HPLC-fluorescence method for the enantioselective analysis of propranolol in rat serum using immobilized polysaccharide-based chiral stationary phase

Article abstract of DOI:10.1002/chir.22296

A stereoselective high-performance liquid chromatographic (HPLC) method was developed and validated to determine S-(-)- and R-(+)-propranolol in rat serum. Enantiomeric resolution was achieved on cellulose tris(3,5- dimethylphenylcarbamate) immobilized onto spherical porous silica chiral stationary phase (CSP) known as Chiralpak IB. A simple analytical method was validated using a mobile phase consisted of n-hexane-ethanol-triethylamine (95:5:0.4%, v/v/v) at a flow rate of 0.6 mL min^-1 and fluorescence detection set at excitation/emission wavelengths 290/375 nm. The calibration curves were linear over the range of 10-400 ng mL^-1 (R = 0.999) for each enantiomer with a detection limit of 3 ng mL^-1. The proposed method was validated in compliance with ICH guidelines in terms of linearity, accuracy, precision, limits of detection and quantitation, and other aspects of analytical validation. Actual quantification could be made for propranolol isomers in serum obtained from rats that had been intraperitoneally (i.p.) administered a single dose of the drug. The proposed method established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology. Molecular modeling studies including energy minimization and docking studies were first performed to illustrate the mechanism by which the active enantiomer binds to the β-adrenergic receptor and second to find a suitable interpretation of how both enantiomers are interacting with cellulose tris(3,5-dimethylphenylcarbamate) CSP during the process of resolution. The latter interaction was demonstrated by calculating the binding affinities and interaction distances between propranolol enantiomers and chiral selector. Chirality 26:194-199, 2014. 2014 Wiley Periodicals, Inc. Copyright

Full text of DOI:10.1002/chir.22296

CHIRALITY 26:194–199 (2014)
HPLC-Fluorescence Method for the Enantioselective Analysis of
Propranolol in Rat Serum Using Immobilized Polysaccharide-Based
Chiral Stationary Phase
1
1
1
1
*
AMER M. ALANAZI, MOHAMED M. HEFNAWY, ABDULRAHMAN A. AL-MAJED, AYMEN K. AL- SUWAILEM,
MOHAMED G. KASSEM,1 GAMAL A. MOSTAFA,1 SABRY M. ATTIA,2 AND MOHAMMED M. KHEDR3
1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
2Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
3Welsh School of Pharmacy, Cardiff University, UK
ABSTRACT
A stereoselective high-performance liquid chromatographic (HPLC) method
was developed and validated to determine S-(À)- and R-(+)-propranolol in rat serum. Enantio-
meric resolution was achieved on cellulose tris(3,5-dimethylphenylcarbamate) immobilized onto
spherical porous silica chiral stationary phase (CSP) known as Chiralpak IB. A simple analytical
method was validated using a mobile phase consisted of n-hexane-ethanol-triethylamine
(95:5:0.4%, v/v/v) at a flow rate of 0.6 mL min-1 and fluorescence detection set at excitation/emission
wavelengths 290/375 nm. The calibration curves were linear over the range of 10–400 ng mL-1
(R = 0.999) for each enantiomer with a detection limit of 3 ng mL-1. The proposed method was vali-
dated in compliance with ICH guidelines in terms of linearity, accuracy, precision, limits of detection
and quantitation, and other aspects of analytical validation. Actual quantification could be made for
propranolol isomers in serum obtained from rats that had been intraperitoneally (i.p.) administered
a single dose of the drug. The proposed method established in this study is simple and sensitive
enough to be adopted in the fields of clinical and forensic toxicology. Molecular modeling studies in-
cluding energy minimization and docking studies were first performed to illustrate the mechanism by
which the active enantiomer binds to the β-adrenergic receptor and second to find a suitable interpre-
tation of how both enantiomers are interacting with cellulose tris(3,5-dimethylphenylcarbamate) CSP
during the process of resolution. The latter interaction was demonstrated by calculating the binding
affinities and interaction distances between propranolol enantiomers and chiral selector. Chirality
26:194–199, 2014. © 2014 Wiley Periodicals, Inc.
KEY WORDS: propranolol; enantioselective; Chiralpak IB; HPLC-FD; molecular modeling
INTRODUCTION
These CSPs are characterized by their facile use, high
enantioselectivity, and high capacity.4 Many factors can be
responsible for the extent of interactions of stereoisomeric
molecules with immobilized polysaccharide-based chiral sta-
tionary phase such as dipole–dipole interactions, electrostatic
forces, hydrogen bonding, hydrophobic bonding, ion–dipole
interactions, steric interferences (size, orientation, and
spacing of groups), and Van der Waals forces. The nature
and effects of some of these factors can influence the chroma-
tography of enantiomers.5,6
Molecular modeling studies have succeeded in interpreting
many ligand-receptor mechanisms depending on the basic
interactions that occur between the ligand and its site of
action. Moreover, in addition, some other factors such as
the binding affinity of the ligand or receptor strain have been
demonstrated. They help to interpret the mechanism by
which the separation of enantiomers takes place and the
reason for the potent biological activity of one enantiomer
over the other.7,8 The analytical methods reported for chiral
separation of propranolol included capillary electrophoresis
The use of a fluorescence detector coupled with high-
performance liquid chromatography (HPLC) technique has
allowed increased sensitivity and selectivity for the determina-
tion of propranolol enantiomers. To the best of our knowledge,
the combinations of Chiralpak IB- HPLC-fluorescence (FL)
detection with pharmacokinetic study have not been repor-
ted in single propranolol enantiomers analysis. Propranolol,
1[Àisopropylamine-3-[1-naphthyloxy]-2-propanol] hydrochloride,
is a nonselective βadrenergic antagonist (β-blocker), which
is widely used in the treatment of several diseases such as car-
diac arrhythmias, angina pectoris, sinus tachycardia, thyrotox-
icosis, hypertrophic subaortic stenosis, and hypertension.1
Propranolol has one chiral center and is administered as a race-
mic mixture. It is reported that the S-(À)- isomer is 100 times
more potent as a β-blocking agent than the R-(+)-isomer.1 Re-
cently, it has been reported that a long term use of propranolol
may cause the hypertensive patients to be a diabetic.2
Polysaccharide derivatives, being coated or immobilized on
silica matrix, have become the first and broadest choice of
selectors to be used as chiral stationary phases (CSPs) for both
liquid and supercritical liquid chromatography.3 Since 2004 three
immobilized polysaccharide-derived CSPs have become com-
mercially available: Chiralpak IA, Chiralpak IB, and Chiralpak
IC. They are based on tris(3,5-dimethylphenylcarbamate) of
amylose, tris(3,5-dimethylphenylcarbamate) of cellulose, and
tris(3,5-dichlorophenylcarbamate) of cellulose, respectively.
© 2014 Wiley Periodicals, Inc.
*Correspondence to: M. Hefnawy, Department of Pharmaceutical Chemistry,
College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451,
Saudi Arabia. E-mail: mhefnawy2003@yahoo.com
Received for publication 18 April 2013; Accepted 18 November 2013
DOI: 10.1002/chir.22296
Published online 3 March 2014 in Wiley Online Library
(wileyonlinelibrary.com).

ENANTIOSELECTIVE ANALYSIS OF PROPRANOLOL
195
methods9–11 and HPLC methods utilizing various chiral selec-
Propranolol Treatment and Serum Sampling
tor CSPs.12–15 However, the reported capillary electrophore-
sis (CE) methods were concerned with the evaluation of
different chiral selectors for resolution of propranolol enantio-
mers using UV detection. Moreover, the chromatography
methods cited for the chiral separation and determination of
propranolol in biological fluids have drawbacks of low sensi-
tivity (UV detection) and deficiency of pharmacokinetic
study. The main advantages of the developed HPLC-FL
method with Chiralpak IB column are to provide a highly sen-
sitive and selective method for the simultaneous analysis of
propranolol enantiomers in rat serum. The developed method
was of high precision, good accuracy, wide linear range of
determination, and lower limit of detection of 3.0 ng mL-1.
The developed method was demonstrated to be applicable
for conducting pharmacokinetic study. The effective separa-
tion of both S-(À)- and R-(+)-enantiomers of propranolol was
studied by a molecular modeling technique, where the
hydroxyl group of propranolol was found to be crucial in
determining the stereochemistry of the structure.
Adult Wistar albino male rats weighing 150–200 g (10–12 weeks old)
were obtained from the Experimental Animal Care Center, College of
Pharmacy, King Saud University. All animal procedures were performed
in accordance with the NIH guidelines and approved by the Ethics Com-
mittee of the Experimental Animal Care Society, College of Pharmacy,
King Saud University, Riyadh, Saudi Arabia. All animals were allowed to
acclimatize in metal cages inside a well-ventilated room for 2 days prior
\to the experiment. The animals were maintained under standard laboratory
conditions (a temperature of 20 3˚C, a relative humidity of 55 10%, and a
12-h light/dark cycle) and were fed a diet of standard commercial pellets
and water ad libitum. The animals were randomly divided into six groups
consisting of five rats each. Five groups were intraperitoneally (i.p.) injected
with propranolol (40 mg kg-1), and the rest group was i.p. injected with
normal saline and considered as the control group to provide the blank rat
serum. Blood samples from propranolol-treated groups were collected
under light ether anesthesia from the orbital plexus 1, 2, 3, 4, and 5 h after
injection. All blood samples were centrifuged (3000 rpm at 4˚C) for 10min
to obtain the serum. The serum samples were placed on ice for immediate
use or stored at À20˚C until analysis.
Assay Method
Assay of rat serum was performed by placing a 300 μL serum into 1.5-mL
Eppendorf tube and accurately measured aliquots of 15, 100, 160μL of the
individual working standard of S-(À)- and R-(+)-propranolol solutions were
added. The internal working standard solution of 25μL was then added to
each tube and sonicated for 5 min then diluted to 1 mL with acetonitrile to
give final concentration of 30, 200, and 320ngmL-1 for each enantiomer.
Each tube was vortexed for 5 min, then centrifuged at 10,000rpm for
5 min. The supernatant solution was evaporated to dryness under gentle
air then reconstituted by mobile phase to 1 mL, sonicated for 5 min, filtered
if necessary through Millipore membrane filter (0.2 μm), then 20μL of the
final solution was injected into the HPLC system. Blank rat serum sample
were processed by the same procedures using acetonitrile instead of
propranolol enantiomers. The absolute recoveries of each enantiomer from
serum was calculated by comparing drug peak area of the spiked analyte
samples to unextracted analyte of stock solution, which had been injected
directly into the HPLC system. Calibration curves were constructed by
diluting stock solutions with pooled rat serum to yield six concentration
points over the range of 10–400 ngmL-1 for each propranolol enantiomer.
EXPERIMENTAL
Apparatus and Reagents
Chromatography was performed on a Shimadzu (Japan) instrument
consisting of one LC–20 AD pump, DGU–20 A3 / DGU–20 A5 on-line
degasser, SIL-20A/20 AC autosampler, RF-10 AXL fluorescence detector,
and CBM–20A system controller. The chiral stationary phase used in this
study was the cellulose tris(3,5-dimethylphenylcarbamate) which is immo-
bilized on 5-μm silica gel known as Chiralpak IB (250 x 4.6 mm i.d.) pur-
chased from Chiral Technologies Europe (Cedex, France). The mobile
phase consisted of n-hexane–ethanol–triethylamine (95:5:0.4%, v/v/v),
which was filtered through a Millipore membrane filter (0.2 μm) from
Nihon, Millipore (Japan), and degassed before use. The flow rate was
0.6 mL min-1 with fluorescence excitation wavelength 290 nm and emis-
sion wavelength 375 nm. ( ) Propranolol, S-(À)- and R-(+)-propranolol
were purchased from Sigma (St. Louis, MO). The internal standard
NAN-190 (1-(2-Methoxyphenyl)-4-(4-phthalimidobutyl)piperazine) is the com-
pound with a selective 5–HT1A receptor antagonistic activity purchased from
Sigma. HPLC-grade n-hexane, ethanol, and analytical grade triethylamine
were purchased from BDH Chemicals (UK). Deionized water was purified
using a cartridge system (Picotech Water Systems RTP, USA). Adult male
Wistar rats were obtained from Experimental Animal Care Center, College
of Pharmacy, King Saud University, Riyadh, Saudi Arabia.
Linearity, Precision, and Accuracy
Linear regression analysis of normalized drug/internal standard peak
area ratio versus concentration gave slope and intercept data for each
analyte, which were used to calculate the concentration of each analyte
in the serum samples. Calibration standards at each concentration were
analyzed in six replicates. The within-run and between-run precision
(reported as RSD, %) and accuracy (reported as relative error, %) of the
assay in serum were determined by assaying six quality control samples
over a period of 3 d. The concentration represented the entire range of
the calibration curve. The lowest level was at 3 times the expected limit
of quantitation (LOQ) for each enantiomer. The second level was the
midpoint of the calibration curve and the third level was 80% of the upper
concentration. The regression equations were used to determine the
concentrations in the quality control samples.
Preparation of Stock and Standard Solutions
Stock solutions containing 1 mg mL-1 of individual S-(À)- and R-(+)-
propranolol were prepared in methanol. Working standard solutions
(2 μg mL-1) were prepared by dilution of an individual aliquot of stock
solution with the same solvent. The internal standard NAN–190 was
prepared in methanol to give a concentration of 0.4 mg mL-1 and was
further diluted with methanol to give a working solution of 40 μg mL-1.
The solutions were stable for at least 7 days if kept in the refrigerator.
Appropriate dilutions of the individual working solutions of propranolol
and internal standard were made and used for constructing the calibra-
tion curves and spiking the rat serum.
MOLECULAR MODELING STUDIES
Propranolol– β2-Adrenergic Receptor Docking
Docking was done by Autodock Vina16 since Autodock
tools serve for visualization and measuring the distances of
interactions.17 Autodock Vina is new software aiming to
improve the accuracy of the binding mode. It can predict
the binding affinity of the ligand (Kcal mol-1) that can be used
for ranking of the resulting poses to predict which conforma-
tion could be the best for fitting and interactions. The crystal
structure of the human β2-adrenergic G-protein-coupled
receptor complexed with carazolol, one of the best known
Chirality DOI 10.1002/chir
Preparation of Standard Serum Sample
The quality control (QC) samples at three concentrations 30, 200, and
320 ng mL-1 were prepared by spiking the drug-free rat serum with appro-
priate volumes of individual S-(À)- and R-(+)-propranolol and stored
frozen until analysis. Before spiking, the drug-free serum was tested to
make sure that there were no endogenous interferences at the retention
times of S-(À)- and R-(+)- propranolol as well as the retention time of the
internal standard.

196
ALANAZI ET AL.
TABLE 1. Chromatographic parameter data for propranolol
enantiomers and NAN-190 as internal standard
β2-blockers, was downloaded from the protein data bank
(pdb code = 2RH1) and saved as pdbqt format after building
the Grid box.[18 This crystal structure gave us the chance
to focus on the site in which carazolol was complexed,
and helped in identification of residues that may be in-
volved in the drug–receptor interactions. Both S-(À)-
and R-(+)-enantiomers of propranolol were built, polar hy-
drogen’s were added, minimized, and saved as pdbqt. A
grid box was built to include all these residues. The sim-
ilarity in structure between carazolol and propranolol, es-
pecially in their side-chains, was shown by alignment of
the two structures, which confirms the similarity in steric
and electrostatic features and probability of same mode of inter-
action. After docking of both S-(À)- and R-(+)-enantiomers of
propranolol, visualizations with Autodock tools were performed
to find a reasonable mechanism by which the S-(À)-enantiomer
could be more potent than the R-(+)-enantiomer. Distances of
interactions were calculated and used with the binding affini-
ties for our interpretation.
a
d
TR
Analyte
Rs
αb
Kc
S-(À)-Propranolol
NAN–190 (IS)
R-(+)-Propranolol
e
3.37
2.74
e
1.24
1.16
2.72 0.04
3.36 0.05
3.88 0.06
19.47 0.06
23.32 0.07
25.55 0.09
aRs = (t2-t1)/0.5 (W2 + W1), where t2 and t1 are the retention of the second and
the first peaks while W2 and W1 are the base peak width of the second and
first peaks.
bSeparation factor, calculated as k2/k1, where k = (tR-t0)/ t0, where tR is the
retention of analyte and t0 is the retention of solvent.
cCapacity factor, where k = (tR-t0)/ t0, where tR is the retention of analyte and t0
is the retention of solvent.
dTR is the retention time, mean + SD, n = 10.
eNot calculated.
Method Validation
A specific method can accurately measure the analyte of
interest even in the presence of potentially sample compo-
nents (sample matrix). A major objective of determining spec-
ificity is to ensure "peak purity" of the main compound to be
determined. Figure. 1A,B shows the chromatograms of a
blank serum sample and the run of serum sample spiked with
1.0 μg mL-1 IS and 10 ng mL-1 of propranolol enantiomers,
respectively, under the optimized conditions. The comparison
of analyte standard solution chromatograms to those of a blank
serum sample (spiked with the same standard solutions)
showed that the matrix effect was minimal. Therefore, the
deproteinization procedure by acetonitrile ensured that there
was no matrix effect between serum samples. No impurity
peaks were overlapped with the peaks of propranolol enantio-
mers and IS. These data collectively and evidently indicated
the specificity of the developed method for the determination
of targeted drug. A good linearity relationship was demonstrated
Propranolol–Cellulose Tris(3,5-Dimethylphenylcarbamate)
CSP Docking
Cellulose
tris(3,5-dimethylphenylcarbamate)
was
constructed from two molecules and minimized to be used
for docking. Energy minimization was done in order to relax
the whole structure and to reduce the strain. Swiss pdb
viewer software was used for the minimization in which the
number of steepest descent = 20 steps, cutoff = 10,000 Å, and
all atoms were selected to be minimized. GROMOS 43B1 is
the force field that is used for minimization, then molecular
docking for both the S-(À)- and R-(+)-enantiomers was done
separately.
RESULTS AND DISCUSSION
Optimization of the Chromatographic Conditions
The resolution of propranolol enantiomers is affected by
several factors. One of these factors is the type of mobile
phase and its components plus the ratio of these components.
The nature of the mobile phase could affect enantioselectivity,
capacity factor, and resolution degree as well as the CSP
stability and column life span, particularly with that based
on polysaccharide derivates. In this study different solvents
(standards and nonstandards)4 and different ratios of these
solvents with or without additive were tested. Finally, the
suitable mobile phase chosen consisted of n-hexane-ethanol-
triethylamine (95:5:0.4%, v/v/v) which gave good resolution
(Rs = 3.37) for both isomers of propranolol (Table 1). The
importance of ethanol in this mobile phase is to improve peak
shape, to shorten retention time and enhance selectivity.
However, further increase in the ethanol ratio in the mobile
phase cause a dramatic decrease in the retention time of the
drug which may be related to hydrogen bonding between
both enantiomers of propranolol and CSP.[4 On the other
hand, addition of 0.4% (TEA) to the mobile phase plays an
essential role for the separation of propranolol isomers
through suppression of the deleterious effect of residual
free silanols on the silica surface which consequently im-
prove the peak symmetry, resolution, and selectivity.19 It
is known that interactive forces such as π–π interactions,
van der Waals forces, and hydrogen bonding may be re-
sponsible for the chiral resolution of propranolol isomers
with Chiralpak IB column.4
Fig. 1. Chromatograms of: (A) blank rat serum and (B) blank rat serum
spiked with 10 ng mL-1 of S-(À)-propranolol (I), R-(+)-propranolol (III), and
1 μg mL-1 of NAN–190 (IS) (II). Chromatographic system column, Chiralpak
IB (250
x 4.6 mm i.d.), Mobile phase consisted of n-hexane-ethanol-
triethylamine (95:5:0.4 %, v/v/v) at a flow rate of 0.6 mL min-1 and fluorescence
detection set at excitation/emission wavelengths 290/375 nm. Column tem-
perature, ambient.
Chirality DOI 10.1002/chir

ENANTIOSELECTIVE ANALYSIS OF PROPRANOLOL
197
TABLE 3. Accuracy and precision data for propranolol
enantiomers in spiked rat serum
between peak area ratios of propranolol enantiomers and
IS corresponding to serum concentrations over a range
of 10–400 ng mL-1 for both enantiomers. The mean linear
regression equation of the peak ratio (y) versus enantiomer
concentration (ng mL-1) in rat serum samples (×) showed
correlation coefficients (R) = 0.9995, y = 0.006× + 0.023 for
S-(À)-propranolol and y = 0.007× + 0.021 for R-(+)-propranolol,
respectively. The good linearity of the calibration graphs and
negligible scatter of experimental points are evident by the
values of correlation coefficient and confidence intervals of
the slope and intercept of the obtained data (Table 2).20 The
sensitivity was expressed as LOQ and limit of detection
(LOD). LOQ is the injected amount that results in a peak with
a height at least 10 times as high as the baseline noise level,
and the LOD as peak height to base line ratio of 3:1.21 Average
values of LOD and LOQ of propranolol enantiomers in spiked
rat serum samples were found to be 3.0 ngmL-1 and 10 ngmL-
1, respectively (Table 2). Blank serum was spiked with standard
solution of propranolol enantiomers at 30, 200, 320 ngmL-1 and
then 40 μg mL-1 of IS was added with the reconstitution step in
the assay method. The sample used for the recovery runs was
prepared as described above. The overall accuracy was
assessed by the relative percentage error, which ranged from
À5.2 to À2.5 for S-(À)-enantiomer and from À6.6 to À2.6% for
R-(+)-enantiomer. The negative of the relative percentage error
clearly exhibited that recoveries of both propranolol enantio-
mers were higher than 93.5% and less than 97.5%, indicating
good overall accuracy. The intraday values of RSD were calcu-
lated based on six replicate runs of three different concentra-
tions in 1 d; the interday values of RSD were calculated by
using three sets from the same three standard solutions
obtained in 5 different days. Results presented in Table 3 indi-
cate that intra-assay RSD of 0.8–1.1% for S-(À)-enantiomer and
0.8–1.2% for R-(+)-enantiomer, while the inter-assay RSD for
both enantiomers was 0.9–1.3 %.
Actual
Found
concentration
concentration RSD Error
Analyte
(ng mL-1)
(ng mL-1)
(%)c
(%)d
Within-daysa
S-(À)- propranolol
30.0
200.0
320.0
30.0
200.0
320.0
30.0
200.0
320.0
30.0
200.0
320.0
28.9 0.2
0.69 À3.57
1.10 À4.52
0.89 À2.46
0.70 À5.06
1.15 À4.82
0.93 À2.59
1.05 À4.80
1.32 À5.23
0.91 À3.43
1.06 À6.06
1.28 À6.55
0.93 À2.00
191
312
2
3
R-(+)-propranolol
28.5 0.2
190
312
2
3
Between-daysb
S-(À)- propranolol
28.6 0.3
190
309
3
3
R-(+)-propranolol
28.2 0.3
187
311
2
3
aMean SD based on n = 6.
bMean SD based on n = 5.
cExpressed as %RSD: (SD/ mean) x 100.
dCalculated as (mean determined concentration / nominal concentration) x 100.
cartridges (55 μm, 70 Å, 100 mg, 1.0 mL), unknown peaks
were observed and specificity of such methods is poor. A
number of solvents (acetonitrile, ethanol, isopropanol, and
trifluoroacetic acid) and a mixture of these solvents were evalu-
ated as deproteinized solvents. Acetonitrile was selected since
it provided the best data in terms of sample cleanup and high
recoveries of both propranolol enantiomers. The mean recover-
ies using acetonitrile were 96.2 1.2 for S-(À)- propranolol and
95.3 1.5 for R-(+)- propranolol (n = 5).
Pharmacokinetic Study
The described method was further applied to a pharmacoki-
netic study of both propranolol enantiomers in rats. Serum
was collected 1, 2, 3, 4, and 5 h after propranolol treatment.
The serum concentration-time curve (AUC) for propranolol
enantiomers is presented in Figure 2, which shows the mean
values of tmax and cmax were 1 h and 755.76 18.97 ng mL-1,
respectively, for S-(À)-propranolol, while for R-(+)-propranolol
the main values of tmax and cmax were 1 h and 362.22
12.93 ng mL-1. The variation in plasma level between the two
Optimization of Rat Serum Sample Procedure
Prior to the determination of propranolol enantiomers in
rat serum samples using the optimized HPLC conditions
obtained, a serum sample pretreatment step had to be
employed. Although many of the available SPE cartridges
have been employed for the extraction of propranolol iso-
mers including C8, C18, phenyl, CN, SCX, and WCX silica
TABLE 2. Validation parameters for the determination of
propranolol enantiomers using the proposed HPLC method
Parameters
S-(À)-propranolol
R-(+)-propranolol
Concentration (ng mL-1)
Intercept (a) CIa
Slope (b) CIa
10.0 – 400.0
0.023 0.001
0.006 0.0005
0.9995
10.0 – 400.0
0.021 0.001
0.007 0.0006
0.9995
Correlation coefficient (R)
b
Sy/x
0.033
0.00003
0.0005
10.0
3.0
0.042
0.00003
0.0005
10.0
3.0
c
Sa
d
Sb
LOQ (ng mL-1)e
LOD (ng mL-1)f
aConfidence intervals at P = 0.05.
bStandard deviation of the residual.
cStandard deviation of the intercept.
dStandard deviation of the slope.
eS/N = 10.
Fig. 2. Concentration-time profile of S-(À)-propranolol and R-(+)-propranolol
in rat serum after intraperitoneal administration of 40 mgkg-1 of propranolol.
Each point represents the mean SD, n = 5.
fS/N = 3.
Chirality DOI 10.1002/chir

198
ALANAZI ET AL.
enantiomers may be related to the lower protein binding of
S(À)-enantiomer and better stereospecific fitting towards its
β-receptor, which consequently increases its plasma level.22
was found to have affinity of À4.8 Kcal mol-1 for the best mode
with an RMSD of 3.819 units. The observed interactions was
mostly for the hydroxyl group of S-(À)-propranolol forming a
hydrogen bond with the –C = O group of carbamate moiety
with a distance of 2.26Å (Fig. 4). On the other hand,
docking of the R-(+)-enantiomer had affinity of À5.1 Kcal mol-1
for the best mode, with RMSD of 1.196. The –OH group is
found in a separate plane away from –NH and the rest of the
structure, and this enabled it to interact freely with most of
the electron acceptor groups found in cellulose tris(3,5-
dimethylphenylcarbamate). Moreover, the measured distances
of the R-(+)-enantiomer were 2.15 Å, which are shorter than
those formed with the S-(À)-enantiomer (Fig. 5A,B). These
short distances will make the bonds stronger and consequently
delay the retention time of the R-(+)-enantiomer, as in Figure 4B.
From this we can observe that the R-(+)-enantiomers have bet-
ter affinity toward cellulose tris(3,5-dimethylphenylcarbamate),
low RMSD, and short distances, which means strong bonds,
and all of these reasons could be the main cause for its higher
retention time than that of S-(À)-enantiomer, allowing effective
separation between the two enantiomers.
Molecular Modeling
The 3D crystal structure of β2-adrenergic receptor in com-
bination with carazolol can be used as guidance for docking
residues and consequently defining key residues that might
be involved in the interactions. By using the former structure,
we were able to investigate all possible docking conforma-
tions for both S-(À)-, R-(+)- and visualized separately looking
for the conformations that might have the best fit as well as
the same orientation like carazolol. The OH group of S-(À)-
enantiomer was believed to be positioned in a centered place
between Asn 312 (L-asparagine amino acid 312) and Asp 113
(aspartic acid 113) allowing for possible hydrogen bond
interaction between OH group of propranolol and –C = O
groups of both Asn 312 and Asp 113. On the other hand, the
R-(+)-enantiomer hydroxyl group positioned only towards
Asn 312 as shown in Figure 3. This difference between the
two enantiomers contributes to the retention time difference
with cellulose in separating the two enantiomers. The effec-
tive separation of both S-(À)- and R-(+)-enantiomers of pro-
pranolol was studied by molecular modeling techniques to
find a suitable relation between the retention time of both
enantiomers and the mechanism by which they could be
separated. The hydroxyl group here is very important for
the interactions simply because of its role in determination
of the stereochemistry of the structure. In the S-(À)-isomer,
OH group is found in the same plane with the –NH and
this will allow both of them to interact with cellulose tris
(3,5-dimethyl phenylcarbamate) but with relatively long
distances and large root of mean square deviation (RMSD).
Energy minimization resulted in removing the steric clashes
that could happen between the ligand and its site of action.
Moreover, it gave the chance to relax the whole structure of
cellulose tris(3,5-dimethylphenyl carbamate), and so makes it
easy for Autodock Vina to calculate the affinities for the docked
enantiomer. Computation of the force field of all bonds, angles,
torsions, nonbonds, and electrostatics resulted in a total energy
of 305.3 kcalmol-1. Upon minimization, the energy was found to
equal 143.9 kcalmol-1. The docking of the S-(À)-isomer against
the minimized cellulose tris(3,5-dimethylphenylcarbamate)
Fig. 4. Possible interactions of S-(À)-propranolol with measured distances
of cellulose tris(3,5-dimethylphenylcarbamate).
Fig. 3. (A) The possible interactions occur by the R-(+)-enantiomer of propranolol with its hydroxyl group toward Asn 312. (B) Orientation of the hydroxyl group
of the S-(À)- enantiomer of propranolol between Asp 113 and Asn 312.
Chirality DOI 10.1002/chir

ENANTIOSELECTIVE ANALYSIS OF PROPRANOLOL
199
Fig. 5. Two different conformations (A,B) of R-(+)-enantiomer of propranolol showing the hydrogen bond formation with cellulose tris(3,5-
dimethylphenylcarbamate) skeleton.
by high-performance liquid chromatography. J Chromatogr A 2007;
1161:242–251.
6. Cavazzini A, Nadalini G, Costa V, Dondi F. Heterogeneity of adsorption
mechanisms in chiral normal-phase liquid chromatography: 2-propanol
and ethyl acetate adsorption equilibria. J Chromatogr A 2007;1143:134–142.
7. Kitchen DB, Decornez H, Furr JR, Bajorath J. Docking and scoring in
CONCLUSIONS
An enantioselective HPLC-fluorescence method using
Chiralpak IB column was developed, for the first time, for
the simultaneous analysis of propranolol enantiomers in rat
serum. The use of a fluorescence detector coupled with an
HPLC technique allowed increased sensitivity and selectivity
for the determination of propranolol enantiomers. To the best
of our knowledge, the combinations of Chiralpak IB-HPLC-FL
detection with a pharmacokinetic study have not been
reported in single propranolol enantiomers analysis. The
developed method was used to remove endogenous interfer-
ence by the simple technique of deproteinization as a sample
cleanup procedure, thereby achieving a high degree of selec-
tivity. Due to the minimal sample preparation, and its good
precision and accuracy, this method appears to be very useful
for the therapeutic and toxicological monitoring of proprano-
lol isomers in clinical practice and for kinetic–metabolic
studies of this drug. Under the optimized conditions, the
developed method was of high precision, good accuracy,
and wide linear range of determination, especially a low limit
of detection. Finally, the developed method was demon-
strated to be applicable for conducting pharmacokinetic
studies. The effective separation of both S-(À)- and R-(+)-
enantiomers of propranolol was studied by molecular modeling
techniques to find a suitable relation between the retention
time of both enantiomers and the mechanism by which they
could be separated. The hydroxyl group of propranolol was
found to be crucial in determining the stereochemistry of
the structure.
virtual screening for drug discovery: methods and applications. Nat Rev
Drug Dis 2004;3:935–949.
8. Kubinyi H, Folkers G, Marti YC. 3D QSAR in drug design. New York:
Kluwer academic publishers; 1997.
9. Li W, Liu C, Tan G, Zhang X, Zhu Z, Chai Y. Molecular modeling study of
chiral separation and recognition mechanism of β-adrenergic antagonists
by capillary electrophoresis. Int J Mol Sci 2012;13:710–725.
10. Yu T, Du Y, Chen B. Evaluation of clarithromycin lactobionate as a novel
chiral selector for enantiomeric separation of basic drugs in capillary
electrophoresis. Electrophoresis 2011;32:1898–1905.
11. Servais AC, Rousseau A, Fillet M, Lomsadze K, Salgado A, Crommen J,
Chankvetadze B. Separation of propranolol enantiomers by CE using
sulfated beta-CD derivatives in aqueous and non-aqueous electrolytes:
comparative CE and NMR study. Electrophoresis 2010;31:1467–1474.
12. Wagdy HA, Hanafi RS, El-Nashar RM, Aboul-Enein HY. Predictability of
enantiomeric chromatographic behavior on various chiral stationary
phases using typical reversed phase modeling software. Chirality 2013;
25:506–513.
13. Bodoki E, Oltean M, Bodoki A, tiufiuc R. Chiral recognition and quantifi-
cation of propranolol enantiomers by surface enhanced Raman scattering
through supramolecular interaction with β-cyclodextrin. Talanta 2012;
101:53–58.
14. Morante-Zarcero S, Sierra I. Simultaneous enantiomeric determination of
propranolol, metoprolol, pindolol, and atenolol in natural waters by HPLC
on new polysaccharide-based stationary phase using a highly selective
molecularly imprinted polymer extraction. Chirality 2012;24:860–866.
15. Morante-Zarcero S, Sierra I. Comparative HPLC methods for β-blockers
separation using different types of chiral stationary phases in normal
phase and polar organic phase elution modes. Analysis of proprano-
lolenantiomers in natural waters. J Pharm Biomed Anal 2012;62:33–41.
ACKNOWLEDGMENTS
16. Trott O, Olson AJ. AutoDock Vina: Improving the speed and accuracy of
The authors thank the Deanship of Scientific Research at
King Saud University for funding the work through the
research group project no. RGP-VPP-037.
docking with
a new scoring function, efficient optimization, and
multithreading. J Comp Chem 2010;31:455–461.
17. Michel F News and views. J Mol Graph Mod 1999;17:55–84.
18. Rasmussen S, Choi HJ, Rosenbaum DM, Kobilka YS, Thian FS, Edwards
PC, Burghammer M, Ratnala V, Sanishvili R, Fischetti RF, Schertler GF,
Weis W, Kobilka BK. Crystal structure of the human β2 adrenergic G-
protein-coupled receptor. Nature 2007;450:383–387.
LITERATURE CITED
1. Silber B, Riegelman S. Stereospecific assay for (À)- and (+)- propranolol in
human and dog plasma. J Pharmacol Exp Ther 1980;215:643–648.
2. Ram CV. Risk of new-onset diabetes mellitus in patient with hypertension
treated with beta blockers. Am J Cardiol 2008;102:242–244.
3. Zhang T, Ngugen D, Franco P. Enantiomer resolution screening strategy
using multiple immobilized polysaccharide-based chiral stationary
phases. J Chromatogr A 2008;1191:214–219.
19. Oxelbark J, Gidlund P. Investigation of a tartaric acid-based linear polyam-
ide and dimer as chiral selectors in liquid chromatography. Chirality
2005;17:79–84.
20. Miller JN, Miller JC. Statistics and chemometrics for analytical chemistry,
5th Ed. Pearson: Tottenham, UK; 2005.
4. Zhang T, Dgugen N, Franco P, Murakani T, Ohnishi A, Kurosawa H.
Cellulose 3,5dimethylphenylcarbamate immobilized on silica: a new chiral
stationary phase for the analysis of enantiomers. Anal Chim Acta
2006;557:221–228.
5. Hoffmann CV, Laemmerhofer M, Linder W. Novel strong cation-exchange
type chiral stationary phase for the enantiomer separation of chiral amines
21. ICH guideline, Q2(R1). Validation of analytical procedures: text and
methodology. London; 2005.
22. Jantarat C, Tangthong C, Songkro S, Martin G, Suedee R. S-propranolol
imprinted polymer nanoparticle-on-microsphere composite porous cellu-
lose membrane for the enantioselectively controlled delivery of racemic
propranolol. Int J Pharm 2008;349:212–225.
Chirality DOI 10.1002/chir