Chromatographic comparison of atenolol separation in reaction media on cellulose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase using ultra fast liquid chromatography

Article abstract of DOI:10.1002/chir.22019

Because chiral liquid chromatography (LC) could become a powerful tool to estimate racemic atenolol quantity, excellent enantiomeric separation should be produced during data acquisition for satisfactory observation of atenolol concentrations throughout the racemic resolution processes. Selection of chiral LC column and analytical protocol that fulfill demands of the ultra fast LC analysis is essential. This article describes the characteristics of atenolol chromatographic separation that resulted from different resolution media and analytical protocols with the use of a Chiralcel OD column. The chromatograms showed quite different characteristics of the separation process. The single enantiomer and racemic atenolol could be recognized by the Chiralcel OD column in less than 20 min. Symmetrical peaks were obtained; however, several protocols produced peaks with wide bases and slanted baselines. Observations showed that efficient enantioresolution of racemic atenolol was obtained at slow mobile phase flow rate, decreased concentration of amine-type modifier but increased alcohol content in mobile phase and highest ultraviolet detection wavelength were required. The optimal ultra fast LC protocol enables to reduce and eliminate the peaks of either the atenolol solvent or the buffers and provided the highest peak intensities of both atenolol enantiomers.

Full text of DOI:10.1002/chir.22019

CHIRALITY 24:356–367 (2012)
Chromatographic Comparison of Atenolol Separation in Reaction
Media on Cellulose tris-(3,5-dimethylphenylcarbamate) Chiral
Stationary Phase Using Ultra Fast Liquid Chromatography
1,2
1
3
JONI AGUSTIAN, AZLINA HARUN KAMARUDDIN, * AND HASSAN Y. ABOUL-ENEIN
1
School of Chemical Engineering, Universiti Sains Malaysia, Seberang Perai Selatan, Pulau Pinang, Malaysia
2
Department of Chemical Engineering, Universitas Lampung, Bandar Lampung 35145, Lampung, Indonesia
3
Pharmaceutical and Medicinal Chemistry Department, National Research Centre, Dokki, Cairo, Egypt
ABSTRACT
Because chiral liquid chromatography (LC) could become a powerful tool to
estimate racemic atenolol quantity, excellent enantiomeric separation should be produced during
data acquisition for satisfactory observation of atenolol concentrations throughout the racemic
resolution processes. Selection of chiral LC column and analytical protocol that fulfill demands of
the ultra fast LC analysis is essential. This article describes the characteristics of atenolol chromato-
graphic separation that resulted from different resolution media and analytical protocols with the use
W
of a Chiralcel OD column. The chromatograms showed quite different characteristics of the
separation process. The single enantiomer and racemic atenolol could be recognized by the
W
Chiralcel OD column in less than 20 min. Symmetrical peaks were obtained; however, several
protocols produced peaks with wide bases and slanted baselines. Observations showed that efficient
enantioresolution of racemic atenolol was obtained at slow mobile phase flow rate, decreased concen-
tration of amine-type modifier but increased alcohol content in mobile phase and highest ultraviolet
detection wavelength were required. The optimal ultra fast LC protocol enables to reduce and elim-
inate the peaks of either the atenolol solvent or the buffers and provided the highest peak intensities
of both atenolol enantiomers. Chirality 24:356-367, 2012. © 2012 Wiley Periodicals, Inc.
W
KEY WORDS: racemic atenolol; Chiralcel OD Column; enantiomers separation; analytical
protocol; organic; buffers media
1
2
INTRODUCTION
operation, the chromatographic technology, which becomes
24–26
a powerful tool in the development of chiral drugs,
has
Atenolol (AT, Fig.1) chemically known as 2-{4-[2-hydroxy-3-
propan-2-ylamino)propoxy]phenyl}acetamide is a famous
b-adrenergic receptor blocker used for maintenance of blood
pressure, angina pectoris, and arrhythmia. This b-blocker
is one of the best-selling drugs in the world
used various chiral selectors to separate the AT active
(
2
7,28
isomer.
1
,2
Because the racemic AT resolutions have been developed,
chromatography could become an effective method to esti-
mate reduction of AT concentration/quantity during the
resolving processes. Thin layer and gas chromatogra-
3
–5
and is mainly
sold in the form of racemate, which has the eudismic ratio of
6
7
4
6. Its active isomer resides on (S)-enantiomer, which is
8
2
1,22,27–29
phy
have been associated with the development,
found to avoid side effects generated by the racemic form,
but high performance liquid chromatography (HPLC) is
the most widely used technique. Many HPLC columns
34
whereas the (R)-AT does not show the b-blocking activity
9
,10
and has not lacked of the side effects.
Because the inactive
W
16–20,30,31
32
33
(
Chiralcel OD,
Amycoat, Eurocel 01, RegisCell,
36,37 38
isomers of the b-adrenergic blockers bring serious health
35
TM
11
Chiral-CBH, Chirex 3022 (S),
and Chirobiotic
V2 )
problems and switching from racemate to single enantio-
1
2
have been examined under various analytical protocols to per-
form the AT enantiomeric separation, which resulted to various
separation factors and chiral chromatograms.
Because conversion, enantiomeric excess (ee), and enan-
tiomeric ratio (E) of the resolution processes are based on
the HPLC areas of the (R)- and (S)-enantiomers, good peak
shapes/chiral chromatograms must be produced during the
separation. To the best of our knowledge, there has been no
mer develops lesser side effects, many pathways have been
constructed to form the optically active AT.
Highlights of the pathways to produce the (S)-AT are given
1
3,14
in previous literatures.
The compound was synthesized
asymmetrically from racemic or achiral raw materials that
required the presence of chiral metal ligand catalysts such
as (R,R)-Co-(salen) complexes to carry out the kinetic resolu-
tion processes.1
5–17
The (S)-enantiomer was also prepared
from catalytic reactions of achiral feeds without involvement
of a chiral catalyst, but a chiral addendum such as (R)- or
Contract grant sponsor: Financial supports from Universiti Sains Malaysia,
Ministry of Science Technology and Innovation (MOSTI Science Fund No.
2
,18–20
(S)-epichlorohydrin must be available.
Further micro-
3
05/227/PJKIMIA/6013337) and Malaysia Technological Development
bial resolution of the racemic AT and its ester with the use
Corporation (MTDC No. 304/PJKIMIA/6053010) for this research are deeply
acknowledged..
*Correspondence to: A.H. Kamaruddin, School of Chemical Engineering,
Universiti Sains Malaysia, 14300 Nibong Tebal, Pulau Pinang, Malaysia.
E-mail: chazlina@eng.usm.my
Received for publication 26 September 2011; Accepted 24 January 2012
DOI: 10.1002/chir.22019
Published online 19 April 2012 in Wiley Online Library
(wileyonlinelibrary.com).
of Rhizopus arrhizus and Geothricum candidum gave the
single enantiomer in high optical purity and chemical yield.21
Enzymatic resolution of the racemate using immobilized
lipases was developed as well through enantioselective
2
2,23
esterification and hydrolysis processes.
Although chro-
matographic resolution is difficult to appear in large-scale
©
2012 Wiley Periodicals, Inc.

RACEMIC ATENOLOL SEPARATION IN REACTION MEDIA
357
crystals into dimethyl sulfoxide with concentrations of 9 mg/ml. All
chemicals were of analytical grade except for analysis (of HPLC grade)
and bought from EOS Scientific (M) Sdn Bhd, Fisher Scientific (M)
Sdn Bhd, Merck Sdn Bhd, and Sigma Aldrich (M) Sdn Bhd. The AT
and other chemicals were used without purification. Candida antarctica
W
lipase fraction B (Lipozyme CALB L LCN02103 supplied by Science
Technics Sdn Bhd, Malaysia), lipase basic kit (62327) from Fluka Analytical
containing Pseudomonas fluorescence lipase (95608), hog pancreatic lipase
(62300), and lipoprotein lipase from Pseudomonas sp. (62336) (supplied by
Sigma Aldrich), and Amano lipase PS from Burkholderia cepacia (supplied
by Sigma Aldrich) catalyzed the enzymatic reactions. All lipases were used
without initial treatment.
Apparatus
Analysis of the AT enantiomers was performed on a Shimadzu UFLC
LC-20A Prominence (Kyoto, Japan) system. The system consist of two
units LC-20 AD dual plunger parallel flow solvent delivery pump, a SIL-
20ACHT auto-sampler, a SPD-20A ultraviolet–visible (UV–vis) detector, a
CTO-20 AC column oven, a DGU-A3 degasser unit, and the CBM-20A system
controller. The UFLC was connected to a personal computer to operate the
equipment using the Shimadzu LCsolution real time application software.
Fig. 1. A mirror image of atenolol enantiomers.
Preparation of Buffer Solution
Several 67 mM Sorensen buffers were prepared by mixing KH
Na HPO solution. A certain quantity of the KH PO (Acros Organics,
Fisher Scientific, Malaysia, 99%+) and Na HPO (Acros Organics, Fisher
Scientific, Malaysia, 98%+) were dissolved in double distilled water to form
133 mM buffers. Both solutions were then adjusted to the desired pH values
and then diluted with the double distilled water to give the 67mM buffers.
2 4
PO and
article mentioning the chiral chromatograms of the racemic AT
generated by the HPLC analysis on this racemate existing in
the reaction media. As part of our project in resolution of the
racemate enzymatically, selection of a column and analytical pro-
tocol that can satisfy observations of the AT concentration during
the reaction time is essential. Because the observations are
based on the AT peaks’ areas, herewith we describe charac-
teristics of the chiral chromatograms that resulted from
different reaction media and general protocols on a cellulose
2
4
2
4
2
4
Sample Preparation and Analysis
The enzymatic reaction of the racemic AT was carried out for 36 h.
During interval times, 1 ml sample was taken out from each reaction flask
ꢀ
and placed inside a 1.5-ml vial then kept in 4 C refrigerator prior to
tris-(3,5-dimethylphenylcarbamate) chiral stationary phase
analysis. Before injection, all samples were either filtered through
W
(
CSP, Chiralcel OD column, Daicel Chemical Industries Ltd.,
0
.45 mm membrane filter using syringe or centrifuged at 2000 g using
Profuge 6k centrifuge (supplied by Interscience Sdn Bhd, Malaysia) for
5–20 min where 500–650 ml of the centrifuged samples were taken out
Osaka, Japan) using an ultra fast liquid chromatography (UFLC)
unit. Because of the racemic AT not being soluble in very hydro-
phobic solvents, which could risk the column, water miscible sol-
vents are decided to be used as the reaction media. Comparisons
of the analytical protocols are made to give useful description on
the analytical protocols applied in the AT resolution studies.
1
and transferred into 1.5-ml clean bottles.
A 1–2 ml of samples were injected automatically at a time into the
W
Chiralcel OD column (250 Â 4.6 mm) with the mobile phase as stated
in Table 1. The UV–vis detector was set at the wavelength of
ꢀ
2
54–276 nm. The UFLC was operated at normal phase at 35 C. Qualita-
tive and quantitative analyses were conducted on the resulted chromato-
grams via the Shimadzu LCsolution post-run analysis software on the
basis of the standard procedure for the instrumentation.
MATERIALS AND METHODS
3
9
Materials
(
R,S)-AT (99% pure), (S)-AT (97% pure), and (R)-AT (99% pure) were
Lipase-catalyzed Acetylation of (R,S)-atenolol
bought from Nanjing Chemlin Chemical Industry Co. Ltd (Nanjing,
China), Tocris Bioscience (Bristol, UK), and Sigma Aldrich (M) Sdn
Bhd (Malaysia), respectively. The racemate was dissolved directly in
the reaction media according to the desired concentrations. Several stan-
dard solutions of pure enantiomers were prepared by dissolving their
Reaction in organic media.
A 5 mg/ml racemic AT was prepared
in dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and tetra-
hydrofurane (THF). 20 ml of each solution were transferred into
100-ml erlenmeyer flasks. Vinyl acetate was added at a ratio of 1:1.5
W
TABLE 1. Characteristics of Chiralcel OD analytical protocols
Mobile phase
B
UFLC results
Protocols
Flow rate
(ml/min)
Wavelength
(nm)
[Ref.]
A
C
Composition (v/v/v)
a
R
S
3
0
01
02
03
04
05
Hexane
Hexane
Hexane
Hexane
Hexane
Ethanol
Ethanol
Ethanol
2-Propanol
Ethanol
Diethylamine
Diethylamine
Diethylamine
Diethylamine
Diethylamine
80 A/20 B/0.6 C
80 A/20 B/0.6 C
75 A/25 B/0.1 C
60 A/40 B/0.1 C
60 A/40 B/(0.2 + 0.2) C
1.00
0.75
0.70
0.50
1.00
254
254
276
275
276
1.33–1.37
n.a
1.36–1.52
1.44–1.93
n.a
0.73–1.07
n.a
0.62–0.82
0.82–0.94
n.a
[**]
3
4
4
1
0
1
+
acetic acid
UFLC, ultra fast liquid chromatography; n.a, complete data are not available; Ref., reference; a, enantioseparation factor; R
*Present study.
S
, enantioresolution.
*
Chirality DOI 10.1002/chir

3
58
AGUSTIAN ET AL.
(
(
3
mole/mole). The mixtures were shaken in an orbital shaker
Max Q4000 Barnstead Lab-Line, Illinois, USA) at 200 rpm and 40 C for
0 min. Lipases were finally added to the mixtures (lipase amino from
DMSO, DMF, and THF where presence of these solvents in
ꢀ
small or residual quantities may destroy the cellulose
42,43
tris-(3,5-dimethylphenylcarbamate) compound.
However,
Burkholderia cepacia: 35 mg; Pseudomonas fluorescence lipase: 8 mg; hog
pancreatic lipase: 8 mg; Candida antarctica lipase fraction B: 200 ml).
Enzymatic acetylations were performed at 200 rpm and 40 C (Protocol-01
these solvents are usually associated with the enzymatic
44–46
reactions.
ꢀ
ꢀ
Our observation on the selection of co-solvent for enzy-
matic reactions of racemic AT solutions showed that racemic
AT solutions in acetone, chloroform, and 2-propanol (the
solvents) formed whitish suspensions when they were mixed
with hexane, heptane, toluene, cyclohexane, or isooctane (the
co-solvents) in proportional or higher mixing ratio as given in
Table 2. It was also found that the solutions of the racemic AT
in DMSO and DMF created clear bilayer mixtures when
cyclohexane, heptane, or isooctane was added to the AT solu-
tions. These combinations would influence the mobile phase
composition with high hexane content in the mixture which
should be considered carefully when acetone and 2-propanol
are used as solvents for AT. This fact could be found on
used 35 C and 25ml AT solutions) in the shaker.
Reaction in phosphate buffers.
A 10 mg/ml of racemic AT was
dissolved in Sorensen buffer (67 mM, pH 6.6–7.8). 40 ml of the AT solu-
tions were placed in reaction flasks (Protocols-03 and -04 used 25 ml)
where vinyl acetate was added (1:5 mole/mole except Protocol-04 that
used 1:1). The flasks were shaken at 40 C and 200 rpm (Protocol-01 used
3
ꢀ
ꢀ
5 C) for 30 min. Then, lipase (Candida antarctica lipase fraction B:
80 ml, Protocol-04 used 665 ml) was added. The enzymatic reactions were
3
conducted in the shaker at conditions as stated previously.
RESULTS AND DISCUSSION
W
Characteristics of Chiralcel OD Column
W
W
The analytical Chiralcel OD column was supplied by
the type of the storage solvent for the Chiralcel OD column.
42,43
Daicel Chemical Industries, Ltd. (Japan). As informed in the
instruction manual,
temperature, pressure, and flow rate of 0–40 C, 5 MPa
As informed in the instruction manual,
the column
4
2
the column has typical operating
storage solvent is a mixture of the hexane/2-propanol
(90/10 v/v). High column flushing with the use of the analyt-
ical mobile phase liquid is required before the column is
flooded with the storage solvent (for its storage). Ethanol
was found to be the best solvent because it is miscible with
the co-solvent and at the same time dissolving racemic AT
as described in Table 2. Because the retention times are
ꢀ
(
maximum), and 1.0 ml/min, respectively. Its mobile phase
is based on mixtures of alkane and alcohol with a limited
4
3
quantity of basic or acidic modifier compound. Unfortu-
nately, this column could not handle solvents such as
acetone, chloroform, ethyl acetate, methylene chloride,
TABLE 2. Combination of AT solutions and their co-solvents
AT solution
concentration
(mg/ml)
(Solvent :
co-solvent)
ratio(v/v)
Solvent
Co-solvent
Condition
Acetone, 2-propanol
Chloroform
Ethanol
DMSO, DMF
Acetone, 2-Propanol
DMSO, DMF
5
9
5
5
5
5
Hexane, cyclohexane, heptane, isooctane
Hexane, cyclohexane, heptane, isooctane, benzene, toluene
Hexane, cyclohexane, heptane, isooctane, toluene, benzene
Cyclohexane, heptane, isooctane, benzene
Toluene, benzene
1:1–1:3
1:1–1:3
1:1–1:3
1:1–1:3
1:1–1:3
1:1–1:3
Whitish suspension
Whitish suspension
Clear solution
Bilayer solution
Clear solution
Toluene
Clear solution
AT, atenolol; DMSO, dimethyl sulfoxide; DMF, dimethylformamide.
W
Hexane, Ethanol, 2-Propanol: the Chiralcel OD mobile phase constituents.
All mixings were conducted at room temperature.
W
TABLE 3. Applications of Chiralcel OD in the atenolol analysis
Analyte(s)
Analytes’ solvent
Racemic chromatogram
Selectivity (a)
Sample origin
Injection volume(ml)
Ref.
1
1
1
3
3
6,17
8
(
(
(
(
(
(
(
(
(
(
(
(
(
(
S)-atenolol
Methanol
Water
Methanol
Methanol
No information
Not available
Not available
Not available
Not available
No information
No data
No data
No data
1.30
Asymmetric synthesis
Asymmetric synthesis
Asymmetric synthesis
Asymmetric synthesis
Commercial drugs
No data
No data
No data
No data
No data
R)-, (S)-atenolol
R)-, (S)-atenolol
S)-atenolol
9,20
0
1
R)-, (S)-atenolol,
R,S)-atenolol
R,S)-atenolol
R)-, (S)-atenolol,
R,S)-atenolol
R,S)-atenolol
R,S)-atenolol
R,S)-atenolol
R,S)-atenolol
R,S)-atenolol
No data
a
4
4
0
1
Mobile phaseb
Mobile phase
Not available
Available
1.86
4.0–4.4
Commercial drugs
Commercial chemicals
20
20
5
5
5
5
5
1
2
3
4
5
No informatioan
Mobile phasea
Mobile phasec
Mobile phase
No information
Not available
Not available
Available
Not available
Not available
1.73
1.54
No data
1.98
Commercial chemicals
Commercial chemicals
Human plasma, urine
Commercial chemicals
Commercial chemicals
8
20
No data
No data
5
1.46
a
Hexane-(2)-propanol-diethylamine.
b
Hexane-ethanol-diethylamine-acetic Acid.
Hexane-ethanol-diethylamine.
c
Chirality DOI 10.1002/chir

RACEMIC ATENOLOL SEPARATION IN REACTION MEDIA
359
shorter when using ethanol in place of 2-propanol, the use of
ethanol as the mobile phase constituent is highly recom-
mended in the chiral separation of racemic AT existing in
the reaction media.
In the separation of AT enantiomers, this column gave
excellent selectivity results (Table 3) as useful HPLC separa-
samples were injected onto the HPLC units manually. As
the UFLC unit uses an auto-injection to inject the samples,
the volumes could be managed lower than these values.
Although the column operated under different mobile phases
that already gave excellent selectivities, a complete chromato-
gram of the racemic AT separation was generally not
presented. This fact made efforts to adopt easily an analytical
protocol through consideration on the representative peak
shapes/chromatograms that could not be conducted using
the available analytical protocols especially for injections of
the analytes existing in the reaction media.
tion have a values of at least 1.2 and resolution factor (R ) of
s
4
7
1
.5. Very high a values were also obtained, but the separa-
tion process required a complex mobile phase (see Ref. 41).
W
Most of the Chiralcel OD column were applied to analyze
the AT dissolved in methanol or the analytical mobile phase.
Alcohols were excellent solvents for dissolving AT especially
4
8,49
methanol and ethanol.
However, none of these liquids
Trial of Protocols
have been associated with the AT in the resolution processes.
Our database on enzymatic resolution of the chiral drugs
showed that methanol was rarely used as solvent, whereas
The highest flow rate used in the AT analysis was
0,41
1
.0 ml/min3
as given in Table 1. The rate was chosen as
the first step in trials. Because the established quantities
1
3,50
of diethylamine ranged between 0.1% and 0.6%, 3
0,31,39
the
hexane does not dissolve the drug under study.
All the previous AT samples originated from the commer-
cial markets, asymmetric synthesis, and biological fluids.
None of the samples were taken from the racemic resolution
processes. The samples were injected directly onto the HPLC
unit except the biological fluids, which required an extraction
process before injection as the AT was dissolved in uncharac-
terized media (plasma and urine). Approximately 5–20 ml
modifier content was set following these values.
High flow rate, high modifier compound (Protocol-01).
This
protocol was based on the study carried out by Mikuldas
30
et al. who worked on direct analysis of the (S)-AT produced
by an asymmetric synthetic pathway. It was detailed in
Table 1. This pathway did not develop the racemic ATE.
ꢀ
Fig. 2. Representative peaks of the experimental solvents (conditions: Protocol-01, 254 nm, 35 C, 1 ml/min flow rate; injection volume: 5 ml; filtered samples).
DMSO, dimethyl sulfoxide; DMF, dimethylformamide; THF, tetrahydrofuran.
Chirality DOI 10.1002/chir

3
60
AGUSTIAN ET AL.
Mikuldas et al. did not develop the protocol for the racemic
AT analysis. Their chromatograms were only related to the
pure AT compounds. The trials obtained the single enantio-
meric AT peaks in less than 20 min.
peaks during data acquisition. Baselines of the peaks were
quite stable where they were parallel to the horizontal axis
but not for the DMF and vinyl acetate, which formed slanted
baselines.
During analysis periods, it was difficult to record chromato-
graphic combination of the AT and solvent peaks clearly
because the solvents’ intensities were high. The AT peaks
appeared to be very small even almost invisible along the data
acquisition. Therefore, to sharpen the AT chromatograms’
view, data acquisition was started from minute 6–7 (the peak
area size did not change). The UFLC representative peaks for
Before separation was carried out on the racemic AT, a set
of “only solvent” chromatograms was prepared. The results
showed that all the “only solvent” peaks were found generally
in less than 5 min (Fig. 2). Intensities of the solvents devel-
W
oped by the Chiralcel OD were high except for the DMF.
The column was able to recognize the peaks sharply, but
the high intensities could preclude observation on the AT
ꢀ
Fig. 3. Chromatograms of the atenolol (AT) in organic media (conditions: 254 nm, 35 C, 1 ml/min; injection volume: 2ml; centrifuged samples). (A) (R)-AT in dimethyl
sulfoxide (DMSO), (B) (S)-AT in DMSO, (C) (R, S)-AT 5 mg/ml, (D) (R,S)-AT in dimethylformamide (DMF), (E) (R,S)-AT in tetrahydrofuran(THF), (F) (R,S)-AT in
3
CHCl 5mg/ml diluted with ethanol (1:1 v/v), (G) (R,S)-AT in acetone 5 mg/ml, and (H) (R,S)-AT in DMSO 5 mg/ml diluted with Toluene (1:1 v/v).
Chirality DOI 10.1002/chir

RACEMIC ATENOLOL SEPARATION IN REACTION MEDIA
7,57
361
the AT dissolved in some organic media resulted from the
(R > 1.20).4
Further works are needed to increase the
S
Protocol-01 were presented in Figure 3. R and a values were
RS values.
S
given in Table 1. Selectivity of the AT separation were moder-
Good peaks’ shapes were generated when these were
injected with the pure AT enantiomers (Fig. 3(A and B)).
The (R)- and (S)-AT peaks appeared faster than this protocol
owner, yet the first eluted enantiomer was the same. The (R)
enantiomer was recognized in 8.5 min, whereas the (S)-AT
eluted at 10.3 min. The UFLC unit was proven able to gener-
ate both enantiomers peaks at faster time. Intensities of the
detector created by each 9 mg/ml single enantiomer were
quite high, although only 2 ml samples were injected. The cel-
lulose-based column could recognize the AT compound at a
low injection volume.
5
6
ate and similar to the Mikuldas et al. results; however, the
resolution factors were lower than their findings and the
minimum recommended value for complete separation
Formation of good peaks was also observed when the
W
Chiralcel OD column was used to separate the racemic AT
dissolved in some common organic solvents as shown in
Figure 3(C–F). Compared with the single enantiomers, a
slight difference in retention time was noted where the race-
mic AT peaks appeared slightly quicker than the single enan-
tiomers. It was found that the detected intensities of the race-
mate were not high (around 3.5–7 mAU). The racemate
concentration in the reaction media was set at 5 mg/ml,
which was around half of the individual AT enantiomer con-
centration. Because the AT does not dissolve in hexane, low
concentrations of the drug were used in the analysis to
W
prevent sedimentation of the AT inside the Chiralcel OD
column. Further observations on the peaks’ characteristics
indicated that the baselines of each enantiomer peak were
almost parallel to the horizontal axis, but most of the base-
lines deviated from the horizontal zero point. Chromato-
graphic readings of the racemic AT gave almost the same
values for both enantiomers as can be observed from tables
inserted in each diagram of the racemate. This means the
Fig. 4. Chromatographic diagrams of atenolol (AT) dissolved in Sorensen
ꢀ
buffers (conditions: 254 nm, 35 C, 1 ml/min; injection volume: 2 ml; centri-
W
Chiralcel OD can recognize the AT concentrations even at
fuged samples). (A) 67 mM buffer pH 6.6 [(R,S)-AT]: 10 mg/ml and (B)
7 mM buffer pH 7.4 [(R,S)-AT]: 10 mg/ml.
6
lower concentrations than the single enantiomers.
ꢀ
Fig. 5. Representative peaks of atenolol (AT) dissolved in dimethyl sulfoxide (DMSO) and Sorensen buffers (conditions: 254 nm, 35 C, 0.75 ml/min; injection
volume: 2 ml; centrifuged samples). (A) (R)-AT in DMSO 9 mg/ml, (B) (S)-AT in DMSO 9 mg/ml, (C) 67 mM buffer pH 6.6 [(R,S)-AT]: 10 mg/ml, and (D)
7 mM buffer pH 7.4 [(R,S)-AT]: 10 mg/ml.
6
Chirality DOI 10.1002/chir

3
62
AGUSTIAN ET AL.
Because the racemic AT has high solubility in water,
very close to each other. Compared with the pure AT chroma-
tograms (Fig. 3(A and B), the results differed greatly,
although the racemic AT had only slightly higher concentra-
tions than the single enantiomers. Two factors could possibly
cause this problem: (1) effect of the water-based analytes
Sorensen phosphate buffers were also selected as alternative
media for the enzymatic reaction. The aqueous solutions
were injected directly to the UFLC unit without purification
or pretreatment. Chromatograms of these racemic AT were
given in Figure 4(A and B).
56–59
where the buffers assisted in the sample separation
and
The chiral column separated and recognized the AT
enantiomers. Higher intensities than the racemic AT in the
organic solvents were obtained. Both peaks’ shapes were
good, but the enantiomeric peaks had wide bases and were
(2) more racemic AT concentrations were diluted by the
mobile phase although only 2 ml samples were injected.
Despite that the analytical mobile phase had high hexane
content (80% v/v), the buffer-based analytes were resolved
ꢀ
Fig. 6. Peaks of pure and racemic atenolol (AT) obtained by Protocol-03 (conditions: 276 nm, 35 C, 0.70 ml/min; injection volume: 1 ml; centrifuged samples).
A) (R)-AT in dimethyl sulfoxide (DMSO) 9 mg/ml, (B) (S)-AT in DMSO 9 mg/ml, (C) Sorensen buffer pH 6.6 [(R,S)-AT]: 10 mg/ml, (D) Sorensen buffer
(
pH 7.4 [(R,S)-AT]: 10 mg/ml, (E) (R,S)-AT in DMSO 5 mg/ml, (F) (R,S)-AT in dimethylformamide (DMF) 5 mg/ml, (G) (R,S)-AT in tetrahydrofuran (THF)
5
mg/ml, and (H) (R,S)-AT in THF (5 mg/ml) diluted with toluene (1:1 v/v).
Chirality DOI 10.1002/chir

RACEMIC ATENOLOL SEPARATION IN REACTION MEDIA
363
highly by the cellulose tris-(3,5-dimethylphenylcarbamate)
chiral stationary phase.
Low flow rate, high modifier compound (Protocol-02).
The
protocol was a modified form of the first protocol. Same
operating conditions were used, but the mobile phase flow
rate was lower than the original value (reduced by 25%). As
the Protocol-01 produced the enantiomeric peaks with wide
bases for the buffer solutions, it was decided to reduce the
flow rate as it has a dramatic influence on retention time
However, the peaks’ baselines were not parallel to the
horizontal axis. These lines deviated severely. The lines were
generated automatically by the UFLC unit, albeit modification
could be carried out using the existing software, but it seems
difficult to change the baselines as the peak areas would also
change. These slanted lines influenced the chromatographic
readings. As shown in the inserted tables, areas of the (R)-
and (S)-AT differed greatly for both buffer types (area of the
44
and selectivity. Protocol-02 was examined on the single
enantiomeric and the racemic AT dissolved in the Sorensen
buffers. Results of the racemic AT chromatograms are
presented in Figure 5.
(R)-enantiomers was almost twice of the (S)-AT area).
Fig. 7. Chromatograms of single enantiomers and racemic atenolol (AT) produced by Protocol-04 (conditions: 275 nm, 35, 0.5 ml/min flow rate; injection volume:
ml; centrifuged samples). (A) (R)-AT in dimethyl sulfoxide (DMSO) 9 mg/ml, (B) (S)-AT in DMSO 4.50 mg/ml, (C) 67 mM buffer pH 6.6 [(R,S)-AT]: 10 mg/ml,
1
(
D) 67 mM buffer pH 7.4 [(R,S)-AT]: 10 mg/ml, (E) (R,S)-AT in DMSO 5 mg/ml, (F) R,S)-AT in dimethylformamide (DMF) 5 mg/ml, (G) (R,S)-AT in tetrahydro-
furan (THF) 5 mg/ml, and (H) (R,S)-AT in THF (5 mg/ml) diluted with toluene (1:1 v/v).
Chirality DOI 10.1002/chir

3
64
AGUSTIAN ET AL.
Single enantiomeric peaks were formed excellently (Fig. 5
A and B)) by the cellulose-based CSP. It recognized the
R)- and (S)-AT areas highly. The peak intensities obtained
than Protocol-01. These peak intensities were obtained from
smaller injection volumes (only 1 ml). This fact indicates that
the used mobile phase gave good environment to the CSP
and analyte so that the CSP recognized the presence of AT
enantiomers even at a small injection volume. It is likely that
the high alcohol content increased solubility of the AT in the
mobile phase. Lower quantity of the diethylamine compound
still provided good peak shapes of the separation process.
The results obtained from Protocol-03 were better than
those from Protocols-01 and À02. High intensities obtained
using Protocol-03 could eliminate or reduce the peaks of
either the solvent or the buffers. As described in each dia-
gram, the DMSO peak disappeared from the chromato-
graphic detection, whereas the Sorensen buffers gave low
peak areas. The peak baselines were parallel to the horizontal
axis. Values of the peak area of the racemic AT were almost
the same (see tables in Fig. 6(C and D)), similar to the chro-
matographic characteristics obtained by Protocol-01 for the
AT dissolved in the organic solvents. The separation factor,
(
(
by Protocol-02 for both enantiomers were higher than the
previous observations (Protocol-01). A large difference in
the peak areas between the single enantiomers was observed.
But a slanted baseline was found on the (R)-enantiomer.
The buffers-based separation produced similar results as
the previous protocol. The peaks’ shapes were good but wide
at their bases. The baselines were heavily nonparallel to
the horizontal axis, which indeed influenced the peaks’ sizes.
Areas of the (R)-AT were found higher than the (S)-enantiomer
as can be observed from tables in Figure 5(A and B). Several
differences to the Protocol-01 results were also observed.
Reduction of the mobile phase flow rate by 25% indeed had
significant effects on the separation process of the AT enan-
tiomers. The new protocol produced both enantiomeric peaks
slower than the Protocol-01. The (R)- and (S)-AT appeared at
minutes 10.1–10.2 and 11.8, respectively, which differed
approximately 2 min from the Protocol-01 results. However,
compared with the previous protocol peaks’ areas, the later
protocol peaks’ sizes were higher (130,000–280,000 peak
areas). These results proved that lowering the flow rate did
increase interaction between the analyte and CSP.
56
as given in Table 1, showed moderate to easy process. But
this protocol produced uncompleted chiral separation as the
RS values were less than 1.2.
Low flow rate, high alcohol content (Protocol-04).
Protocol-
Low flow rate, low modifier compound (Protocol-03).
This
04 was developed to determine the optical purity and
separate b-blocking substances available in commercial
drug chemicals. The protocol used 2-propanol instead of
ethanol as the mobile phase constituent. It has higher
alcohol content than the previous protocols, but the modi-
fier composition was the same as the third protocol. Be-
cause maximum allowable flow rate of 2-propanol is lower
than ethanol,43 the Chiralcel OD column was operated
using the lowest mobile phase flow rate (Table 1).
Symmetrical peaks were produced during the chiral
separations (Fig. 7). Quite stable peaks’ baselines and
high peaks’ intensities were observed. This protocol gave
the highest enantioselectivity factors than the methods
described previously (Table 1). However, similar to Proto-
procedure was applied because lower flow rate gave quite
higher peaks’ areas. The protocol was adopted from Santoro
31
W
et al. that used the Chiralcel OD column to separate and
quantify the AT content in drug tablets. Compared with the
previous protocols, Protocol-03 was not only lower in the flow
rate but also significantly lower in the modifier quantity
W
(
see Table 1). But it used higher alcohol content and a higher
UV detection wavelength. Results of the data acquisition
of the single enantiomers (in DMSO) and racemate (in
Sorensen buffers) are shown in Figure 6(A–D).
The peaks appeared at minutes 9.9 and 11.7, which were
not too different from the previous procedures. Although
concentrations of the enantiomers were the same, this proto-
col gave higher enantiomeric peak intensities (2–3 times)
cols 01–03, the produced R were lower than the minimum
S
TABLE 4. Performance of the enzymatic processes
Initial peak area Final peak area
Reaction
time (h)
UFLC
result
Protocol
Media
Enzyme
CALB
(R)-AT
(S)-AT
(R)-AT
(S)-AT
Conversion (%)
a
a
0
1
Buffer pH 6.6
Buffer pH 7.4
DMSO
404,565
365,272
364,639
386,184
859,347
962,174
336,419
311,406
405,557
387,844
719,011
599,518
4
4
6
6
4
4
4
4
575,084
479,770
283,940
273,886
834,251
811,644
848,275
869,367
59,744
550,784
550,784
295,047
275,667
656,905
692,252
821,430
825,555
70,809
Figure 8(A)
Figure 8(B)
CALB
LAPS
LAPS
CALB
CALB
CALB
CALB
PFL
X
X
X
X
X
X
X
X
X
X
X
X
R
R
R
R
R
R
R
R
R
R
R
R
S
:26.12
:27.67
DMF
S
0
2
3
Buffer pH 6.6
Buffer pH 7.4
Buffer pH 7.0
Buffer pH 7.4
THF
Figure 8(C)
Figure 8(D)
a
0
937,008 1,001,900
902,967
382,653
514,793
1,486,801 1,526,610
1,485,625 1,535,572
592,618
711,706
961,598
398,041
539,598
4
:91.78
Figure 8(E)
Figure 8(F)
DMF
HPL
4
10–12
10–12
6
393,648
1,405,513 1,339,520
1,522,099 1,125,854
375,060
S
:31.67
0
4
Buffer pH 6.6
Buffer pH 7.4
THF
CALB
CALB
Lipoprotein
CALB
611,694
731,471
0
0
Figure 8(G)
Figure 8(H)
DMF
6
152,396
255,771
UFLC, ultra fast liquid chromatography; CALB, Candida antarctica lipase fraction B; LAPS, Lipase amino from Burkholderia cepacia; PFL, Pseudomonas fluorescence
lipase; HPL, hog pancreatic lipase; DMSO, dimethyl sulfoxide; DMF, dimethylformamide; THF, tetrahydrofuran.
a
Conversions cannot be calculated.
Chirality DOI 10.1002/chir

RACEMIC ATENOLOL SEPARATION IN REACTION MEDIA
365
Fig. 8. Ultra fast liquid chromatography chromatograms of the enzymatic process solutions. (A) Sorensen buffer pH 7.4 [(R,S)]-atenolol (AT): 5 mg/ml; injection:
ml, (B) Sorensen buffer pH 7.4 [(R,S)]-AT: 5 mg/ml; injection: 2 ml, (C) Sorensen buffer pH 6.6 [(R,S)]-AT: 10 mg/ml; injection: 2 ml, (D) Sorensen buffer pH 7.4
2
[
(R,S)]-AT: 10 mg/ml; injection: 2 ml, (E) (R,S)-AT in tetrahydrofuran (THF) 5 mg/ml; injection: 1 ml, (F) (R,S)-AT in dimethylformamide (DMF) 5 mg/ml; injection:
1
ml, (G) (R,S)-AT in THF 5 mg/ml; injection: 1 ml, and (H) (R,S)-AT in DMF 5 mg/ml; injection: 1 ml.
recommended chiral resolution for a complete chromato-
graphic separation.
Protocol-03 (Fig. 6(A)) and was three times higher than
the first protocol for the same enantiomer (Fig. 3(A)).
The racemic AT dissolved well in the mobile phase con-
taining high 2-propanol composition. Although Protocol-
04 produced higher peak areas, the protocol was limited
by maximum allowable working pressure (5 MPa) in
which 2-propanol tends to develop higher column pres-
Compared with the earlier protocols, the AT peaks
appeared slower. The (R)-AT appeared at minute 12,
whereas the (S)-enantiomer was eluted at minute 15. How-
ever, this flow rate developed the highest time difference
between the AT enantiomers during the separation pro-
cess. Excellent peaks’ areas were also generated by this
protocol. As described in Figure 7(A), the (R)-AT peak
area was slightly higher than the result produced by
4
3
sures than ethanol. With the use of this protocol, the
W
Chiralcel OD column recognized a very hydrophobic
compound highly as drawn in Figure 7(H).
Chirality DOI 10.1002/chir

3
66
AGUSTIAN ET AL.
Protocol-05.
Because high flow rate and alcohol con-
to easy separation factors, but the resolution values
required further improvement.
tent develop high pressure in the column, it is decided
not to observe the chiral AT separation operated by the
last protocol. Although Protocol-05 has high ethanol com-
High mobile phase flow rate and increased concentra-
tion of modifier compound in mobile phase but low alco-
hol content were used by Protocol-01, which gave smooth
peaks for the solvents and AT. At these conditions, separa-
tion of the AT (racemate and single enantiomers) dis-
solved in organic solvents was found better than the
buffer-based analytes. Although Protocol-02 had lower flow
rate but higher area than the first protocol, the column
still could not separate the racemic AT peaks satisfactorily.
Increased peak intensities of the organic solvents were
produced by both protocols.
Reduced flow rate and decreased concentration of modifier
compound but high alcohol content and increased UV detection
wavelength (Protocols -03 and À04) gave the best separation
results. These protocol produced higher intensities obtained
from smaller injection volumes than the previous protocols,
although the AT concentrations were the same. It reduced or
eliminated peaks of either the solvent or buffer and provided
peaks with baselines parallel to the horizontal axis.
W
position, which is useful to clean-up the Chiralcel OD
4
2
column, this compound is related to low mobile phase
flow rate. As described in the instruction manual, the
maximum flow rate for 100% ethanol is 0.5 ml/min.
4
2,43
Our observation on Protocols 01–04 indicated that when
the cleaning process was carried out at this flow rate,
the column pressure could increase to more than its max-
imum allowable value. Hence, same as to Protocol-04,
high alcohol composition would risk the column, espe-
cially when it is operated under the typical flow rate for
W
the Chiralcel OD column (1.0 ml/min).
Enzymatic acetylation of racemic AT.
The enzymatic pro-
cesses were aimed to resolve the (R,S)-AT to yield the
active isomer of the AT compound. Because its racemic
compound shows hydrophilic behavior, it cannot be
processed in hydrophobic solvents. Hence, water miscible
compounds (DMSO, DMF, and THF) and buffer solutions
were chosen as the reaction media. Results of the
processes are given in Table 4, whereas the UFLC chro-
matograms are presented in Figure 8. It was observed that
the AT enantiomers reacted with the acetate compound in
both media. Some reactions still required longer reaction
times to increase their conversions. High conversions
were obtained once the reactions were conducted in
organic media using Pseudomonas fluorescence lipase
ACKNOWLEDGMENTS
Joni Agustian thanks the MTCP scholarship from Ministry
of Higher Education and Universiti Sains Malaysia Graduate
Assistant Scheme for assisting his study.
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