Fast liquid chromatography for racemic atenolol acetate separation—The analytical protocol

Article abstract of DOI:10.1002/chir.22769

Kinetic resolution of (R,S)-atenolol is a faster strategy to produce (S)-atenolol. Since this racemate is a less soluble compound, resolution of its ester offers high concentrations in the process. A good analytical method is required to observe the enantiomer concentrations. This paper described application of ultra-fast liquid chromatography on the atenolol ester separation using different resolution media and analytical procedures. Chiralcel OD column resolved the ester. The chromatograms indicated different characteristics of the process. The enantiomers could be recognized by the column in less than 1 (one) hour. Symmetrical peaks were obtained, but several procedures produced peaks with wide bases and slanted baselines. Efficient enantioresolution was obtained at high mobile phase flow rate, decreased concentration of amine-type modifier, but increased alcohol content in the mobile phase. High UV detection wavelength was required. At 1.0?mL/min, the (90/10/0.5) composition resulted α?=?1.46 and R_S?=?0.9998 that were good separation.

Full text of DOI:10.1002/chir.22769

Received: 13 August 2017
DOI: 10.1002/chir.22769
Revised: 31 August 2017
Accepted: 3 September 2017
R E G U L A R A R T I C L E
Fast liquid chromatography for racemic atenolol acetate
separation––The analytical protocol
Joni Agustian1 | Azlina Harun Kamaruddin2 | Hassan Y. Aboul‐Enein3
1 Department of Chemical Engineering,
Abstract
Universitas Lampung, Bandar Lampung,
Kinetic resolution of (R,S)‐atenolol is a faster strategy to produce (S)‐atenolol.
Lampung, Indonesia
2 School of Chemical Engineering,
Engineering Campus, Universiti Sains
Malaysia, Penang, Malaysia
3 Pharmaceutical and Medicinal Chemistry
Department, Pharmaceutical and Drug
Industries Division, National Research
Centre, Cairo, Egypt
Since this racemate is a less soluble compound, resolution of its ester offers high
concentrations in the process. A good analytical method is required to observe
the enantiomer concentrations. This paper described application of ultra‐fast
liquid chromatography on the atenolol ester separation using different resolu-
tion media and analytical procedures. Chiralcel OD column resolved the ester.
The chromatograms indicated different characteristics of the process. The
enantiomers could be recognized by the column in less than 1 (one) hour.
Symmetrical peaks were obtained, but several procedures produced peaks with
wide bases and slanted baselines. Efficient enantioresolution was obtained at
high mobile phase flow rate, decreased concentration of amine‐type modifier,
but increased alcohol content in the mobile phase. High UV detection wave-
length was required. At 1.0 mL/min, the (90/10/0.5) composition resulted
α = 1.46 and RS = 0.9998 that were good separation.
Correspondence
Azlina Harun Kamaruddin School of
Chemical Engineering, Engineering
Campus, Universiti Sains Malaysia
14300 Nibong Tebal, Penang, Malaysia.
Email: chazlina@usm.my
Hassan Y. Aboul‐Enein, Pharmaceutical
and Medicinal Chemistry Department,
Pharmaceutical and Drug Industries
Division, National Research Centre,
Dokki, Cairo, 12622, Egypt.
KEYWORDS
Email: haboulenein@yahoo.com
atenolol, Chiralcel OD, enantiomers separation, kinetic resolution, ultra‐fast liquid chromatography
Funding information
Financial supports from Universiti Sains
Malaysia (PRGS: 1001/PJKIMIA/
8044030), MOSTI (Science Fund: 305/227/
PJKIMIA/6013337), MTDC (304/
PJKIMIA/6053010) were deeply
acknowledged.
1 | INTRODUCTION
(R,R)‐Co‐(salen) complexes, (R)‐ or (S)‐epichlorohydrin
being present during the syntheses.4-10 Enzymatic reso-
Switching (R,S)‐atenolol to (S)‐atenolol would develop
lesser side effects as the single enantiomer avoids the side
effects generated by the racemate,1 and (R)‐atenolol
has not lacked of the side effects.2,3 Many pathways
were studied to form the (S)‐atenolol either synthesis or
resolution routes.
Assymetric syntheses of the (S)‐atenolol were con-
ducted using chiral or achiral raw materials, which
required the chiral catalysts or addendums such as
lutions of the racemic compound by immobilized
lipases were developed via enantioselective esterifica-
tion or and hydrolysis.11-15 Kinetic resolutions of the
(R,S)‐atenolol to give the (S)‐atenolol could be done
microbially using Rhizopus arrhizus or Geothricum
candidum; however, the (R,S)‐atenolol acetate was used
as well to give the single enantiomeric atenolol.11
Since the racemic atenolol acetate could develop
the single enantiomeric compound, it is important
Chirality. 2017;1–7.
wileyonlinelibrary.com/journal/chir
© 2017 Wiley Periodicals, Inc.
1

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AGUSTIAN ET AL.
to estimate both enantiomers' concentration during
the resolution processes.
2 | MATERIALS AND METHODS
Although high‐performance liquid chromatography
(HPLC) is the most widely used method to quantify the
atenolol enantiomers using columns such as Chiralcel
OD,6-10,16-18 Amycoat,19 Eurocel 01,20 RegisCell,21
Chiral‐CBH,22 Chirex 3022 (S),23,24 and Chirobiotic V2,25
which were operated at various protocols, only some
articles were related to the atenolol esters measurement.
The racemic atenolol caproate and acetate were
analyzed using infrared spectrometer or nuclear magnetic
resonance or thin layer chromatography.11,26,27
Previously, Enquist and Hermansson28 observed atenolol
diacetate concentrations using HPLC column of α1‐AGP
working under water‐based mobile phase. As the
enzymatic reactions mostly occurred in organic solvents,
direct measurement of the (R,S)‐atenolol acetate
during the reactions would reduce the analytical time.
To the best of our knowledge, there has been no article
mentioning chromatograms of the (R,S)‐atenolol
acetate generated by ultra‐fast liquid chromatography
(UFLC) where the racemate exists in organic media.
Hence, selection of a column and an analytical protocol
that can satisfy observations of the (R,S)‐atenolol acetate
concentrations during the reaction using UFLC is
essential.
2.1 | Materials
(R,S)‐atenolol (99%), (S)‐atenolol (97%), and (R)‐atenolol
(99%) were bought from Nanjing Chemlin Chemical
Industry Co Ltd (China), Tocris Bioscience (England),
and Sigma‐Aldrich (M) Sdn Bhd (Malaysia), respec-
tively. 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.
Atenolols and other chemicals were used without
purification.
2.2 | Synthesis of (R,S)‐atenolol acetate,
(S)‐atenolol acetate, and (R)‐atenolol
acetate
Synthesis of the ester was conducted through chemical
acetylation of (R,S)‐atenolol (Figure 1). It was started by
dissolving 8 g racemate and 3.4 mL acetic anhydride into
180 mL acetone under vigorous stirring for 5 minutes. The
mixture was refluxed at 333 to 338 K for 3 hours. After
acetylation, solvent was removed under vacuum. The
collected residue was diluted with 25 mL deionised water
and stirred for around minutes at room temperature.
Later, 100 mL dichloromethane was added to the solution
and the stirring was continued for 30 minutes. The mix-
ture was transferred to a separating funnel and left for
half an hour at room temperature. After the aqueous
phase was removed, the organic layer was placed into
250 mL glass beaker and then extracted with 50 mL
sodium carbonate solution (1% w/v) with stirring for
30 minutes. After that, the mixture was then settled for
half an hour, and the organic phase was collected. These
steps were repeated 5 times, but the last settling was con-
ducted overnight. Once the aqueous phase was removed,
the organic layer was then placed under constant air flows
and stirring to evaporate the solvent. The crystals were
finally dried overnight at 318 to 323 K. After drying, the
crystals were crushed into powder and were stored at
FIGURE 1 Formation of the racemic atenolol acetate
TABLE 1 Characteristics of (R,R)‐Whelk O1 chiral column
Mobile Phase
Operating Conditions
Injection (5 μL)
(R,S)‐atenolol
(R,S)‐ester
Peak separation
Chromatogram
Figure 2A
Figure 2B
Hex/EtOH/DEA (75/25/0.1)
Hex/EtOH/DEA (75/25/0.1)
Hex/EtOH/DEA (75/25/0.1)
Hex/EtOH/DEA (75/25/0.1)
Hex/EtOH/DEA (80/20/0.1)
Hex/IPA (90/10)
35°C/275 nm/0.75 mL/min
35°C/275 nm/0.75 mL/min
35°C/254 nm/0.75 mL/min
35°C/254 nm/0.75 mL/min
35°C/254 nm/0.75 mL/min
35°C/254 nm/0.75 mL/min
No
No
No
No
No
No
(R,S)‐ester
Figure 2C
Figure 2D
Figure 2E
(R,S)‐atenolol
(R,S)‐ester
(R,S)‐ester
Figure 2F

AGUSTIAN ET AL.
3
(A)
(C)
(E)
(B)
(D)
(F)
FIGURE 2 Chromatograms racemic atenolol and its ester (A: racemic atenolol; B: racemic atenolol ester; C: racemic atenolol ester;
D: racemic atenolol; E: racemic atenolol; F: racemic atenolol ester. Chromatographic conditions are presented in Table 1
TABLE 2 Protocols for (R,S)‐atenolol ester
Protocol
Mobile Phase
60a/40b/0.1c
75a/25b/0.1c
80a/20b/0.1c
85a/15b/0.1c
85a/15b/0.5c
90A/10B/0.1C
90a/10b/0.4c
90A/10B/0.4C
90a/10b/0.1c
90a/10b/0.5c
Flow Rate, mL/min
α
RS
Chromatogram
Figure 3C
Figure 3D
Figure 3E
Figure 3F
Figure 3G
Figure 3H
Figure 3I
A
B*
C
D
E
F
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.85
1.00
1.00
No data
1.18
1.14
1.16
1.18
1.17
1.18
1.17
1.17
1.46
No data
0.3838
0.3135
0.7163
0.5389
0.9260
0.7843
0.8013
0.7766
0.9998
G
H
I
Figure 3J
Figure 3K
Figure 3L
J
aHexane;
bEthanol;
cDiethylamine; UFLC: 35°C, 275 nm;
*The starting protocol.

4
AGUSTIAN ET AL.
(A)
(B)
(C)
(D)
(F)
(E)
(H)
(G)
(J)
(I)
(K)
(L)
FIGURE 3 Chromatograms of atenolol acetate. Chromatographic conditions are shown in Table 2

AGUSTIAN ET AL.
5
FIGURE 4 Peaks of racemic atenolol (left) and mixture of racemic atenolol and its ester (right) developed by using a mobile phase
composed of hexane:ethanol :diethylamine (90:10:0.5 (v.v.v)
room temperature before used. These steps were copied
for synthesis of (S)‐atenolol acetate and (R)‐atenolol
acetate.
racemic atenolol, and its ester was similar with the mobile
phase applied in the transesterification process.
The chromatogram for the racemic atenolol was not
high, but the column provided a good measurement of
the racemic atenolol ester (Figure 2A,B. When the
wavelength was reduced to 254 nm, the column identi-
fied the racemic atenolol better than the previous
method and no change on the ester peak was observed.
Similar results were found when the mobile phase was
changed to a lower alcohol content (80/20/0.1% (v/v)).
But the mobile phase with the lowest alcohol content
gave a peak with a very wide base. In general, the
racemic atenolol and its ester appeared at minute 7
and minute 16, respectively. The column can identify
the racemic atenolol and its ester but cannot separate
the enantiomers.
2.3 | Apparatus
Analysis of the enantiomers was performed on a
Shimadzu UFLC LC‐20A Prominence system. The system
consist of 2 units LC‐20 AD dual plunger parallel flow
solvent delivery pump, a SIL‐20ACHT auto‐sampler, a
SPD‐20A UV‐VIS detector, a CTO‐20 AC column oven,
a DGU‐A3 degasser unit, and the CBM‐20A system
controller. A personal computer is linked to the UFLC
using the Shimadzu LC solution Real Time Application
software.
Since no peak separations were found with the
(R,R)‐Whelk O1 column, Chiralcel OD was used to
separate the racemic atenolol ester in the same man-
ner as the racemic atenolol. Comparison of α and RS
developed by various mobile phase compositions and
flow rates using the available UFLC unit were given in
Table 2.
In general, each mobile phase composition and flow
rate gave a moderate selectivity. The resulted values were
found in the range of 1.14 to 1.18. Although the alcohol
content had been decreased to the same with the storage
mobile phase composition and the flow rate was increased
to the typical column flow rate (1.0 mL/min), the selectiv-
ity values were almost the same. But the big difference
was observed from the RS.
2.4 | Samples preparation and analysis
About 1 to 5 μL of 25 mM (R,S)‐atenolol acetate dissolved
in organic solvents was injected automatically at a time
into the HPLC column with certain a mobile phase
composition (described later). UV/Vis detector was set at
the wavelength of 254 to 276 nm. The UFLC was operated
at normal phase at 35°C. Qualitative and quantitative
analysis were conducted on chromatograms via Shimadzu
LC solution postrun analysis software based on the
standard procedure for this instrumentation.29
3 | RESULTS AND DISCUSSION
The first column tested to identify the racemic atenolol
ester was (R,R)‐Whelk O1 column made by Regis
Technologies Inc (Illinois). The column has been used
extensively to measure the nonsteroidal anti‐inflamma-
tory drugs such as ibuprofen and ketoprofen and has
the same mobile phase component as Chiralcel OD. The
experimental results using the column were shown in
Table 1. The first mobile phase used to identify the
As described in Table 2, the RS differed greatly. The
separation factor for the starting protocol produced
the lowest RS value amongst the tested protocols
(α = .3838). However, this protocol was better than the
protocol that used the highest alcohol content (40%),
which gave no racemic ester separation (Figure 3C).
Lower alcohol content indeed developed higher RS than
the starting method under the same mobile phase flow

6
AGUSTIAN ET AL.
rate (0.5 mL/min, Protocol A‐G). At this flow rate, the
highest RS was found at the mobile phase composition
of 90%‐hexane + 10%‐ethanol (Protocol G). This composi-
tion was applied during all the racemic ester analysis as it
will risk the column if the mobile phase has lower alcohol
content than the column storage composition because the
atenolol does not dissolve in hexane.
Although the flow rate of 0.5 mL/min offered higher
RS, the typical flow rate for the column was chosen as
the analytical mobile phase flow rate (Protocol I, J) since
this flow rate gave faster enantiomeric separation
time (shown in Figure 3) and higher flow rates tended
to provide better enantiomeric resolution factors. Obser-
vation on the modifier compound indicated that lower
modifier quantity developed higher RS at low flow rate,
but higher flow rate produced higher RS.
4 | CONCLUSION
The Chiralcel OD column could separate the (R,S)‐ateno-
lol acetate enantiomers, which required almost 1 hour
separation time. Both enantiomeric peaks appeared sym-
metrically, but several protocols gave peaks with wide
bases and slanted baselines. The experiments indicated
that efficient enantioresolution of (R,S)‐atenolol acetate
was obtained at high mobile phase flow rate, decreased
concentration of amine‐type modifier, but increased alco-
hol content in mobile phase. High UV detection wave-
length was required. At 1.0 mL/min, the (90/10/0.5)
mobile phase composition resulted α = 1.46 and
RS = 0.9998 that were good for enantiomers' separation.
ACKNOWLEDGMENTS
Based on the starting protocol, the (S)‐atenolol ester
appeared faster than its opponent as given in Figure 3A,
B. Its retention time was 14.044 minutes, while the (R)‐
atenolol ester was detected at 15.052 minute. The mobile
phase with higher alcohol content than the starting proto-
col indeed gave quicker retention time as shown in
Figure 3C, but no racemic atenolol ester separation was
developed. Other UFLC protocols produced the racemic
atenolol ester retention times slower than the starting pro-
tocol, but some of these protocols gave better enantio-
meric separation.
Financial supports from Universiti Sains Malaysia (PRGS:
1001/PJKIMIA/8044030), MOSTI (Science Fund: 305/
227/PJKIMIA/6013337), MTDC (304/PJKIMIA/6053010)
were deeply acknowledged. Joni Agustian thanks to the
MTCP scholarship from MOHE, USM Graduate Assistant
Scheme, and USM Graduate Research Assistant Scheme
for assisting his study.
The authors declare no conflict of interest.
ORCID
The starting protocol developed an overlapping situa-
tion of enantiomeric peaks similar to the Protocol C,
although both enantiomers' peaks appeared faster. This
was not good as the racemic ester could not be separated
finely. Although the alcohol content of 15% (Protocol
D, E) provided faster retention time than the storage
mobile phase composition under the same flow rate, the
later protocol developed better racemic ester separation.
Hence, the (90/10) content was selected as the
mobile phase composition. This content was able to
provide peaks of the racemic atenolol and its ester
vmixture (Figure 4). Separation of the racemic atenolol
enantiomers produced by this mobile phase composition
using 0.5% DEA content gave a bigger difference on the
peaks retention time than the mobile phase used in
the transesterification stage (ie, the starting protocol for
the hydrolysis stage). Further observations on this content
suggested that the flow rate of 1.0 mL/min offered faster
retention time than its rivals (0.5 and 0.85 mL/min).
Under this flow rate, the (90/10/0.5) composition resulted
α = 1.46 and RS = 0.9998 that were good for enantiomers
separation. But overlap peaks were observed between the
(S)‐atenolol and (S)‐atenolol ester when both racemates
were mixed. The modifier content also influenced
chromatographic intensity where higher modifier
created higher peaks area.
Hassan Y. Aboul‐Enein
7009
http://orcid.org/0000-0003-0249-
REFERENCES AND NOTES
1. Kumar A, Vyas KD, Singh D, et al. Novel diasteriomeric salts of
atenolol and their use in the production of optically active aten-
olol. WIPO Patent WO 2006/046252 A2. 2006.05.04.
2. Stoschitzky K, Lindner W, Kiowski W. Stereoselective vascular
effects of the (R)‐ and (S)‐enantiomers of propranolol and
atenolol. J Cardiovasc Pharmacol. 1995;25:268‐272.
3. Stoschitzky K, Kahr S, Donnerer J, et al. Stereoselective increase
of plasma concentrations of the enantiomers of propranolol and
atenolol during exercise. Clin Pharmacol Ther. 1995;57:543‐551.
4. Mehta SR, Bhawal BM, Deshpande VH, Gurjar MK. Process for
producing atenolol of high optical purity. US Patent US
6,982,349B1. 2006.01.03.
5. Bose DS, Narsaiah AV. An efficient asymmetric synthesis of
(S)‐atenolol: using hydrolytic kinetic resolution. Bioorg Med
Chem. 2005;13:627‐630.
6. Kawthekar RB, Bi WT, Kim GJ. Synthesis and application of
bimetallic chiral [Co(salen)]‐type complexes: a new catalytic
approach to synthesis of optically pure β‐blockers via kinetic
resolution of epichlorohydrin. Appl Organomet Chem.
2008;22:583‐591.

AGUSTIAN ET AL.
7
7. Kawthekar RB, Kim GJ. Enantioselective synthesis of β‐blockers
via hydrolytic kinetic resolution of terminal oxiranes by
using bimetallic chiral {{2,2′‐[Cyclohexane‐1,2 diylbis
(nitrilomethylidyne)] bis[phenolato]}(2‐)}cobalt([co (salen)])‐
type complexes. Helv Chim Acta. 2008;91:317‐332.
dimethyl phenylcarbamate) chiral stationary phase using ultra
fast liquid chromatography. Chirality. 2012;24(5):356‐367.
19. Ali I, Saleem K, Gaitonde VD, Aboul‐Enein HY, Hussain I. Chi-
ral separations of some β‐adrenergic agonists and antagonists on
AmyCoat column by HPLC. Chirality. 2010;22:24‐28.
8. Kitaori K, Takehira Y, Furukawa Y, Yoshimoto H, Otero J. A
practical synthesis of optically active atenolol from chiral
epichlorohydrin. Chem Pharm Bull. 1997;45(2):412‐414. 1997
20. Herbert Knauer GmbH. Applications journal V01/2011.
Available: http://www.knauer.net/dwnld_fls/a_e_applications_
journal.pdf (2011.08.28).
9. Takehira Y, Saragai N, Kitaori K. Process for producing optically
active atenolol and intermediate thereof. US Patent
US005,130,482A. 1992.07.14.
21. Regis Technologies Inc. Atenolol. Available: http://www.registech.
com/Applications/ChiralApplications/atenolol.html (2011.08.28).
22. Regis Technologies Inc. Regis chiral user's guide. Available:
http://www.registech.com/Library/Regis_Chiral_Users_Guide.
pdf (2011.08.28).
10. Takehira Y, Saragai N, Kitaori K. Process for producing optically
active atenolol and intermediate thereof. US Patent
US005,223,646A. 1993.06.29.
23. Torul H. Tamer U. Determination of enantiomers of atenolol
and propranolol in pharmaceutical formulations by HPLC.
JAOAC Inter. 2011;94(3):833‐838.
11. Damle SV, Patil PN, Salunkhe MM. Chemoenzymatic synthesis
of (R)‐ and (S)‐atenolol and propranolol employing lipase
catalysed enantioselective esterification and hydrolysis. Synth
Comm. 1999;29(22):3855‐3862.
24. Phenomenex Inc. Applications: atenolol. Available: http://www.
phenomenex.com/Application/Detail/13824?returnURL=/Sear-
ch (2011.08.28).
12. Barbosa O, Ortiz C, Torresa T, Fernandez‐Lafuente R. Effect of
the immobilization protocol on the properties of lipase B from
Candida antarctica in organic media: enantiospecific production
of atenolol acetate. J Mol Cat B: Enzymatic. 2011;71:124‐132.
25. Deeb SE. Evaluation of a vancomycin‐based LC column in enan-
tiomeric separation of atenolol: method development,
repeatability study and enantiomeric impurity determination.
Chromatographia. 2010;71:783‐787.
13. Agustian J, Kamaruddin AH, Aboul‐Enein HY. Factors
screening to statistical experimental design of racemic atenolol
kinetic resolution via transesterification reaction in organic
solvent using free Pseudomonas fluorescens lipase. Chirality.
2017;29:376‐385.
26. Anroop B, Ghosh B, Parcha V, Kumar A, Khanam J. Synthesis
and comparative skin permeability of atenolol and propranolol
esters. J Drug Del Sci Tech. 2005;15(2):187‐190.
27. Anroop B, Ghosh B, Parcha V, Khanam J. Transdermal delivery
of atenolol: effect of prodrugs and iontophoresis. Curr Drug
Deliv. 2009;6:280‐290.
14. Agustian J, Kamaruddin AH. The reaction mechanism and
kinetics data of racemic atenolol kinetic resolution via enzymatic
transesterification process using free Pseudomonas fluorescence
lipase. Inter J Chem Kinetics. 2016;48(5):253‐265.
28. Enquist M, Hermansson J. Separation and quantitation of (R)‐
and (S)‐atenolo1 in human plasma and urine using an α‐AGP
column. Chirality. 1989;1:209‐215.
15. Agustian J, Kamaruddin AH. Enantio‐conversion and ‐selectiv-
ity of racemic atenolol kinetic resolution using free
Pseudomonas fluorescens lipase (Amano) conducted via
transesterification reaction. RSC Adv. 2016;6:26077‐26085.
29. Shimadzu (Asia Pacific) Pte. Ltd. LCsolution Operation
Guide (Basic). Singapore: Shimadzu (Asia Pacific) Pte. Ltd.;
2009:25 p.
16. Mikuldas H, Cepanec I, Sporec A, Litvic M, Vinkovic V. Use of
enantioselective liquid chromatography for preparation of pure
atenolol enantiomers. J Sep Sci. 2005;28:251‐256.
How to cite this article: Agustian J, Kamaruddin
AH, Aboul‐Enein HY. Fast liquid chromatography
for racemic atenolol acetate separation––The
analytical protocol. Chirality. 2017;1–7. https://doi.
org/10.1002/chir.22769
17. Santoro MI, Cho HS, Kedor‐Hackmann ER. Enantiomeric sepa-
ration and quantitative determination of atenolol in tablets by
chiral high‐performance liquid chromatography. Drug Dev Ind
Pharm. 2000;26(10):1107‐1110.
18. Agustian J, Kamaruddin AH. Chromatographic comparison of
atenolol separation in reaction media on cellulose tris‐(3,5‐