen
atoms, and saving in PDBQT file format. Docking was
done with AutoDock 4.2 allowing for all the rotatable
bonds of the ligands as a rotatable and rigid receptor.35
Using the same tool, an enantiomer (ligand) was edited
to be saved in PDBQT format. The lattice box size of
60 Å × 80 Å × 110 Å with spacing (0.375 Å) was used.
After saving both files in PDBQT format, Vina software
was used to obtain the binding energy/affinity between
the receptor tris‐(3,5‐dimethylphenyl carbamate) amylose
and ligand (enantiomer). After using Vina software, the
output file was opened in PyMOL to do virtual screening,
molecular docking, and a binding site study and to obtain
an image of interaction and the bond length of the
hydrogen bond between crown ether and enantiomer.
The plugin represents a crossing point among PyMOL
and two docking programs of AutoDock Vina and
AutoDock (4.2). The joint effect of the two softwares
completed wide use of a Python script compilation
(AutoDock Tools) for fixing docking experiments. Addi-
tionally, Ligplot software was applied for the assessment
of hydrophobic interactions. Fifty self‐determining
docking experiments were done for all the enantiomers
separately and the chiral selector for low free energy of
fastening conformation from a large cluster, which was
written and saved in PDBQT layout. The PDBQT files
had been transformed to the PDB file format.

ALI ET AL.
389
3 | RESULTS AND DISCUSSION
3.1 | Chromatography
The HPLC measurements of retention (k), separation (α),
and resolution (Rs) factors were ascertained for the enantio-
mers of baclofen, bupropion, and etodolac on amylose
derivatized chiral stationary phase. The magnitude of the
enantioselectivity for S‐ and R‐enantiomers of baclofen,
bupropion, and etodolac were 1.37, 2.62, 2.25, 3.25, 1.8,
and 3.0. The magnitudes of separation and resolution
factors were 1.90, 1.44, and 1.67 and 2.77, 2.35, and
2.04. These magnitudes are given in Table 1. The magni-
tude of separation and resolution factors were >1, showing
acceptable chiral separation of all three racemic drugs. The
separated peaks of HPLC are shown in Figure 3. Further, a
serious examination of HPLC measurements and the peaks
established good chiral separation of all the enantiomers
of the three racemic drugs.
3.2 | Chiral HPLC method optimization
The different compositions of the eluents were tried in order
to optimize the HPLC conditions. The various organic sol-
vents tried with water were methanol, ethanol, acetonitrile,
and propanol in different amounts. The various pHs of these
combinations were also adjusted and tested. The ultraviolet
wavelength varied was 210–300 nm. The concentrations of
injection used were 5–25 μL. The optimization was fixed
by using temperatures from 15–40 °C. As a consequence of
comprehensive testing, the best chiral chromatographic con-
ditions were produced and described. Chiral chromato-
graphic methods were validated via accuracy, selectivity,
precision, robustness, limits of detection and quantitation,
and linearity.
3.3 | Simulation study of dipeptides on
AmyCoat chiral selector
FIGURE 3 Chiral resolution of (a): baclofen, (b): bupropion and (c)
etodolac on AmyCoat RP column (150 x 4.6 6 mm, 5.0 μm)
The chiral mechanism was determined by a docking study
of the enantiomers with amylose chiral selector. The
chiral selector showed various functionalities, which
interacted with the enantiomers of the profens. The
docking affinities (energies) of R‐ and S‐enantiomers of
baclofen, bupropion, and etodolac were −3.4 and −3.3,
−3.4 and −3.2, −3.8 and −3.9, K cal/mol (Table 2). It
is apparent from Table 2 that the various interactions
were hydrogen bondings and hydrophobic interactions.
The elution order may be predicted by considering the
binding affinities (kcal mol−1) as reported. The binding
energies of R‐enantiomers are higher that S‐enantiomers
for all three drugs. Therefore, it may be assumed that
S‐enantiomers eluted first following R‐enantiomers. The
different interactions among various residues of the chiral
selector and enantiomers are outlined below.
TABLE 1 The capacity (k), separation (α), and resolution (Rs) fac-
tors of the enantiomers of of baclofen, bupropion and etodolac on tris‐
(3,5‐dimethylphenyl carbamate) amylose chiral selector
Profens
k1 S‐enantiomer k2 R‐enantiomer
α
Rs
Baclofen
Bupropion
Etodolac
1.37
2.25
1.8
2.62
3.25
3.00
1.90 2.77
1.44 2.35
1.67 2.04

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ALI ET AL.
TABLE 2 Modeling results of the enantiomers of baclofen, bupropion, and etodolac with tris‐(3,5‐dimethylphenyl carbamate) amylose chiral
selector
Binding affinity No. of hydrogen Residues involved in H‐bonding
Residues involved in
hydrophobic interactions
S. no. Enantio‐mers
(kcal mol−1
)
bonds
(Bond length in Å)
Baclofen
1.
2.
R
S
‐3.4
2
UNK.390::H of NH2 group(2.8)
UNK.390::H of NH2 group(1.9)
Unk 159,161,163::C2, Unk 159::C3,
Unk 163::C4, Unk 157,159::C5,
Unk 157,287:: C7, Unk 287::C8,
Unk 287,288::C9, Unk 157,287::C10,
Unk 155,158::N, Unk 284,285::O1,
Unk 155::O2
‐3.3
2
UNK.348::H of NH2 group (2.0)
UNK.388::H of NH2 group (2.7)
Unk 158,159::C1, Unk 191::C2,
Unk 162::C3, Unk 191::C4, Unk
158::C5, Unk 156,157::C7, Unk
Unk 129,314,316::C9, Unk 157,316,
287::C10, Unk 157,158::N, Unk 128,
192::O1, Unk 313,315::O2
Bupropion
3.
R
‐3.4
‐3.2
2
2
UNK`.388::H of NH group (2.5)
UNK`.289::H of ‐OH group (2.3) 158::C3, Unk 157,316::C4, Unk 155,287,
288::C5, Unk 157,159,
Unk 188::C1, Unk 158::C2, Unk 191,
161:: C7, Unk 157,158::C8, Unk 158,192:
:C10, Unk 287,316::C12,
Unk 287::C13
4.
S
UNK`.390::H of NH group (2.0)
Unk 185,189::C1, Unk 185,131::C2,
UNK`.289::H of ‐OH group (2.4) Unk 189::C3, Unk 188::C4, Unk 307,
308::C5, Unk 182,184,185,186,308,
310::C6, Unk 131::C8, Unk 188,
189::C10, Unk 310::C11, Unk
310::C12, Unk 310::C13, Unk
131::O
Etodolac
5.
R
‐3.8
‐3.7
2
2
UNK`.353::H of ‐OH group (2.8) Unk 163::C1, Unk 158,162::C2,
UNK`.647::H of ‐OH group (2.4) Unk 158, 159::C5, Unk 153::C6,
Unk 161,157:: C7, Unk 158,159::C8,
Unk 155,287::C9, Unk 157,158,
316::C10, Unk 157,158,159::C11,
Unk 157,158::C12, Unk 316::C17,
Unk 158::N, Unk 284::O3
6.
S
UNK`.353::H of ‐OH group (2.8) Unk 159,162::C1, Unk 162::C2, Unk
UNK`.647::H of ‐OH group (2.1)
159::C4, Unk 157,287::C6, Unk 158,
159::C7, Unk 162::C8, Unk 157::C9,
Unk 158,159::C11, Unk 88,190,191,
192::C14, Unk 128,129,191,192,
313,315::O3
3.4 | Baclofen
3.4.1 | R‐enantiomer
Oxygen of the –OH group in tris‐(3,5‐dimethylphenyl carba-
mate) amylose::H of –NH2 group in baclofen (2.8 Å). Oxygen
of –OH group in tris‐(3,5‐dimethylphenyl carbamate) amy-
lose::H of –NH2 group in baclofen (1.9 Å).
3.4.2 | S‐enantiomer
Oxygen of the –OH group in tris‐(3,5‐dimethylphenyl
carbamate) amylose::H of –NH2 group in baclofen
(2.7 Å). Oxygen of (−O‐) group in tris‐(3,5‐
dimethylphenyl carbamate) amylose::H of –NH2 group
in baclofen (2.0 Å).

ALI ET AL.
391
3.5 | Bupropion
3.7 | Mechanisms of resolution at the
supramolecular level
3.5.1 | R‐enantiomer
The chiral recognition mechanism is explained by chro-
matographic and modeling results. Amylose derivatized chi-
ral selector is helical in shape (Figure 2).36-40 It was seen
that the amylose based chiral selector has wide range chiral
separation capacity.41-43 The chiral separation of profen
enantiomers on amylose derivatized chiral selector is due
to the chiral grooves in the structure of the stationary phase.
The antipodes of these profens enantiomers set
enantioelectively. The setting of the antipodes is controlled
hydrogen, hydrophobic, π–π, van der Waal's, and steric
effects interactions. It is attractive to report that the reported
profens have a benzene ring facilitating π–π interactions
among the antipodes and chiral amylose derivatives. It is
already established that π–π interactions are crucial in the
chiral separation of many aromatic racemates.44-51 The set-
ting of all the antipodes of profens in the derivatized amy-
lose grooves is depicted in Figures 4–9. These were
developed using modeling analyses. Two antipodes of each
profen are involved in chiral grooves by electrostatic forces
of attraction between amino groups, and carboxylic acid
groups of profens and amino groups, amide groups,
hydroxyl groups, and oxide groups of the derivatized amy-
lose. Briefly, the enantiomers of the reported profens get
set enantioelectively in derivatized amylose chiral grooves.
Contrarily, the eluent attempted to take forward these anti-
podes. Consequently, the competitive forces of stationary
and mobile phase resulted in the different retention of the
antipodes. Low retained enantiomer eluted first and more
retained enantiomer eluted last.
Oxygen of –OH group in tris‐(3,5‐dimethylphenyl carba-
mate) amylose::H of –NH group in bupropion (2.5 Å). Oxy-
gen of (−CO‐) group in bupropion::H of –OH group in tris‐
(3,5‐dimethylphenyl carbamate) amylose (2.3 Å).
3.5.2 | S‐enantiomer
Oxygen of –OH group in tris‐(3,5‐dimethylphenyl carba-
mate) amylose::H of –NH group in bupropion (2.0 Å). Oxy-
gen of (−CO‐) group in bupropion::H of –OH group in tris‐
(3,5‐dimethylphenyl carbamate) amylose (2.4 Å).
3.6 | Etodolac
3.6.1 | R‐enantiomer
Oxygen of –O‐ group in etodolac::H of –OH group in tris‐
(3,5‐dimethylphenyl carbamate) amylose (2.8 Å). Oxygen of
(−COOH) group in etodolac::H of –OH group in tris‐(3,5‐
dimethylphenyl carbamate) amylose (2.4 Å).
3.6.2 | S‐enantiomer
Oxygen of –O‐ group in etodolac::H of –OH group in tris‐
(3,5‐dimethylphenyl carbamate) amylose (2.8 Å). Oxygen of
(−COOH) group in etodolac::H of –OH group in tris‐(3,5‐
dimethylphenyl carbamate) amylose (2.1 Å).
FIGURE 4 Docking model of R‐baclofen
with tris‐(3,5‐dimethylphenyl carbamate)
amylose chiral selector

392
ALI ET AL.
FIGURE 5 Docking model of S‐baclofen
with tris‐(3,5‐dimethylphenyl carbamate)
amylose chiral selector
FIGURE 6 Docking model of R‐
bupropion with tris‐(3,5‐dimethylphenyl
carbamate) amylose chiral selector
Therefore, the conclusion pointed to good specificity of the
chiral separation. The accuracy was determined by interpola-
tion of replicates (n = 5) peak areas. The percent error was
estimated and observed from 0.55–1.0% for each enantiomer.
This range indicated a good accuracy of the developed
method. The validation calculations were carried out with
Excel software. The outcomes of the enantiomeric chromato-
graphic method were determined in terms of percent relative
standard deviation (%RSD), correlation coefficient (CC), and
confidence limit (CL). The validation magnitudes showed
good reproducible results.
3.8 | Validation of the chiral HPLC method
The developed chiral chromatographic method was validated
by five sets of experiments (n = 5) in similar conditions. The
validation was carried out by accuracy, precision, linearity
(2.0–200.0 μg/mL), limit of detection and quantification,
specificity, and robustness. The validation was carried out
as per standard methods. The produced chiral HPLC methods
were fairly good specific, as shown in Figure 3. No effect of
the supplemented impurities (in standard solution) was seen
on the retention time chromatograms of these profens.

ALI ET AL.
393
FIGURE 7 Docking model of S‐
bupropion with tris‐(3,5‐dimethylphenyl
carbamate) amylose chiral selector
FIGURE 8 Docking model of R‐etodolac
with tris‐(3,5‐dimethylphenyl carbamate)
amylose chiral selector
3.9 | System suitability test
3.10 | Specificity
Test of suitability was done with five loadings (n = 5) of
the standard solution onto the HPLC injector. The calcu-
lated values were for tailing of the peaks, separation, per-
cent RSD of the area of the peaks, and chromatographic
times (Table 3). The values in Table 3 are indicative of
low RSD, <1.5% (peak area) and <1.5% (chromatographic
time). The peaks tailing of S‐ and R‐enantiomers of bac-
lofen and bupropion were 0.90 and, 1.0. Contrarily, the
S‐ and R‐enantiomers of etodolac showed 1.2 and 1.45
magnitudes.
The specificity of the methods was ascertained for the impu-
rities and degradation of profen. The eluent was loaded onto a
machine as blank. It was observed that no peak was seen in
the blank situation. It confirmed these methods to be specific.
3.11 | Linearity
The linearity was ascertained by plotting the areas of the
peaks versus various amounts of the profens. The slope,
y intercepts, regression coefficients (r2), and correlation

394
ALI ET AL.
FIGURE 9 Docking model of S‐etodolac
with tris‐(3,5‐dimethylphenyl carbamate)
amylose chiral selector
TABLE 3 Summary of the system suitability and linearity parameter
Baclofan
Bupropion
Etodolac
HPLC method
Parameters
S
R
S
R
S
R
System suitability
Tailing factor
Resolution
0.90
—
0.80
0.50
1.0
0.90
6.43
0.71
0.60
1.0
1.2
—
1.45
2.67
1.4
2.88
0.70
0.30
7.90
1.00
0.90
% RSD peak area
% RSD Retention time
Linearity
1.0
0.35
0.95
Slope
4237.2
−5730.0
0.9985
0.9866
1.0
435.3
−750
0.9986
0.9883
1.0
355.2
−730.1
0.9967
0.9854
1.0
395.0
−955
0.9988
0.9996
1.0
464.0
3136
0.9980
0.9856
1.1
422.1
−750.2
0.9978
0.9867
2.0
Y‐intercept
Corr. coeff. (r)
Regression coeff (r2)
LOD (μg/mL)
LOQ (μg/mL)
5.1
5.1
5.1
5.1
5.5
10.0
coefficients (r) were estimated (Table 4). Superior linearity
was seen for all the enantiomers of the reported profens in
2.0–200.0 μg/mL concentrations. The regression constant
(r2) was greater than 0.98.
these were 1.1 and 2.0 μg/mL for the S‐ and R‐ enantiomers
of etodolac (Table 3).
3.13 | Precision
The intraday and interday precision was ascertained by
standard procedures. The obtained data are summarized
in Tables 4 and 5. The intraday precision was ascertained
in a single day, while interday precision was done on the
next day. The percent assay for intraday precision was cal-
culated for the S‐ and R‐ enantiomers of baclofen,
bupropion, and etodolac. These percent mean assays and
3.12 | Limit of detection (LOD) and
quantification (LOQ)
The limits of detection and quantification were ascertained as
a signal‐to‐noise ratio of 3 for limit of detection and 10 for
limit of quantitation. Limit of detection of R‐ and S‐enantio-
mers of baclofen and bupropion was 1.0 μg/mL. Contrarily,

ALI ET AL.
395
TABLE 4 Intraday precision
Baclofen
1.0 mg
1.5 mg
% Mean assay
2.0 mg
% Mean assay
% Mean assay
100.0
%RSD
0.55
%RSD
0.55
%RSD
S
99.9
99.7
99.0
99.0
0.56
0.56
R
99.9
0.55
0.55
Bupropion
S
100.0
99.9
0.55
0.55
99.9
99.7
0.55
0.55
98.0
99.0
0.56
0.56
R
Etodolac
S
99.00
98.0
0.58
0.65
98.6
97.5
0.58
0.65
98.0
97.0
0.60
0.69
R
TABLE 5 Interday precision
TABLE 7 Robustness study for the new chiral HPLC method
Baclofen
Bupropion
Etodolac
Baclofen (variation in % RSD)
(mean % assay) (mean % assay) (mean % assay)
S
R
Samples
S
R
S
R
S
R
RT
Area
0.75
0.78
0.75
0.71
RT
0.53
0.55
0.53
0.50
Area
0.75
0.78
0.75
1.0
Sample ‐ 1 100.0
Sample ‐ 2 100.0
Sample ‐ 3 100.1
Sample ‐ 4 100.0
Sample ‐ 5 100.1
100.0
100.0
100.1
100.0
100.1
0.55
100.0
100.0
100.1
100.0
100.1
0.55
100.0
100.0
100.1
100.0
100.1
0.55
99.99
99.98
99.99
99.98
99.99
0.70
98.9
98.9
98.8
98.9
99.9
0.74
Flow rate (1.0 mL/min)
0.53
0.55
0.53
0.50
Flow rate (0.8 mL/min)
Column temperature (27 °C)
Column temperature (20 °C)
Bupropion (variation in % RSD)
% RSD
0.55
Flow rate (1.0 mL/min)
0.54
0.57
0.54
0.51
0.77
0.80
0.76
0.73
0.54
0.57
0.54
0.52
0.77
0.80
0.76
1.10
Flow rate (0.8 mL/min)
Column temperature (27 °C)
Column temperature (20 °C)
%RSD are given in Table 4. Similarly, the values of
interday percent mean assays and %RSD for these enantio-
mers are given in Table 5. These values of intraday and
interday assays were fine.
Etodolac (variation in % RSD)
Flow rate (1.0 mL/min)
0.56
0.57
0.56
0.60
0.78
0.79
0.78
0.80
0.60
0.62
0.60
0.60
0.84
0.86
0.84
1.20
Flow rate (0.8 mL/min)
Column temperature (27 °C)
Column temperature (20 °C)
3.14 | Recovery
The recovery was ascertained using three different spiked
amounts of baclofen, bupropion, and etodolac profens. The
spiked concentration of each drug was 50–100.0 μg/mL.
The recoveries of the antipodes are given in Table 6. The per-
cent recoveries were 99–101% for all six enantiomers.
3.15 | Robustness
The robustness was ascertained by minor variation of one
experimental variable at a time, while maintaining the other
variables fixed. The changes were observed constant in the
peaks, which might be affected by the presentation of the
method. These values are presented in Table 7. The changes
in run times (Rt) and the areas of the peaks were greater than
2, confirming a robust method.
TABLE 6 Recovery study
Concentration
Baclofen
Bupropion
Etodolac
(μg/mL)
(% recovery)
(% recovery)
(% recovery)
S
R
S
R
S
R
50
100.0 101.0 100.0 101.0 100.0 99.0
100.0 101.0 100.0 101.0 100.0 99.0
100.0 101.0 100.0 101.0 100.0 99.0
100.0 101.0 100.0 101.0 100.0 99.0
100.0 101.0 100.0 101.0 100.0 99.0
100.0 101.0 100.0 101.0 100.0 99.0
4 | CONCLUSION
60
70
The chiral separation of baclofen, bupropion, and etodolac
profens was achieved successfully on derivatized amylose
reversed stationary phase. Limits of detections and quantita-
tion were 1.0–2.0 and 5.1–10.0 μg/mL. Chiral recognition
mechanisms were recognized by simulation and HPLC
80
90
100
% RSD
0.55
0.56
0.55
0.56
0.71
0.75

396
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How to cite this article: Ali I, Suhail M, Alothman
ZA, Alwarthan A. Chiral separation and modeling of
baclofen, bupropion, and etodolac profens on
amylose reversed phase chiral column. Chirality.
2017;29:386–397. https://doi.org/10.1002/chir.22717
42. Ali I, Sanagi MM, Aboul‐Enein HY. Advances in chiral separations
by non‐aqueous capillary electrophoresis in pharmaceutical and bio-
medical analysis. Electrophoresis. 2014;35:926‐936.
43. Ali I, Hussain A, Saleem K. Determination of stereo‐selective bind-
ings of racemic propranolol with β2‐AD‐GPCR in human plasma. J
Liq Chromatogr Rel Technol. 2013;36:792‐806.