Enantiomeric separation and simulation studies of pheniramine, oxybutynin, cetirizine, and brinzolamide chiral drugs on amylose-based columns

Article abstract of DOI:10.1002/chir.22276

Solid phase extraction (SPE)-chiral separation of the important drugs pheniramine, oxybutynin, cetirizine, and brinzolamide was achieved on the C ₁₈ cartridge and AmyCoat (150 x 46 mm) and Chiralpak AD (25 cm x 0.46 cm id) chiral columns in hum

Full text of DOI:10.1002/chir.22276

CHIRALITY 26:136–143 (2014)
Enantiomeric Separation and Simulation Studies of Pheniramine,
Oxybutynin, Cetirizine, and Brinzolamide Chiral Drugs on
Amylose-Based Columns
2
2
1
IMRAN ALI,1,2 ZEID A. AL-OTHMAN, ABDULRAHMAN AL-WARTHAN, SYED DILSHAD ALAM, AND JAVED A. FAROOQI3
*
1Department of Chemistry, Jamia Millia Islamia, (Central University) New Delhi, India
2Department of Chemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia
3Department of Chemistry, School of Sciences, Indira Gandhi National Open University, New Delhi, India
ABSTRACT
Solid phase extraction (SPE)-chiral separation of the important drugs
pheniramine, oxybutynin, cetirizine, and brinzolamide was achieved on the C18 cartridge and
AmyCoat (150 x 46 mm) and Chiralpak AD (25 cm x 0.46 cm id) chiral columns in human plasma.
Pheniramine, oxybutynin, cetirizine, and brinzolamide were resolved using n-hexane-2-PrOH-DEA
(85:15:0.1, v/v), n-hexane-2-PrOH-DEA (80:20:0.1, v/v), n-hexane-2-PrOH-DEA (70:30:0.2, v/v),
and n-hexane-2-propanol (90:10, v/v) as mobile phases. The separation was carried out at
25 1 ºC temperature with detection at 225 nm for cetirizine and oxybutynin and 220 nm for
pheniramine and brinzolamide. The flow rates of the mobile phases were 0.5 mLmin-1. The reten-
tion factors of pheniramine, oxybutynin, cetirizine and brinzolamide were 3.25 and 4.34, 4.76 and
5.64, 6.10 and 6.60, and 1.64 and 2.01, respectively. The separation factors of these drugs were
1.33, 1.18, 1.09 and 1.20 while their resolutions factors were 1.09, 1.45, 1.63 and 1.25, and 1.15, respec-
tively. The absolute configurations of the eluted enantiomers of the reported drugs were determined
by simulation studies. It was observed that the order of enantiomers elution of the reported drugs
was S-pheniramine > R-pheniramine; R-oxybutynin > S-oxybutynin; S-cetirizine > R-cetirizine; and
S-brinzolamide > R-brinzolamide. The mechanism of separation was also determined at the supra-
molecular level by considering interactions and modeling results. The reported SPE-chiral high-
performance liquid chromatography (HPLC) methods are suitable for the enantiomeric analyses
of these drugs in any biological sample. In addition, simulation studies may be used to determine
the absolute configuration of the first and second eluted enantiomers. Chirality 26:136–143,
2014. © 2014 Wiley Periodicals, Inc.
KEY WORDS: SPE-chiral-HPLC; human plasma; modeling; mechanism of separation
INTRODUCTION
marketing cost of the chiral drugs rose to U.S. $172 billion in
2002. Bingyun and Haynie12 described an enhancement in
the consumption cost of enantiomeric drugs as U.S. $200 billion
in 2008. Furthermore, it was expected to increase about U.S.
$350 billion in 2013 followed by U.S. $1 trillion in 2020.1,13
Pheniramine and cetirizine and (Fig. 1) are H1 receptor
antagonist antihistamines used for curing hey fever, allergy,
seasonal and perennial allergic rhinitis, and chronic
idiopathic urticaria.14–16 The enantiomers of cetirizine are
R-levocetirizine and S-dextrocetirizine, with former active
antipode (2-fold higher affinity for the H1-receptor).17–19 Fur-
thermore, both enantiomers have different pharmacological
effects. For example, S-dextrocetirizine is used for the treat-
ment of urticaria, while R-levocetirizine is given for curing
allergic disorders.20,21 The dextro form of pheniramine is
pharmaceutically active.22 Another popular drug, oxybutynin,
(Fig. 1) has been widely prescribed for the treatment of
urinary tract disorders. The R-enantiomer is a more potent
anticholinergic than the S-isomer.23–25 Brinzolamide (Fig. 1)
is a racemic drug widely used for curing open-angle glaucoma
Due to health awareness and advancement of pharmaceuti-
cal science the need of optically active pure drugs (single
enantiomer) is felt globally.1 It is essential to know the stereo-
chemistry of the chiral drugs before launching them into the
market. In last few decades, enantioseparations and the need
of chiral drugs have been active areas of research.2 It is a
great challenge to scientists and industrialists to separate
and ensure optical purity of the enantiomers.3 Various factors
such as drug concentration, pH, ionic concentration, temper-
ature, etc., are responsible for controlling the racemization
process.4 It is a well-known fact that one of the enantiomers
is pharmaceutically active while the other may be inactive or
toxic or ballast, leading to various side effects, toxicity, and
problems in the human body.5,6 US-FDA, European Commit-
tee for Proprietary Medicinal Products, Health Canada, and
Pharmaceutical and Medical Devices Agencies of Japan have
banned the marketing of all racemic drugs.7–9 The enantio-
meric differences at the pharmacokinetic level are mainly be-
cause of stereoselective drugs binding, absorption, clearance,
excretion, etc. The interactions mainly occurred with different
human plasma proteins.10 Hence, pharmacokinetic studies
and chiral purity testing need analytical methods with high
resolution power and good efficiency. Chiral high-performance
liquid chromatography (HPLC) fulfill all these needs easily.11
Globally, the consumption of chiral drugs was U.S $33 billion
in 1993, which reached U.S. $73 billion in 1997. Later on, the
© 2014 Wiley Periodicals, Inc.
*Correspondence to: Imran Ali, Department of Chemistry, Jamia Millia
Islamia, (Central University) New Delhi, India and Department of Chemistry,
College of Science, King Saud University, Riyadh 11451, Kingdom of Saudi
Arabia. E-mail: drimran_ali@yahoo.com; drimran.chiral@gmail.com
Received for publication 11 July 2013; Accepted 24 October 2013
DOI: 10.1002/chir.22276
Published online 26 January 2014 in Wiley Online Library
(wileyonlinelibrary.com).

STUDIES OF PHENIRAMINE, OXYBUTYNIN, CETIRIZINE, AND BRINZOLAMIDE CHIRAL DRUGS
137
(a):
(b):
(c):
(d):
Fig. 1. Chemical structures of R- and (S)- enantiomers of (a): pheniramine, (b): oxybutynin (c): cetirizine, and (d): and brinzolamide.
or ocular hypertension. R-brinzolamide is pharmaceutically
active. Generally, brinzolamide is a racemic mixture in pharma-
ceutical preparation, which needs chiral separation. These
drugs are being used frequently for different health problems.
A thorough search of the literature was carried out and it was
observed that a few papers are available on the chiral separation
of cetirizine and pheniramine.14,26–30 On the other hand, there
is only one publication each on the chiral separation of
oxybutynin and brinzolamide.25 Furthermore, it was observed
that these molecules were resolved on classical chiral columns.
These methods have a high limit of detection with a long run
time. Taking into consideration the importance of chiral analy-
ses in human plasma, attempts have been made to develop
solid phase extraction methods; 80% of chromatographers are
using solid phase extraction (SPE) for sample preparation of
the reported drugs in human plasma.1,31–33 In addition, the
chiral-HPLC method was also developed and used to monitor
the enantiomeric ratio of these drugs in human plasma. Also,
efforts have been made to ascertain the enantioseparation
mechanism on chiral columns using amodeling approach.
The results of these experiments are given herein.
Instrumentation
The experiments were carried out on an HPLC system of ECOM
(Prague, Czech Republic) consisting of a solvent delivery pump (model
Alpha 10), manual injector, absorbance detector (Indtech. Instrument,
Bombay, India) and Winchrome software. The columns used were
AmyCoat [tris-(3,5-dimethylphenyl carbamate)] (150 x 46 mm id) and
Chiralpak AD (25 cm x 0.46 cm id). These columns were purchased from
Kromasil (Sweden) and Daicel Chemical Industries (Japan), respectively.
An SPE unit was purchased from Varian (Palo Alto, CA). C18 Cartridges
were obtained from Waters (Milford, MA). A pH meter from Control
Dynamics (Model APX175 E/C) and centrifuge from Remi (model C-30BL)
were used. Millipore water (Millipore-Q, Bedford, MA) was used to carry
out these experiments.
Solid Phase Extraction (SPE)
To optimize sample preparation conditions, 1.0 mL each of
pheniramine, oxybutynin, cetirizine, and brinzolamide of 1.0 mg mL-1
concentration were mixed with 5.0 mL fresh-frozen human plasma, sepa-
rately. The spiked human plasma samples were vortexed for 2.0 min
and kept for 30.0 min. Then 15.0 mL acetone was mixed with each
vortexed sample and kept for 30 min. These samples were centrifuged
at 10,000 rpm (11,180 g) for 10.0 min to separate the supernatant. The
supernatant was evaporated to dryness under vacuum and the residue
was redissolved in 10.0 mL phosphate buffer (50 mM, pH 7.0). Sep-Pac
C18 cartridges (1.0 mL Waters) were preconditioned with 2.0 mL metha-
nol and 5.0 mL Millipore water. Then 10.0 mL phosphate buffer (50 mM,
pH 7.0) having pheniramine, oxybutynin, cetirizine, and brinzolamide
were passed through the cartridge with a 0.1 mLmin-1 flow rate, followed
by cartridges washing with 2.0 mL Millipore water at the same flow rate.
The cartridges were dried with hot air and the reported drugs eluted by
10.0 mL methanol containing 0.1% trifluoro acetic acid at a 0.1 mLmin-1
flow rate. The eluted methanol solutions for the reported drugs were
concentrated under vacuum to 0.5 mL separately. These samples were
further used for Chiral-HPLC analyses. To optimize SPE conditions, vari-
ous experimental factors were optimized.
EXPERIMENTAL
Chemicals and Reagents
Methanol, ethanol, n-hexane, 2-propanol, and diethyl amine of HPLC
grade and sodium dihydrogenphosphate (NaH2PO4.2H2O) of AR grade
were supplied by Merck (Mumbai, India). Trifluoro acetic acid was obtained
from Qualigens (Mumbai, India). Frozen Human Plasma (Mfg. License
No. 504) was purchased from the Rotary Blood Bank (New Delhi, India).
The standards of pheniramine, oxybutynin, cetirizine, and brinzolamide
were purchased from Sigma-Aldrich (St. Louis, MO). The solution of these
drugs of 1.0, 0.5, and 0.1 mgmL-1 concentrations were prepared in methanol
separately, respectively.
Chirality DOI 10.1002/chir

138
ALI ET AL.
High-Performance Liquid Chromatography (HPLC)
experiments. The percentage recoveries of pheniramine,
oxybutynin, cetirizine, and brinzolamide were 99.1, 99.0, 99.0,
and 99.1%, respectively. The experimental errors were consid-
ered during the calculation of percentage binding of the
reported drugs with human plasma proteins. The optimization
of SPE was achieved by varying different experimental condi-
tions such as concentration and pHs of phosphate buffer, flow
rates of human plasma samples, phosphate buffer, and eluting
solvents. In addition, various eluting solvents tried were metha-
nol, ethanol, acetone, ethyl acetate, and dichloromethane. In
order to achieve the maximum recoveries, 0.1 to 1% acetic acid
or trifluroacetic acid was also added in the above-cited eluting
solvents. As a result of exhaustive experimentation, the best
experimental conditions are reported herein. The maximum
percentage recoveries of pheniramine, oxybutynin, cetirizine,
and brinzolamide from C18 cartridge were achieved using phos-
phate buffer (50.0 mM, pH 5.0, 7.0, and 9.0) at 0.1 mL min-1 as
the flow rate. The best eluting solvent was methanol containing
0.1% trifluoro acetic acid for pheniramine, oxybutynin,
cetirizine, and brinzolamide at a 0.1 mLmin-1 flow rate. The
calculated percentage recoveries of the reported drugs on the
C18 cartridge and percentage bindings with human plasma
are given in Table 1.
The percentage recoveries of pheniramine enantiomers on
the C18 cartridge were 25.18, 20.67, 32.59, 36.40, 39.00, and
42.10, respectively. These values clearly depicted maximum
recovery at pH 9.0. Therefore, the human plasma bindings
of the enantiomers were calculated by considering percent-
age recoveries at pH 9.0. Similarly, the percentage recoveries
of oxybutynin enantiomers on the C18 cartridge were 11.24
and 13.17, 10.82 and 16.20, and 12.17 and 14.89 at pH 5.0,
7.0, and 9.0, respectively. These values clearly indicated max-
imum recovery at pH 9.0. Therefore, the human plasma bind-
ings of the enantiomers were calculated by considering
percentage recoveries at pH 9.0. The percentage recoveries
of cetirizine enantiomers on the C18 cartridge were 17.59
and 20.68, 22.20 and 28.61, and 25.91 and 27.02 at pH 5.0,
7.0, and 9.0, respectively. These values clearly indicated
maximum recovery at pH 9.0. Therefore, the human plasma
bindings of the enantiomers were calculated by considering
percentage recoveries at pH 9.0. The percentage recoveries
of brinzolamide enantiomers on the C18 cartridge were
19.00 and 20.40, 6.28 and 17.59, and 4.70 and 15.90 at
pH 5.0, 7.0, and 9.0, respectively. These values clearly showed
maximum recovery at pH 9.0. Therefore, human plasma
The experiments were carried out on an HPLC system as described
above. The standard solutions of 1.0 mgmL-1 of pheniramine, 0.5 mgmL-1
of oxybutynin, 1.0 mgmL-1 of cetirizine, and 0.1 mgmL-1 of brinzolamide
were prepared in methanol. Then 5.0μL of each solution was injected into
the HPLC system. The mobile phases used in this study were n-hexane-2-
PrOH-DEA (85:15:0.1, v/v), n-hexane-2-PrOH-DEA (80:20:0.1, v/v), n-hex-
ane-2-PrOH-DEA (70:30:0.2, v/v), and n-hexane-2-propanol (90:10, v/v) for
pheniramine, oxybutynin, cetirizine, and brinzolamide drugs, respectively.
The separation was carried out at 25 1 ºC with detection at 225 nm for
cetirizine and oxybutynin and 220nm for pheniramine and brinzolamide.
The flow rates of the mobile phases were 0.5 mLmin-1. The mobile phase
was filtered and degassed before use daily. The capacity (k), separation
(α) and resolution (Rs) factors were calculated.
In Silico Studies
To determine the chiral recognition mechanism, the interactions of
enantiomers of the reported drugs on AmyCoat [tris-(3,5-dimethylphenyl
carbamate) amylose] chiral selector (Fig. 2) were determined by
modeling studies.
Methodology
The modeling studies were carried out on a computer having these
configurations: Intel Core i3 CPU (2.3 GHz) with XP-based operating sys-
tem (Windows 2003). Marvin Sketch (v. 5.8.2) was used to draw the enan-
tiomeric structures of the drugs. Marvin Sketch (v. 5.8.2) was used to
draw the enantiomeric structures of the reported drugs. The structures
were cleaned to 3D and saved in pdb format. The docking studies was
performed with AutoDock 4.2 (Scripps Research Institute, La Jolla, CA)
considering all the rotatable bonds of ligand as rotatable and tris-(3,5-
dimethylphenyl carbamate) amylose as rigid.34 Grid box size of
50 × 50 × 50 with 0.375 spacing was used. Docking was performed using
an empirical-free energy function and Lamarckian Genetic Algorithm,
with an initial population of 150 randomly placed individuals, a maximum
number of 2,500,000 energy evaluations, a mutation rate of 0.02, and
crossover rate of 0.80. Fifty independent docking runs were performed
for each ligand and tris-(3,5-dimethylphenyl carbamate) amylose for low-
est free energy of binding conformation from the largest cluster, which
was written and saved in PDBQT format. These PDBQT files were
converted to PDB file format.
RESULTS AND DISCUSSION
Solid Phase Extraction
To determine the efficiency of the reported SPE method,
the percentage recoveries of the enantiomers of pheniramine,
oxybutynin, cetirizine, and brinzolamide were calculated.
The percentage recoveries of pheniramine, oxybutynin,
cetirizine, and brinzolamide were determined by running blank
(a):
(b):
Fig. 2. 2D (a) and 3D (b) structures of tris-(3,5-dimethylphenyl carbamate) amylose chiral selector.
Chirality DOI 10.1002/chir

STUDIES OF PHENIRAMINE, OXYBUTYNIN, CETIRIZINE, AND BRINZOLAMIDE CHIRAL DRUGS
139
TABLE 1. Percentage bindings of pheniramine, oxybutynin, cetirizine, and brinzolamide with human plasma protein
Pheniramine
% Recovery
Oxybutynin
% Recovery
Cetirizine
% Recovery
Brinzolamide
% Recovery
% Binding
with plasma
protein
% Binding
with plasma
protein
% Binding
with plasma
protein
% Binding
Sl.
pH of
0.1%TFA
in MeOH
0.1%TFA
in MeOH
0.1%TFA
in MeOH
0.1%TFA
in MeOH
with plasma
protein
no. buffer
1
2
3
5.0
7.0
9.0
25.18 (20.67) 30.00 (36.51) (13.17) 11.24 (36.33) 38.26 20.68 (17.59) 28.82 (31.91) (20.40) 19.00 (29.10) 30.50
32.59 (36.40) 25.90 (31.40) (16.20) 10.82 (33.30) 38.68 28.61 (22.20) 20.89 (27.30) (17.59) 6.28
39.00 (42.10) 32.50 (37.67) (14.89) 12.17 (34.61) 37.33 27.02 (25.91) 22.48 (23.59) (15.90) 4.70
(31.91) 43.22
(33.60) 44.80
The values outside and inside of the parentheses belong to the R– and S–enantiomers, respectively.
bindings of the enantiomers were calculated by considering
percentage recoveries at pH 9.0. Hence, human plasma
bindings of the enantiomers of pheniramine, oxybutynin,
cetirizine, and brinzolamide were 32.50 and 37.67, 34.61 and
37.33, 22.48 and 23.59, and 33.60 and 44.80, respectively.
experiment was carried out using mobile phases n-hexane-2-
PrOH-DEA (85:15:0.1, v/v), n-hexane-2-PrOH-DEA (80:20:0.1, v/v),
n-hexane-2-PrOH-DEA (70:30:0.2, v/v), and n-hexane-2-
propanol (90:10, v/v) for pheniramine, oxybutynin, cetirizine,
and brinzolamide drugs, respectively. The developed SPE-
Chiral-HPLC methods were validated by carrying out five sets
(n= 5) of the chromatographic procedure under identical condi-
tions. The HPLC method was validated with respect to various
parameters including linearity, limit of detection (LOD), limit of
quantitation (LOQ), precision, accuracy, selectivity, recovery,
and robustness and ruggedness.
Chromatography
The chromatographic parameters such as retention (k),
separation (α), and resolution (Rs) factors were calculated
for the enantiomers of pheniramine, oxybutynin, cetirizine,
and brinzolamide using both AmyCoat and Chiralpak AD col-
umns. The values of retention factors for the first and second
enantiomers were calculated and are given in Table 2. It is
clear from this table that the retention factors of pheniramine,
oxybutynin, cetirizine, and brinzolamide were 3.25 and 4.34,
4.76 and 5.64, 6.10 and 6.60, and 1.64 and 2.01, respectively.
The separation factors of these drugs were 1.33, 1.18, 1.09
and 1.22 while the values of resolution factors were 1.09,
1.45, 1.63 and 1.20, and 1.15, respectively. The values of separa-
tion and resolution factors were greater than one indicating
complete resolution of the enantiomers of the reported drugs.
Furthermore, it was observed that the chiral separations of
these drugs were complementary to each other on AmyCoat
and Chiralpak columns (Table 2). This is due to the fact that
both these columns have the same chiral selector, i.e., tris-
(3,5-dimethylphenyl carbamate) amylose. The chromatograms
for these drugs in standard and human plasma samples using
AmyCoat are shown in Figures 3 to 6, respectively.
MECHANISM OF SEPARATION AT THE
SUPRAMOLECULAR LEVEL
Among various chiral selectors, the best one is polysaccharide-
based chiral selectors. AmyCoat and Chiralpak AD columns
have amylose polysaccharide (Fig. 7), which is more helical
than cellulose and others.35 The separation of the reported
drugs on AmyCoat and Chiralpak AD columns is due to the
presence of chiral grooves. The enantiomers of these drugs
get fitted stereospecifically to different extents. The fittings of
the enantiomers are stabilized by various forces such as π-π
interactions, hydrogen bondings, van der Waal’s forces, steric
effects, etc. A perusal of Table 2 indicates a poor resolution fac-
tor of brinzolamide. This may be due to the fact that this mole-
cule does not have a phenyl ring avoiding the possibility of π-π
interactions. Therefore, it may be concluded that π-π interac-
tions are the major controlling forces in the chiral separation.
This fact has already been published by our group.36–38 Briefly,
the enantiomers of these drugs fitted stereospecifically in the
chiral groove. The mobile phase tends to carry these enantio-
mers along with it. As a result of the competition between
stationary and mobile phases, the less retained enantiomer
eluted first followed by the more retained enantiomer. Efforts
HPLC Method Optimization
To optimize the chromatographic conditions, various com-
binations of solvent systems were tried. The alteration in flow
rate, detection wavelength, and amount injected was also car-
ried out. As a result of exhaustive experimentation, the best
HPLC conditions were developed and reported herein. The
TABLE 2. Separation (α) and resolution (Rs) factors for pheniramine, oxybutynin, Cetirizine, and brinzolamide on AmyCoat and
Chiralpak AD columns in standard and in human plasma samples
Separation
factors (α)
Resolution
factors (Rs)
Sl. no.
1.
Chiral drugs
Mobile phase
Pheniramine (standard)
Pheniramine (human plasma)
Oxybutynin (standard)
Oxybutynin (human plasma)
Cetirizine (standard)
Cetirizine (human plasma)
Brinzolamide (standard)
Brinzolamide (human plasma)
1.33 (1.38)
1.30 (1.32)
1.18 (1.17)
1.15 (1.15)
1.09 (1.10)
1.06 (1.08)
1.22 (1.22)
1.20 (1.19)
1.09 (1.10)
1.08 (1.09)
1.45 (1.45)
1.43 (1.44)
1.63 (1.62)
1.60 (1.60)
1.25 (1.25)
1.25 (1.25)
n-hexane-2-Propanol-DEA (85:15:0.1,v/v)-do-0.5 mL/min at 220 nm
2.
3.
4.
n-hexane-2-Propanol-DEA (80:20:0.1,v/v)-do-0.5 mL/min at 225 nm
n-hexane-2-propanol-DEA (70:30:0.2,v/v)-do-0.5 mL/min at 225 nm
n -hexane-2-propanol (90:10,v/v)-do-0.5 mL/min at 220 nm
The values outside and inside of the parentheses correspond to AmyCoat and Chiralpak AD column, respectively.
Chirality DOI 10.1002/chir

140
ALI ET AL.
Fig. 3. HPLC chromatograms of pheniramine standard and in human plasma sample.
Fig. 4. HPLC chromatograms of oxybutynine standard and in human plasma sample.
Fig. 5. HPLC chromatograms of cetirizine standard and in human plasma sample.
Fig. 6. HPLC chromatograms of brinzolamide standard and in human plasma sample.
Chirality DOI 10.1002/chir

STUDIES OF PHENIRAMINE, OXYBUTYNIN, CETIRIZINE, AND BRINZOLAMIDE CHIRAL DRUGS
141
Fig. 7. 3D structure of amylase polymer.
have also been made to determine the interaction of the enan-
TABLE 3. Modeling results of R- and S-enantiomers of
tiomers with chiral selectors by a modeling approach. In silico
studies of the chiral recognition mechanism is discussed in
the following section.
pheniramine, oxybutynin, cetirizine, and brinzolamide drugs
with tris-(3,5-dimethylphenyl carbamate) amylose chiral
selector
Simulation Studies of the Drugs on AmyCoat Chiral Selector
Name of
In silico study is an attractive tool to assess the binding of
molecule to receptor. In the present work, attempts were
made to establish the enantiomeric interactions on a chiral
stationary phase. Amylose-based columns have different
types of functional groups responsible for various types of
bondings. The docking energies of R- and S-enantiomers of
pheniramine, oxybutynin, cetirizine, and brinzolamide were
À6.55 and À5.25, –4.90 and À6.10, –7.95 and À7.43, and
À8.55 and À8.13kcal/mol, respectively. The differences of
docking energies (ΔE) of R- and S-enantiomers of pheniramine,
oxybutynin, cetirizine, and brinzolamide drugs were 1.30, 1.20,
0.52, and 0.42 kcal/mol, respectively (Table 3). It is clear
from Table 3 that the energy values of R-enantiomers are
greater than S-enantiomers of pheniramine, cetirizine, and
brinzolamide. Contrarily, the S-enantiomer of oxybutynin has
greater energy than the R-antipode. Therefore, it may be
predicted that the first eluted enantiomers of pheniramine,
cetirizine, and brinzolamide were of S- configuration. In the
case of oxybutynin drug the first eluted enantiomers were of
R- configuration.
enantiomeric
drugs
Binding
energy
Total
Internal
Docking
energy
*ΔE = RDE- SDE
Pheniramine
(R)
(S)
Oxybutynin
(R)
(S)
Cetirizine
(R)
(S)
À5.85
À4.57
À0.7
À6.55
À5.25
1.30
À0.68
À3.83
À4.69
À1.07
À1.41
À4.90
À6.10
1.20
0.52
0.42
À5.57
À5.15
À2.38
À2.28
7.95
À7.43
Brinzolamide
(R)
(S)
À8.09
À7.62
À0.46
À0.51
À8.55
À8.13
*| RDE- SDE |= Difference in the docking energies of enantiomers.
hydrophobic interactions were also playing key roles. The
docking models of the enantiomers of these drugs are shown
in Figure 8.
The chromatographic experimental results (retention
times) of the enantiomeric resolution can be correlated with
the docking output. ΔtR of enantiomeric resolution was on
the order of pheniramine (1.30 min) > oxybutynin (1.05 min)
cetirizine (0.59 min) > brinzolamide (0.45 min). These results
were verified by the order of docking energy differences (ΔE),
i.e., pheniramine (1.30 kcal/mol) > oxybutynin (1.20 kcal/mol) >
cetirizine (0.52 kcal/mol) > brinzolamide (0.42 kcal/mol). There-
fore, close chiral resolution of brinzolamide is because of the low
binding energy difference of the enantiomers (due to the ab-
sence of π-π interactions). On the other hand, the binding energy
differences were quite good for enantiomers of pheniramine,
oxybutynin, and cetirizine and, hence, well resolution.
R-pheniramine had one H-bond (2.99 Å), between nitrogen
and oxygen atoms of pheniramine and amylose, respectively,
while S-pheniramine had no H-bond with the chiral receptor.
Similarly, R-Cetirizine showed one H-bond (2.073 Å) between
the oxygen and nitrogen atoms of cetirizine and amylose,
respectively, while H-bonds was absent in S-cetirizine. The
S-enantiomer of brinzolamide was able to form one H-bond
(1.947 Å) between the oxygen and nitrogen atoms of
brinzolamide and amylose, respectively, while it lacked in
R-antipode. In the case of both R- and S-enantiomers of
oxybutynin, H-bonding was absent. On the other hand, the
bondings such as van der Waal’s forces, electrostatic, and
VALIDATION OF CHIRAL HPLC AND SPE METHODS
The developed chiral HPLC and SPE methods were vali-
dated by carrying out five sets (n = 5) of the chromatographic
and SPE extraction procedures under identical experimental
conditions. It is interesting to note that the values of chro-
matographic parameters for the reported drugs (Table 2)
were almost equal in standard solutions and human plasma
extracts, which indicated the robustness of the chiral HPLC
method. The validation of the developed method was
ascertained by regression analysis using the Excel Microsoft
program. The results for chiral chromatographic and SPE
methods regression analysis are given in Table 4. A perusal
of Table 4 indicates that the values of standard deviation
(SD), correlation coefficient (CC), and confidence limit (CL)
of chiral HPLC varied from 0.10 to 0.11, 0.9996 to 0.9999,
and 99.0 to 99.2, respectively. On the other hand, these values
for SPE method ranged from 0.11 to 0.13, 0.997 to 0.9998,
and 99.0 to 99.1, respectively. The data of the peak area ver-
sus drug concentrations were treated by linear least square
regression analysis. The linearity was calculated according to
DIN 32645 with B.E.N. v. 2.039 and the values are given in
Table 5. It is clear from Table 5 that the linearity ranges were
1.0–50μg/L for cetirizine, pheniramine, and brinzolamide. On
Chirality DOI 10.1002/chir

142
ALI ET AL.
R-Oxybutynin
S-Oxybutynin
R-Pheniramine
S-Pheniramine
R-Cetirizine
S-Cetirizine
R-Brinzolamide
S-Brinzolamide
Fig. 8. Docking model of R- and S-enantiomers of pheniramine, oxybutynin, cetirizine, and brinzolamide with amylose chiral selector.
and brinzolamide drugs in human plasma by SPE-HPLC. The
TABLE 4. Regression analysis data for the chiral chromato-
graphic and SPE methods of pheniramine, oxybutynin,
cetirizine, and brinzolamide drugs
qualitative and quantitative analyses of the reported drugs were
carried out using the above-mentioned HPLC conditions
(Figs. 3–6). The reported method is new for the determination
of the order of elution of the R- and S-enantiomer of
pheniramine, oxybutynin, cetirizine, and brinzolamide in hu-
man plasma by HPLC. The reported method is also confirmed
by in silico studies.
Chiral HPLC
(peak area)
SPE (percentage
recoveries)
Compounds
SD
CR
CL
SD
CR
CL
Pheniramine
Oxybutynin
Cetirizine
0.11 0.9997 99.0
0.11 0.9998 99.1
0.12 0.9996 99.0
0.10 0.9999 99.2
0.12
0.12
0.13
0.11
0.9998
0.9998
0.9997
0.9998
99.0
99.0
99.0
99.1
CONCLUSION
An eco-friendly, reproducible, accurate, inexpensive, and
effective SPE-Chiral-HPLC method was described for the
chiral separations of pheniramine, oxybutynin, cetirizine,
and brinzolamide drugs in human plasma. For calculation
of the percentage recoveries of pheniramine, oxybutynin,
cetirizine, and brinzolamide drugs, five sets (n = 5) of SPE
experiments were carried out under identical experimental
conditions. The percentage recoveries were carried out at
different pHs, i.e., 5.0, 7.0, and 9.0. The maximum percentage
recoveries of pheniramine, oxybutynin, cetirizine, and
brinzolamide were 32.50 and 37.67, 34.61 and 37.33, 22.48
and 23.59, and 33.60 and 44.80, respectively, at pH 9.0. The
values for LOD for pheniramine, oxybutynin, cetirizine, and
brinzolamide ranged from 1.0–2.5 ng/mL, while these values
for LOQ were 5.0–10.0 ng/mL, respectively. The experimen-
tal results of the proposed method were compared with
modeling output and both were in good agreement. There-
fore, the reported method can be used for monitoring the chi-
ral ratio of these drugs in any biological samples. In addition,
the modeling approach is useful to ascertain the configura-
tion of the separated enantiomers.
Brinzolamide
CC: correlation coefficient, CL: confidence limit. and SD: standard deviation.
TABLE 5. Linearity of pheniramine, oxybutynin, cetirizine,
and brinzolamide drugs
Compounds
Lin. range
CC
CL (%)
Pheniramine
Oxybutynin
Cetirizine
1.0–50 μg/L
1.0–40 μg/L
1.0–50 μg/L
1.0–50 μg/L
0.9998
0.9998
0.9997
0.9999
99.0
99.1
99.0
99.2
Brinzolamide
CC: correlation coefficient and CL: confidence limit.
the other hand, the values of linearity range of oxybutynin were
1.0–40 μg/L. The values of correlation coefficients varied from
0.9997 to 0.9999 while the values of confidence limit were
99.0–99.2% for all four drugs studied. The limits of detection
of cetirizine, pheniramine, oxybutynin, and brinzolamide were
1.5, 1.6, 1.0, and 1.4 μg/L, respectively. On the other hand,
limits of quantification of these drugs were 6.5, 6.8, 5.2, and
6.8 μg/L, respectively. These validation values indicated good
reproducibility of the reported chiral HPLC and SPE methods.
ACKNOWLEDGMENT
The authors thank King Saud University for funding this pro-
ject through the Visiting Professor Program.
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