Chiral HPLC Separation and Modeling of Four Stereomers of DL-Leucine-DL-Tryptophan Dipeptide on Amylose Chiral Column

Article abstract of DOI:10.1002/chir.22624

Chiral high-performance liquid chromatography (HPLC) separation and modeling of four stereomers of DL-leucine-tryptophan DL-dipeptide on AmyCoat-RP column are described. The mobile phase applied was ammonium acetate (10?mM)-methanol-acetonitrile (50:5:45, v/v). The flow rate of the mobile phases was 0.8?mL/min with UV detection at 230?nm. The values of retention factors for LL-, DD-, DL-, and LD- stereomers were 2.25, 3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging from 1.0–2.3 and 5.6–14.0?μg/mL. The simulation studies established the elution orders and the mechanism of chiral recognition. It was seen that π–π connections and hydrogen bondings were the main forces for enantiomeric resolution. The reported chiral HPLC method may be applied for the enantiomeric separation of DL-leucine-DL-tryptophan in unknown matrices. Chirality 28:642–648, 2016.

Full text of DOI:10.1002/chir.22624

CHIRALITY (2016)
Chiral HPLC Separation and Modeling of Four Stereomers of DL-
Leucine-DL-Tryptophan Dipeptide on Amylose Chiral Column
MOHAMMAED F. ALAJMI,1 AFZAL HUSSAIN,1 MOHD. SUHAIL,2 SOFI DANISH MUKHTAR,2 DIBYA RANJAN SAHOO,2
2
LEONID ASNIN3 AND IMRAN ALI
*
1Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia
2Department of Chemistry, Jamia Millia Islamia, New Delhi, India
3Perm National Research Polytechnic University, Perm, Russia
ABSTRACT
Chiral high-performance liquid chromatography (HPLC) separation and
modeling of four stereomers of DL-leucine-tryptophan DL-dipeptide on AmyCoat-RP column
are described. The mobile phase applied was ammonium acetate (10 mM)-methanol-acetoni-
trile (50:5:45, v/v). The flow rate of the mobile phases was 0.8 mL/min with UV detection
at 230 nm. The values of retention factors for LL-, DD-, DL-, and LD- stereomers were 2.25,
3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60,
1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging
from 1.0–2.3 and 5.6–14.0 μg/mL. The simulation studies established the elution orders and
the mechanism of chiral recognition. It was seen that π–π connections and hydrogen
bondings were the main forces for enantiomeric resolution. The reported chiral HPLC
method may be applied for the enantiomeric separation of DL-leucine-DL-tryptophan in un-
known matrices. Chirality 00:000–000, 2016. © 2016 Wiley Periodicals, Inc.
KEY WORDS: AmyCoat-RP; chiral resolution; dipeptide; DL-Leucine-DL-tryptophan; HPLC;
modeling studies
It is well recognized that one of the stereomers may be
pharmaceutically dynamic while the other may be dormant
or toxic or ballast.1 Therefore, all persons involved, i.e., acade-
micians, scientists, clinicians, and government regulatory au-
thorities are very much concerned about the optically active
stereomers for biologically important pharmaceuticals and
drugs. The diverse biological activities of stereomers are
due to their diverse stereoselective metabolisms, excretions,
clearances, and distribution rates in the human body. The
US Food and Drug Administration (FDA), European Commit-
tee for Proprietary Medicinal Products, Health Canada, and
the Pharmaceutical and Medical Devices Agencies of Japan
have certain guiding principles for controlling the racemic
drugs to be sold in the market.2,3 Dipeptides are important
chiral compounds owing to their participation in several bio-
logical processes such as protein synthesis, neurotransmis-
sion, fertility, vital activities of pathogenic microbes, and
control of inflammation processes. Therefore, the various di-
peptides are being used in drug development4–7 and as bio-
logical markers.8
The dipeptides are chiral molecules with two asymmetric
centers and four stereomers, with both amino acids in DL-
configurations. Consequently, a single dipeptide may behave
as a mixture of four dipeptides in our bodies. The dipeptides
with aromatic amino acids are important for their roles in pro-
tein and nucleic acid recognition. A thorough search of the lit-
erature shows some articles on the chiral separation of
derivatized dipetides,9 which is a tedious and time-consuming
and costly solvent-consuming job. However, recently only one
article on the chiral separation of aromatic underivatized di-
peptides was published by this laboratory.10 Chiral high-
performance liquid chromatography (HPLC) is considered
the best technique for this purpose due to its reproducibility,
ease of operation, efficiency, and selectivity.11–14 Keeping
these facts into mind, attempts have been made to separate
four stereomers of DL-leucine-DL-tryptophan dipeptides
(Fig. 1). An HPLC column used was amylose polysaccharides
(AmyCoat-RP column). The results of these findings are re-
ported herein.
MATERIALS AND METHODS
Chemicals and Reagents
DL-Leucine-DL-tryptophan and its optically active pure stereomers
were purchased from Sean Fisher Peptide 2.0 (Chantilly, VA). Acetoni-
trile and methanol (HPLC grade), glacial acetic acid, and ammonium ac-
etate (AR grade) were purchased from Merck (Mumbai, India).
Deionized water was prepared by a Millipore Milli-Q (Bedford, MA) system.
The solutions (100 μg/mL) of DL-leucine-DL-tryptophan and their
stereomers were prepared in water–methanol-acetonitrile (50:40:10, v/v/v).
Instrumentation
An HPLC system of Shimadzu (Japan) consisting of a solvent delivery
pump (LC-10 AT VP), manual injector, UV-Visb.detector (SPD-10 A),
and Class-VP software was used to carry out the experiments. The col-
umn used was AmyCoat-RP [tris-(3,5-dimethylphenyl carbamate)] (150
x 46 mm id, 5.0 μm), supplied by Kromasil (Bohus, Sweden). A pH meter
(Model APX175 E/C) of Control Dynamics was used.
High-Performance Liquid Chromatography
The experiments were done on HPLC equipment as discussed above.
20.0 μL of each solution of dipeptide and pure stereomers was loaded
onto the HPLC system. The mobile phases used were ammonium acetate
(10 mM)-methanol-acetonitrile (50:5:45,v/v,v). The flow rate, detection
wavelength, and temperature were 0.8 mL/min, 230 nm, and 25 1 °C,
respectively. The mobile phases were filtered through nylon membrane
*Correspondence to: Imran Ali, Department of Chemistry, Jamia Millia Islamia,
Jamia Nagar, New Delhi, 110025, India. E-mail: drimran_ali@yahoo.com;
drimran.chiral@gmail.com
Received for publication 6 May 2016; Accepted 24 June 2016
DOI: 10.1002/chir.22624
Published online in Wiley Online Library
(wileyonlinelibrary.com).
© 2016 Wiley Periodicals, Inc.

ALAJMI ET AL.
Figure 3. A critical inspection of chromatographic parameters
and chromatograms established first-class chiral separation
of all four stereomers of DL-leucine-DL-tryptophan dipeptide.
The individual peak of the separated stereomers was identi-
fied by running pure stereomers under matching experimen-
tal conditions. The order of elution observed was LL > DD-
> DL- > LD-stereomers.
Chiral HPLC Method Optimization
HPLC experimental conditions were optimized using vari-
ous varieties of mobile phases. Ammonium acetate and aceto-
nitrile were used in the different ratios such as 10:90, 20:80,
30:70, 60:40, and 50:50. Likewise, ammonium acetate, metha-
nol, and acetonitrile were used in 50:10:40, 40:30:30, 50:10:40,
50:20:30, etc. ratios. The different concentrations of ammo-
nium acetate ranged from 5 to 50 mM. The alteration in mo-
bile phase flow rates were varied from 0.2 to 2.0 mL/min. It
was observed that the peaks were broad at 0.2 nd 0.5 mL/
min flow rates. On the other hand, the peaks were partially re-
solved at 1.0 and 1.5 mL/min flow rate. Moreover, there was
no separation at 2.0 mL/min flow rate. The detection wave-
lengths varied from 210 to 350 nm. The detection and quanti-
fication limits were poor at higher wavelengths than 20 nm.
The amounts of injection were 5 to 25 μL. The optimization
was ascertained by controlling the temperature from 10 to
50 °C. As a result of comprehensive HPLC experiments, the
best conditions were developed and are reported in this arti-
cle. The chromatographic method was validated using di-
verse variables, viz. linearity, limit of detection (LOD, S/N
1:5), and limit of quantification (LOQ, S /N 1:10), precision,
accuracy, selectivity, robustness, and ruggedness.
Fig. 1. The structures of four stereomers of DL-leucine-DL-tryptophan
dipeptide.
of 25 mm diameter and 0.45 μm pore size and degassed daily before use.
The capacity (k), separation (α), and resolution (Rs) factors were
calculated.
Simulation Studies
For determination of the chiral resolution mechanism, the interactions
of dipeptide stereomers with AmyCoat column [tris-(3,5-dimethylphenyl
carbamate) amylose] chiral selector (Fig. 2) were calculated by modeling
approach. The results of modeling were used to ascertain the chiral reso-
lution mechanism.
Procedure
The modeling studies were done on a computer with configurations of
Intel(R) Core i3 CPU (2.3 GHz) with XP-based operating system (Win-
dows 2003). The Marvin Sketch (5.8.2 version) was adopted to sketch
structures of the stereomers of DL-leucine-DL-tryptophan. The structures
were cleaned to 3D and saved in PDB format. The modeling was done
with AutoDock 4.2 Vina (Scripps Research Institute, La Jolla, CA) and
Pymol software; taking into account all the bonds of ligand as rotatable
and tris-(3,5-dimethylphenyl carbamate) amylose as rigid.15 The X, Y,
and Z axes with 80 × 80 × 60 Å with 0.375 Å spacing were adopted. The
modeling was done by an empirical free energy function and Lamarckian
Genetic Algorithm, with an initial population of 150 randomly placed indi-
viduals. In addition, a maximum number of 2,500,000 energy evaluations,
mutation rate of 0.02, and crossover rate of 0.80 were also used. In this ar-
ticle, a plugin for PyMOL is described, which allowed doing the molecu-
lar docking, virtual screening, and binding site analysis with PyMOL. The
plugin represented an interface between PyMOL and two popular
docking programs, i.e., AutoDock (4.2)16 and AutoDock Vina.17 The com-
bined effect of these two softwares furnished wide use of a Python script
compilation (AutoDock Tools) for the setup of docking runs. Additionally,
Ligplot software was applied for the assessment of hydrophobic interac-
tions. Fifty independent docking runs were applied for each ligand
(stereomer) and tris-(3,5-dimethylphenyl carbamate) amylose for lowest
free energy of binding conformation from the largest cluster, which was
written and saved in PDBQT format. These PDBQT files had been con-
verted to PDB file format.
Simulation Study of Dipeptides on AmyCoat Chiral Selector
The modeling study is one of the most important parapher-
nalia for ascertaining the bindings and mechanism of chiral
separation of the stereomers with the chiral selector. Hence,
efforts were made to ascertain the stereomeric interactions
on a chiral stationary phase. AmyCoat-RP column contains
the tris-(3,5-dimethylphenyl carbamate) amylose chiral selec-
tor. It has different types of functional groups for interacting
stereoselectively with four stereomers. The docking energies
of L-leucine-L-tryptophan, D-leucine-D-tryptophan, D-leucine-
L-tryptophan, and L-leucine-D-tryptophan stereomers were
À3.0, À3.3, À3.9, and À4.2 kcal/mol, respectively (Table 2).
It was due to different hydrogen bondings, hydrophobic inter-
actions, π–π interactions, and steric effects among stereomers
and chiral selectors. The number of hydrogen bonds was one
among each stereomer and chiral stationary phase. These hy-
drogen bonds were found among oxygen atoms of the car-
boxylic group of dipeptide, nitrogen atom of amine group of
dipeptide, and different hydrogen atoms of the chiral selec-
tors. The residues involved in hydrogen bonding of receptors
were UNK236, UNK255, and UNK186. Similarly, the residues
involved in hydrophobic interactions were UNK386 and
UNK403. The 3D docking pose of four stereomers of DL-leu-
cine-DL-tryptophan dipeptide with tris-(3,5-dimethylphenyl
carbamate) amylose chiral selector is shown in Figure 4. It
is clear from this figure that all four stereomers interacted
with tris-(3,5-dimethylphenyl carbamate) amylose chiral se-
lector in a different fashion. Therefore, the elution order of
these stereomers was explained by considering the above-
cited forces among stereomers and chiral stationary phase.
RESULTS AND DISCUSSION
Chromatography
Chiral HPLC parameters viz. retention (k), separation (α),
and resolution (Rs) factors were calculated for the resolved
stereomers of DL-leucine-DL-tryptophan dipeptide on an
AmyCoat-RP column. The values of retention factors for LL-,
DD-, DL-, and LD-stereomers were 2.25, 3.60, 5.00, and 6.50,
respectively. The values of separation and resolution factors
were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19, correspond-
ingly. These values are given in Table 1. A perusal of this ta-
ble indicates that the values of α and Rs were greater than
one, representing baseline separation of all four stereomers.
The chromatogram of the separated stereomers is shown in
Chirality DOI 10.1002/chir

MODELING OF FOUR STEREOMERS
Fig. 2. 2D (a) and 3D (b) structures of tris-(3,5-dimethylphenyl carbamate) amylose chiral selector.
Chirality DOI 10.1002/chir

ALAJMI ET AL.
TABLE 1. Chromatographic parameters for chiral resolution of DL-leucine-DL-tryptophan dipeptide on AmyCoat-RP column
Retention factors (k)
Separation factors (α)
Resolution factors (Rs)
Sl.
No
Dipeptide
k1
k2
k3
k4
α1
1.60
α2
α3
1.30
Rs1
7.76
Rs2
Rs3
7.19
1.
DL-Leucine-DL-tryptophan
2.25
3.60
5.00
6.50
1.39
8.05
k1 = LL-k2 = DD-k3 = DL-k4 = LD-stereomers.
Experimental conditions:
Column: Amycoat-RP (150 x 4.6 mm, 5 μm).
Mobile Phase: Ammonium Acetate (10 mM)-Methanol-Acetonitrile (50:5:45, v/v).
Flowrate: 0.8 mL/min.
Column oven temp: 25 1 °C.
Wavelength: 230 nm.
Sample conc.: 100 μg/mL.
Injection volume: 20 μL.
Diluent: Water–Methanol-Acetonitrile (50:40:10, v/v/v).
Run time: 20 min.
Fig. 3. Chromatogram of the chiral resolution of DL-leucine-DL-tryptophan dipeptide. LL: 6.5, DD: 9.0, DL: 12.0 and LD: 14.3 min.
TABLE 2. Modeling results of different stereomers of DL-leucine-DL-tryptophan dipeptide with tris-(3,5-dimethylphenyl carbamate)
amylose chiral selector
Leucine-
tryptophan
Binding affinity
No. of H
bonds
Residues involved in H-bonding
Residues involved in hydrophobic
interactions
S.NO.
1.
(kcal molÀ1
)
(Bond length)
L-Leu-L-trp
D-Leu-D-trp
D-Leu-L-trp
L-Leu-D-trp
À3.0
1
1
1
1
UNK236/O:: HO17 of NH group(3.6) Unk386:: C3,C13,C15, O2 &O3.
Unk403::N2
Unk386::C3,C5,C6,C8,C9 & C12.
Unk403::C11&C13
Unk386::C11, C14 & C15.
Unk403::C4,C8 &C13
Unk386::C11,C15 & O3.
2.
3.
4.
À3.3
À3.9
À4.2
UNK186/HO4:: O of CO group(3.4)
UNK236/O::HO4 of NH group(3.4)
UNK255/O::HO4 of NH group(3.2)
C = O: Carbonyl group, H; Hydrogen, L: Ligand, N: Nitrogen, O: Oxygen.
The modeling studies results were in agreement with the
chromatographic elution order of the stereomers.
controlled by diverse interactions. The most significant inter-
actions are π–π interactions, hydrogen bondings, van der
Waal’s forces, steric effects, etc.25–29 It is interesting to note
that the reported dipeptide is aromatic, providing π–π interac-
tions with chiral selectors. It is prominent that π–π forces are
stronger than others participating in chiral separation.30–37
Therefore, these forces are accountable for the best chiral
separation of the stereomers. Nevertheless, the other interac-
tions as cited above are also in performance in the chiral sep-
aration of stereomers.
Mechanisms of Resolution at the Supramolecular Level
The chiral separation mechanism was explained by taking
into consideration the docking results and the chromato-
graphic elution order. It is well known that the best chiral se-
lector is derivatized amylase.18–21 AmyCoat-RP column is
based on derivatized amylose polysaccharide (Fig. 2). Amy-
lose is more helical than cellulose and others polysaccha-
rides.22–24 The chiral separation of dipeptide stereomers on
AmyCoat-RP column was due to the attendance of chiral
grooves. The stereomers of these dipeptides set stereospecif-
ically with diverse extents. The setting of the stereomers is
Chirality DOI 10.1002/chir
Validation of Chiral HPLC Method
System suitability test. HPLC suitability test was done using
six replicate injections (n = 6) of the standard solution. The

MODELING OF FOUR STEREOMERS
The LOD and LOQ were calculated as signal-to-noise ratio
of 5 and 10, respectively. The limits of detection of LL-, DD-,
DL-, and LD-stereomers were 2.3, 1.0, 2.0, and 2.7 μg/mL, re-
spectively. The limits of quantitation of LL-, DD-, DL-, and LD-
stereomers were 11.6, 5.6, 10.8, and 14.0 μg/ mL, respec-
tively. The values confirmed satisfactory limits of detection
and quantification.
Precision. The precision of HPLC method was estimated by
considering intraday and interday precision. These are
discussed below.
Intraday and interday precision were determined and the
results are summarized in Tables 4 and 5, respectively. The
intraday precision was estimated within a single day. On the
other hand, the interday precision was estimated on the other
day. The % assay for intraday precisions were calculated for
LL-, DD-, DL-, LD-stereomers. The concentrations of 0.08,
0.10, and 0.12 mg were selected for this. The values for LL-,
DD-, DL-, LD-stereomers for 0.08 mg were 98.9, 99.0, 99.7,
and 99.5, respectively. These values for 0.1 and 0.12 mg were
100.1, 100.6, 100.3, and 99.9, and 101.4, 101.2, 100.2, and
101.0, respectively. The % assay for interday precisions for
LL-, DD-, DL-, and LD-stereomers were calculated for six sam-
Fig. 4. 3D docking pose of four stereomers of DL-leucine-Dr.-tryptophan
dipeptide with tris-(3,5-dimethylphenyl carbamate) amylose chiral selector.
tailing factor, %RSD of the peak area, and %RSD of retention
times were calculated (Table 3). These results showed low
RSD values, <2% for peak area, and <1% for retention time.
The tailing factors of LL-, DD-, DL-, LD-stereomers of DL-ala-
nine-DL-tyrosine were 1.0, 1.21, 1.15, and 1.11, repectively.
TABLE 4. Intraday precision data
0.08 (mg)
0.10 (mg)
0.12 (mg)
Specificity. Specificity of HPLC was done for any interference
of other peak along with main peaks. The diluent was loaded
onto HPLC as a blank solution. No peak was observed from
the blank solution. It indicated the specificity of the system.
% Mean
Assay
% Mean
Assay
% Mean
Assay
%RSD
%RSD
%RSD
LL
98.9
99.0
99.7
99.5
1.3
1.1
0.80
1.2
100.1
100.6
100.3
99.9
0.80
0.40
0.70
0.30
101.4
101.2
100.2
101.0
0.90
0.50
0.60
0.90
DD
DL
LD
Linearity. Linearity was tested by plotting peak area against
diverse concentrations of dipeptide. The slope, y-intercepts,
correlation coefficient (r), and regression coefficient (r2)
were calculated. These values are given in Table 3. The line-
arity range was good for both the dipeptides in a concentra-
tion range of 50.0–400 μg/mL for this dipeptide. The
regression coefficients (r2) were 0.9985, 0.9967, 0.9989, and
0.9980 for LL-, DD-, DL-, and LD stereomers.
TABLE 5. Interday precision data
Samples
LL
DD
DL
LD
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
% RSD
100.7
100.9
100.2
99.7
99.4
100.8
99.4
99.2
99.6
99.3
99.9
99.4
0.25
100.6
100.2
100.4
100.1
100.5
99.9
98.2
98.8
98.4
98.2
99.1
99.5
0.54
Limit of detection (LOD) and quantification (LOQ). The LOD
and LOQ were calculated as a signal-to-noise ratio of 5 and
10, respectively. The limits of detection of LL-, DD-, DL-,
and LD-stereomers were 2.3, 1.0, 2.0, and 2.7 μg/mL, respec-
tively. The limits of quantitation of LL-, DD-, DL-, and LD-
stereomers were 11.6, 5.6, 10.8, and 14.0 μg/ LOD and LOQ.
0.62
0.26
TABLE 3. System suitability and linearity parameters
DL-leucine-DL-tryptophan
HPLC method
TABLE 6. Percentage recovery data for dipeptide
Parameters
LL
DD
DL
LD
Level
LL
DD
DL
LD
System suitability
Tailing factor
% RSD peak area
% RSD retention time
Linearity
Corr. coeff. (r)
Regression coeff. (r2)
LOD (μg/mL)
LOQ (μg/mL)
0.08
0.08
0.08
0.1
0.1
0.1
0.12
0.12
0.12
99.1
99.5
99.0
100.4
100.8
100.3
99.4
98.6
98.0
98.3
100.5
100.1
99.8
100.4
99.5
99.5
99.8
99.2
100.0
98.6
99.8
100.5
100.9
101.5
98.9
99.6
98.8
100.6
100.3
100.1
99.9
1.00
1.0
0.94
1.21
0.30
0.80
1.15
0.90
0.98
1.11
0.50
0.90
0.9993
0.9985
2.3
0.9999
0.9967
1.0
0.9994
0.9989
2.0
0.9994
0.9980
2.7
99.6
99.9
99.3
99.8
11.6
5.6
10.8
14.0
99.8
Chirality DOI 10.1002/chir

ALAJMI ET AL.
TABLE 7. Robustness data for chiral HPLC
LL-
DD-
DL-
LD-
Parameters
RT
Area
RT
Area
RT
Area
RT
Area
Flowrate (0.8 mL/min.)
Flowrate (0.5 mL/min.)
Column temp. (25 °C).
Column temp. (20 °C).
0.55
0.71
1.00
0.90
0.78
0.70
0.60
0.95
0.68
0.75
1.02
0.95
0.87
0.80
0.73
1.16
0.62
0.75
1.10
1.00
0.83
0.84
0.77
1.16
0.66
0.67
1.15
1.05
0.93
0.86
1.00
1.20
biomarkers for discrimination among different forms of liver disease. J
Hepatol 2011; 55: 896–905.
8. Ali I, Al-Othman ZA, Alwarthan A, Asnin L, Chudinov A. Advances in chi-
ral separations of small peptides by capillary electrophoresis and chroma-
tography. J Sep Sci 2014; 37: 2447–2466.
ples. The values were in the range of 99.4–100.9, 99.2–99.9,
99.9–100.6, and 98.2–99.5, respectively. The % RSD for all
the assays in intraday precision were 0.62, 0.25, 0.26, and
0.54 for LL-, DD-, DL-, LD-stereomers, respectively.
9. Yagasaki M, Hashimoto S. Synthesis and application of dipeptides; cur-
rent status and perspectives. Appl Microbiol Biotechnol 2008; 81: 13–22.
Recovery. The recovery was estimated at three diverse
spiked concentrations (250–350 μg/mL). The results are
given in Table 6. The % recoveries were ranged between
98–100.9% for all four stereomers.
10. Ali I, Sahoo DR, Al-Othman ZA, Alwarthan A, Asnin L, Larsson B. Vali-
dated chiral HPLC separation method and simulation studies of dipep-
tides on amylose chiral column. J Chromatogr A 2015; 1406: 201–209.
11. Aboul-Enein HY, Ali I. Studies on the effect of alcohols on the chiral dis-
crimination mechanisms of amylose stationary phase on the
enantioseparation of nebivolol by HPLC. J Biochem Biophys Methods
2001; 48: 175–188.
12. Ali I, Saleem K, Hussain I, Gaitonde VD, Aboul-Enein HY. Polysaccha-
rides chiral stationary phases in liquid chromatography. Sep Purif Rev
2009; 38: 1–51.
Robustness. The robustness of chiral HPLC was estimated
by slightly changing one parameter at a time while keeping
the others steady and observing the changes in the chromato-
grams, which may influence the performance of chiral HPLC.
The results of flow rate and temperature changes are given in
Table 7. The changes in retention time (Rt) and peak area
were <1.5, confirming the robustness of HPLC.
13. Ali I, Gupta VK, Aboul-Enein HY, Singh P, Sharma B. Role of racemization
in optically active drugs development. Chirality 2007; 19: 453–463.
14. Ali I, Naim L, Ghanem A, Aboul-Enein HY. Role of racemization in opti-
cally active drugs development. Talanta 2006; 69: 1013–1017.
CONCLUSION
15. Ali I, Al-Othman ZA, Alwarthan A, Alam SD, Farooqi JA. Enantiomeric
separation and simulation studies of pheniramine, oxybutynin, cetirizine,
and brinzolamide chiral drugs on amylose based columns. Chirality
2014; 26: 136–143.
16. Huey R, Morris GM, Olson AJ, Goodsell DS. A semiempirical free energy
force field with charge-based desolvation. J Comput Chem 2007; 6:
1145–1152.
The reported chiral HPLC method for separation of four
stereomers of DL-leucine-DL-tryptophan was reproducible,
accurate, inexpensive, and effective. The limits of detections
and quantitation ranged from 1.0–2.3 and 5.6–14.0 μg/mL.
The simulation studies established the elution orders and
the mechanism of chiral recognition. It was seen that π–π con-
nections and hydrogen bondings were the main forces for en-
antiomeric resolution. The reported chiral HPLC method may
be applied to the enantiomeric separation of DL-leucine-DL-
tryptophan in unknown matrices.
17. Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of
docking with
a new scoring function, efficient optimization, and
multithreading. J Comput Chem 2010; 2: 455–161.
18. Morris GM, Goodsell GS, Halliday RS, Huey R, Hart WE, Belew RK,
Olson AJ. Automated docking using a Lamarckian genetic algorithm
and an empirical binding free energy function. J Comput Chem 1998;
19: 1639–1662.
19. Ali I, Alam SD, Al-Othman ZA, Farooqi JA. Recent advances of chiral
drugs by SPE-HPLC in biological samples. J Chromatogr Sci 2013; 51:
645–654.
ACKNOWLEDGMENTS
The authors are thankful to the Deanship of Scientific Re-
search at King Saud University, Riyadh, Saudi Arabia for its
funding to this research group No. RGP-150.
20. Aboul-Enein HY, Ali I. Applications of polysaccharide based chiral station-
ary phases for resolution of different compound classes. Methods Mol
Biol 2004; 243: 183–196.
21. Aboul-Enein HY, Ali I. Optimization strategies for HPLC
enantioseparation of racemic drugs using polysaccharides and macrocy-
clic glycopeptide antibiotic chiral stationary phases. Farmaco 2002; 57:
513–529.
LITERATURE CITED
1. Aboul-Enein HY, Ali I. Chiral separations by liquid chromatography and
related technologies. New York: Marcel Dekker; 2003.
2. Simonyi M, Fitos I, Visy J. Chirality of bioactive agents in protein binding
storage and transport processes. Tips 1986; 7: 112–116.
22. Ali I, Kümmerer K, Aboul-Enein HY. Mechanistic principles in chiral sep-
arations using LC and CE. Chromatographia 2006; 63: 295–307.
3. FDA policy statement for the development of new stereomeric drugs. Chi-
rality 1992; 4: 338–340.
23. Ali I, Sanagi MM, Aboul-Enein HY. Advances in chiral separations by non-
aqueous capillary electrophoresis in pharmaceutical and biomedical anal-
ysis. Electrophoresis 2014; 35: 926–936.
4. Ali I, Gaitonde VD, Aboul-Enein HY, Hussain A. Chiral separation of β-
adrenergic blockers on cellucoat column by HPLC. Talanta 2009; 78:
458–463.
24. Ali I, Hussain A, Saleem K. Determination of stereo-selective bindings of
racemic propranolol with β2-AD-GPCR in human plasma.
Chromatogr Rel Technol 2013; 36: 792–806.
25. Aboul-Enein HY, Ali I. HPLC enantiomeric resolution of nebivolol on nor-
mal and reversed amylose based chiral phases. Pharmazie 2001; 56:
214–216.
26. Ali I, Aboul-Enein HY. Enantioseparation of some clinically used drugs by
HPLC using cellulose tris-(3, 5-dichlorophenylcarbamate) chiral station-
ary phase. Biomed Chromatogr 2003; 17: 113–117.
J Liq
5. Prasad C. Bioactive cyclic dipeptides. Peptides 1995; 16: 151–164.
6. Humphrey MJ, Ringrose PS. Peptides and related drugs: A review of their
absorption, metabolism, and excretion. Drug Metab Rev 1986; 17:
283–310.
7. Soga T, Sugimoto M, Honma M, Mori M, Igarashi K, Kashikura K, Ikeda
S, Hirayama A, Yamamoto T, Yoshida H, Otsuka M, Tsuji S, Yatomi Y,
Sakuragawa T, Watanabe H, Nihei K, Saito T, Kawata S, Suzuki H, Tomita
M, Suematsu M. Serum metabolomics reveals γ-glutamyl dipeptides as
Chirality DOI 10.1002/chir

MODELING OF FOUR STEREOMERS
27. Aboul-Enein HY, Ali I. Comparative study of the enantiomeric resolution
32. Ali I, Saleem K, Gaitonde VD, Aboul-Enein HY, Hussain I. Chiral separa-
tions of some β-adrenergic agonists and antagonists on AmyCoat column
by HPLC. Chirality 2010; 22: 24–28.
33. Ali I, Al-Othman ZA, Hussain A, Saleem K, Aboul-Enein HY. Chiral sepa-
ration of β-adrenergic blockers in human plasma by SPE-HPLC.
Chromatographia 2011; 73: 251–256.
34. Ali I, Hussain I, Saleem K, Aboul-Enein HY. Enantiomeric resolution of
ibuprofen and flurbiprofen in human plasma by SPE-chiral HPLC
methods. Comb Chem High Througput Screen 2012; 15: 509–514.
35. Al-Othman ZA, Ali I. Rapid and economic chiral-HPLC method of
nebivolol enantiomers resolution in dosage formulation. Biomed
Chromatogr 2012; 26: 775–778.
of chiral antifungal drugs econazole, miconazole and sulconazole by
HPLC on various cellulose chiral columns in normal phase mode. J Pharm
Biomed Anal 2002; 27: 441–446.
28. Aboul-Enein HY, Ali I, Gübitz G, Simons C, Nicholls PJ. HPLC enantio-
meric resolution of novel aromatase inhibitors on cellulose-and amylose-
based chiral stationary phases under reversed phase mode. Chirality
2000; 12: 727–733.
29. Aboul-Enein HY, Ali I. Comparison of the chiral resolution of econazole,
miconazole, and sulconazole by HPLC using normal-phase amylose CSPs.
Fres J Anal Chem 2001; 370: 951–955.
30. Aboul-Enein HY, Ali I, Laguerre M, Felix G. Molecular modeling of enan-
tiomeric resolution of methylphenidate on different polysaccharide based
36. Ali I, Haque A, Hussain A, Sanagi MM, Hussain I, Aboul-Enein HY. Su-
pramolecular chiro-biomedical aspect of β-blockers in drug development.
Curr Drug Target 2014; 15: 729–741.
chiral stationary phases.
J Liq Chromatogr Rel Technol 2002; 25:
2739–2748.
31. Ali I, Hussain A, Aboul-Enein HY, Bazylak G. Supramolecular systems
based HPLC for chiral separation of β-adrenergics and β-adrenolytics in
drug discovery scheme. Curr Drug Discov Technol 2007; 4: 255–274.
37. Al-Othman ZA, Al-Warthan A, Ali I. Advances in enantiomeric resolution
on chiral monolithic phases in liquid chromatography and
electrochromatography. J Sep Sci 2014; 37: 1033–1057.
Chirality DOI 10.1002/chir