FARMACIA, 2017, Vol.

65, 3
REVIEW

ALTERNATIVE THERAPIES IN GASTRIC HYPERSECRETION

IRINA MIHAELA MATRAN, ANDREEA FARCA*, MARIUS BOJI2, DAN L. DUMITRACU

Iuliu Haieganu University of Medicine and Pharmacy, 8 Victor Babe Street, 400012, Cluj-Napoca, Romania
Iuliu Haieganu University of Medicine and Pharmacy, Drug Information Research Center, 6 Pasteur Street, 400349,
Cluj-Napoca, Romania
Iuliu Haieganu University of Medicine and Pharmacy, 2nd Medical Department, 2-4 Clinicilor Street, 400013, Cluj-
Napoca, Romania

*corresponding author: afarcas@umfcluj.ro

Manuscript received: November 2016

Abstract
In addition to its advantages (technological progress, high-performance medical treatment, multiple information sources, new
communication systems), the 21st century brings many disadvantages, such as: professional stress due to the uncertainty of
the future and to the increasingly higher professional performance indicators, the quality of interpersonal relationships, an
imbalance between professional and personal life, a lack of interest in self-knowledge. Besides all these stress factors, certain
drugs, inadequate nutrition both in terms of quantity and quality, a small number of meals eaten per day, a late last meal, as
well as late bedtime cause an increase of gastric secretion. Depending on the psychosomatic profile of patients, these can be
divided into two main categories: patients who accept drug therapy and patients who prefer alternative therapies. This review
aims to present all types of alternative therapies, for which preclinical studies are available.

Rezumat
Pe lng avantajele secolului XXI (progresul tehnologiei, tratamente medicale performante, surse multiple de informaii, noi
sisteme de comunicare), acesta aduce i numeroase dezavantaje, ca de exemplu: stresul profesional datorat nesiguranei zilei
urmtoare, a indicatorilor profesionali de performan tot mai ridicai, calitatea relaiilor interpersonale, dezechilibrul ntre
viaa profesional i cea personal, lipsa de interes a cunoaterii de sine. Pe lng toi aceti factori de stres, anumite
medicamente, alimentaia necorespunztoare, att din punct de vedere al cantitii, calitii, numrului mic de mese servite
ntr-o zi, ora trzie de servire a ultimei mese, precum i ora trzie de culcare, duc la creterea secreiei gastrice. n funcie de
profilul psihosomatic al pacienilor, acetia pot fi mprii n dou mari categorii: pacieni care accept tratamente
medicamentoase i pacieni care prefer terapii alternative. n acest sens, prezentul articol ncearc prezentarea tuturor
tipurilor de terapii alternative pentru care exist studii preclinice.

Keywords: alternative therapies, gastric hypersecretion, phytotherapy

Introduction PPIs treatment, there is an increasingly high number

of patients who prefer alternative therapies.
Over the past years, the incidence of gastrointestinal
Alternative therapies used for the treatment of gastric
diseases has continuously increased in Europe, and
secretion are: acupuncture, phytotherapy, breathing
particularly in Eastern Europe. The most frequent
exercises, acupressure, hypnotherapy, electromagnetic
gastric disorders are upper digestive haemorrhage,
resonance, and electro-acupuncture. Also, in the
Barretts oesophagus, esophagitis, eosinophilic
treatment of gastroesophageal reflux, relaxation as
esophagitis, gastroesophageal reflux disease, and
a psychological factor and lifestyle changes have
Helicobacter pylori infection [11]. For their treatment,
been applied.
numerous drugs have been developed, such as: anti-
In recent years, increasing clinical and preclinical
secretory agents histamine (H2) receptor blockers,
evidence in favour of phytotherapy has been obtained.
antacids, proton pump inhibitors (PPIs), prokinetics
Another promising alternative therapy is represented
or topical drugs for mucosal protection. Although
by special nutritional food products. According to
PPIs are the most effective in the treatment of these
Directive 2009/39/CE of 6th May 2009, changed
diseases, due to their adverse events or reactions (e.g.
and/or completed, this type of food products will be
hypersensitivity reactions, iron deficiency anaemia,
designed and produced to meet the special nutritional
increased risk of fractures, hypomagnesemia and B12
needs of persons for whom they are mainly intended.
vitamin deficiency, acid-base imbalance), but also
Thus, these persons may have disturbed digestive
due to the fact that many patients do not respond to
or metabolic processes or a certain physiological

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 FARMACIA, 2017, Vol. 65, 3
condition, and they can particularly benefit from studies, and articles published in all languages in
the controlled consumption of certain substances in the period 1996-2015 were selected. The search
these foods. According to the Directive, these food terms used were the gastroprotective effects of
products can be characterized as health food of phytotherapy in mice, Wistar rats, rabbits and cats.
diet food. This study reviews alternative phyto-
therapeutic treatments and the results obtained in Results and Discussion
preclinical studies.
The results of the analysis of phytotherapeutic
treatment used in preclinical studies are shown in
Materials and Methods
Table I.
The PubMed, Medline, SpringerLink, EbscoHost and
Elsevier databases were searched for preclinical
Table I
Phytotherapy used in preclinical studies for the treatment of gastric secretion
Phytotherapeutic remedy, Preclinical study Main action monitored and demonstrated
bibliographic source Rabbits/Cats Mice Rats
Acacia ataxcantha - - X Increase of gastric pH
(methanolic leaf extract), [2]
Aparisthmium cordatum - X X Antiulcerogenic activity of the diterpenoid aparisthman: gastric
(extract), [17] acid , mucus production , prostaglandin synthesis
Artocarpus obtusus Jarret - - X Gastric mucosal protection through: MDA , GSH , NO ,
(extract), [10] COX-2 inhibition
Bacopa monniera Wettst - - X Gastric mucosal defensive factors
(fresh juice), [30]
Benincasa hispida (fruit - - - Efficiency in dyspepsia
juice), [44]
Berberis lyceum (raw extract X X - Mediation of the spasmolytic effect through Ca2+ channels
and methanolic extract), [25, 42]
Bidens aurea (flower extract), - - X Protective effects of a flavonoid fraction against gastric lesions;
[3] gastric mucus , PGE2
Boswellia serrata (extract), - - X Antiulcer action: increased gastric mucosal resistance,
[34] prostaglandin synthesis
Carica candamarcensis - - X Increased mucus content
(fruit), [20]
Citrullus lanatus (juice), [27] - - X Reduction of gastric lesions; inhibition of gastric acid secretion
Croton cajucara Benth - X X PGE2 ; stimulation of gastric mucus secretion
(bark), [16]
Curcuma longa/turmeric - X - Anti-inflammatory; antioxidant; antimicrobial; antiplatelet;
(powder), [19, 38] anticancer; antisecretory; - iNOS
Emblica officinalis (alcoholic - - X Antisecretory and antiulcer activity; cytoprotective property
extract), [4]
Enantia chlorantha (alcoholic - - X Increased mucus production
bark extract), [23]
Eucalyptus citriodora - - X Reduction of gastric lesions; increased mucin content; anti-
(extract), [9] inflammatory activity (reduction of pro-inflammatory markers:
IL-1, TNF-, 5-LO and COX-2); absence of haemorrhage and
necrosis
Gynostemma pentaphyllum - - X Maintenance of gastric mucus production
(whole plant extract), [29]
Lobaria pulmonaria (L) Hoffm - - X Anti-inflammatory and antiulcerogenic effects
(aqueous extract/tea), [35]
Mahonia bealei, [36] - - X Peptic activity ; mucin levels ; H+, K+, ATP-ase ; gastrin
levels
Mammea Americana L./ - X - Reduction of ulcerative lesions and increase of pH by EtOH and
Guttiferae (fruit extracts: DCM extracts; no antiulcer action of MeOH extract
EtOH, MeOH, DCM), [41]
Mikania laevigata Schultz - - X Antisecretory and cytoprotective activity
Bip (hydro-alcoholic leaf
extract), [7]
Momordica cymbalaria - - X Reduction of gastric secretion volume

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Phytotherapeutic remedy,
bibliographic source
(unripe fruit), [6]
Moringa oleifera (fine
aqueous leaf extract), [32]
Neurolaena lobate (hydro-
alcoholic extract of aerial
parts), [14]
Ocimum gratissimum
(extract), [26]
Peganum harmala (seeds),
[43]
Portulaca oleracea (aqueous
and ethanol extracts) [13]
Rubus idaeus (lyophilized
fruit), [1]
Solanum paniculatum L.
(Jurubeba) (aqueous extract
of flowers, fruit, leaves, roots,
stems), [22]
Stachytarpheta cayennensis
(lyophilized aqueous extract of
flowers, fruit, whole plant), [21]
Strychnos potatorum Linn
(aqueous seed extract and
powder), [31]
Terminalia chebula (fruit),
[24]
Tinospora cordifolia Miers
(extract), [28]
Trichopus zeylanicus Gaertn.
(alcoholic extract), [33]
Usnea longissimi (extract), [5]
Vernonia kotschyana sch. bip.
(aqueous extract), [12]
Voacanga Africana (leaf
extract), [37]
Xylocarpus granatum (fruit),
[18]
IL-1 interleukin 1 , TNF- tumour necrosis factor, 5-LO 5-lipoxygenase, iNOS inducible nitric oxide synthase; MDA
malondialdehyde; GSH glutathione; NO nitric oxide; COX-2 cyclooxygenase 2; PGE2 prostaglandin; LPO lipid peroxidation; SOD
superoxide dismutase, GPx glutathione peroxidase, CAT catalase, MPx myeloperoxidase, CNOS constitutive nitric oxide synthase,
EtOH ethanol, MeOH methanol, DCM dichloromethane, 5-HT serotonin, 5-HT3 serotoninergic receptors
FARMACIA, 2017, Vol. 65, 3
Preclinical study Main action monitored and demonstrated
Rabbits/Cats Mice Rats
pH ; total acidity and free acidity
- - X Prevention of ulcer by the modulation of 5-HT secretion through
5-HT3 receptors in the gastrointestinal tract
- - X Alteration of gastric juice parameters: pH and the amount of
gastric juice ; increase of prostaglandin synthesis and mucus
amount
- - X Mucus amount ; gastric acid secretion
- - X Anti-inflammatory activity; total acidity and free acidity ;
mucin secretion; inhibition of H+, K+, ATP-ase in vitro
- X - Reduction of gastric acidity
- - X Increase of cellular antioxidant enzymes levels; reduction of
lipid peroxidation levels
- X - Inhibition of gastric acid secretion by the aqueous root, stem and
flower extracts; no alteration of gastric secretion by the aqueous
leaf extract; stimulation of gastric acid secretion by the aqueous
fruit extract
- X X Stimulation of intestinal motility by the flower and leaf extracts;
no analgesic and anti-inflammatory effect of the whole plant
extract
- - X Antiulcer, antisecretory and mucoprotective activity
- - X Total acidity and free acidity ; mucin secretion; inhibition of
K+, ATP-ase in vitro
- - X Gastroprotective effect: increased levels of
PGE2, anti-inflammatory cytokines and pro-angiogenic factors
- X - Reduction of stress
- - X LPO, SOD , GPx , GSH , CAT , MPx , iNOS , CNOS
- - - Reduction of the severity of ethanol-induced ulcers
- - X Cytoprotective, antisecretory and ulcer healing effects
- - X Inhibition of H+, K+, ATP-ase in vitro; antiulcer, antisecretory
activity

This study was carried out in order to evaluate the
state of knowledge on the alternative therapies in
gastric secretion. Specifically, medicinal plants, used
so far in the treatment of this type of secretion, for
which preclinical studies are available, were identified
and presented in the table above. Their antiulcerogenic
activity was demonstrated by studies on mice, Wistar
rats, rabbits and cats. The arguments in favour of
the medicinal plants were: increased gastric acid and
mucus secretion, prostaglandin synthesis, decrease
of MDA, GSH, NO, inhibition of COX-2 activity,
increase of pH, reduction of the free and total
acidity. A decrease of gastric lesions, an elevation of
mucin levels and an inhibition of H+, K+, ATP-ase,
as well as a modulation of 5-HT secretion through
5-HT3 receptors in the gastrointestinal tract were
also found.
For the preclinical evaluation of medicinal plants,
certain extracts (MeOH, EtOH, water/tea) of flowers/
seeds/whole plants/bark/leaves, as well as fresh juices,
powders and lyophilized fruit such as black raspberry
were used. In some cases, the antisecretory/antiulcer
effects of certain substances in the composition of
medicinal plants, such as the diterpenoid aparisthman
from the Aparisthmium cordatum extract [17] and a
certain flavonoid fraction obtained from the Bidens
aurea flower extract [3], were monitored.
In addition to the treatment of gastric secretion, the
phytotherapeutic remedies presented in the table are
recommended also for other disorders. Thus, in
Romania, due to their anti-inflammatory action and

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 FARMACIA, 2017, Vol. 65, 3
analgesic effect, Boswellia serrata tablets are
recommended in rheumatoid polyarthritis, chronic
degenerative rheumatism, muscle pain, neuralgia,
ulcerative colitis and Crohn disease. Some others
species are intensively studied in Romania for their
antioxidant, antimicrobial, and antiinflammatory
properties that would allow further development
and use of natural products [39, 40]. Others are
used for special zootechnical treatments to decrease
cholesterol in broiler chickens - Berberis lycium
root bark [8].
Over the past years, aside the preclinical studies, also
the number of clinical studies on phytotherapeutic
preparations has increased and the results of these
studies have been disseminated in scientific meetings
(e.g. the Congress of the Romanian Association of
Phytotherapists in 2015) or published in specialized
journals. Some of these clinical studies assessed the
efficacy of indigenous alfalfa in oncology, of Rumex
carbo in Wilsons syndrome, or the metabolic efficacy
and adherence to phytotherapy in a group of patients
with early type 2 diabetes mellitus. The phyto-
therapeutic approach of genital inflammatory disorders,
of non-alcoholic steatohepatitis, of depression and
other mental disorders, and the gemmotherapy in skin
diseases were also assessed in different clinical studies.
These phytotherapeutic preparations are available
in several member states of the European Union.
For the protection of public health, the collection,
processing and storage of phytotherapeutic intermediary
and end products, as well as their distribution and
marketing, should take place under the best hygiene
and quality conditions, with the observance of the
best practice guidance. Regardless of the type of
phytotherapeutic products conventional use/well-
established use or stand alone drugs, manufacturers
have the legal obligation to demonstrate at any time
the quality of the plant preparation. Also, alike for
allopathic drugs, the safety of the phytotherapeutic
products should be permanently monitored and
assessed; for this, it is important that adverse events
and reactions, and especially serious adverse reactions
are reported to regulatory agencies when suspected.
Phytotherapeutic products represent a valuable
alternative therapy for patients who do not respond
to conventional drug therapy or for those with mild/
moderate gastric hypersecretion or without other
more severe associated gastrointestinal disorders.
However, phytopreparations have both advantages
and disadvantages compared to allopathic drugs.
Some of the advantages of these products consist of
the fact that they can be better tolerated than
conventional drugs, do not have so many drug
interactions, are frequently less expensive, and many
of them can be stored under the same conditions as
allopathic drugs. Moreover, most phytotherapeutic
326
products are available in various forms, such as simple
or compound tinctures, fatty oils and compound
classic syrups, creams and gels, soft capsules, tablets,
soluble preparations, ovules and suppositories or teas,
all these natural remedies being readily available to
patients. The main disadvantage of phyto-preparations
probably is that this type of products is regarded
with scepticism by certain doctors or users. This might
be probably due to the lack of extensive clinical
evaluations to demonstrate efficacy and safety,
compared to allopathic drugs. Another disadvantage
is the lack of standardised and reproducible products.
Conclusions
The present review outlines the significant gastric
antisecretory effects of medicinal plants, demonstrated
in numerous preclinical studies. This provides scientific
validation for their use in various forms (extracts,
tinctures, powders, juices etc.). While preclinical
research on medicinal plants in gastric secretion is
extensive, we consider that appropriate clinical
studies of these phytotherapeutic remedies should
be further developed to demonstrate efficacy.
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American Journal of Analytical Chemistry, 2013, 4, 488-495
http://dx.doi.org/10.4236/ajac.2013.49062 Published Online September 2013 (http://www.scirp.org/journal/ajac)

Isolation and Characterization of

R-Enantiomer in Ezetimibe
Kameswararao Chimalakonda*, Venugopal Kamani, Madhusudhan Gutta,
Srinivasulu Polisetty, Sai Venkata Srinivas Koduri
Inogent laboratories Pvt Ltd., IDA-Nacharam, Jawaharlal Nehru Technological University Hyderabad, Hyderabad, India
Email: *Kameswararao_scientist@yahoo.co.in

Received June 22, 2013; revised July 22, 2013; accepted August 28, 2013

Copyright 2013 Kameswararao Chimalakonda et al. This is an open access article distributed under the Creative Commons Attri-
bution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

ABSTRACT
A simple and rapid Supercritical Fluid Chromatography (SFC) method has been developed to isolate and characterize
R-Isomer of Ezetimi be by using normal phase Chiral Cel OD-H with 250 mm 30 mm, 5 microns column using a mo-
bile phase system containing super critical fluid carbondi oxide (Co2) and the percentage of 2-Propanol as a mobile
phase (85:15) and detection at 230 nm. The isolated R-Isomer is characterized by using UV-vis, FT-IR, ESI-MS,
HPLC1H and 13C NMR. The purity of isolated R-Isomer is about 98%.

Keywords: Isolation; Characterization; (R)-Isomer; Ezetimibe; Supercritical Fluid Hromatography (SFC)

1. Introduction cholesterol through the intestinal wall. This reduces the

Introduction: Enantiomers of racemic drugs often show
different behaviors in pharmacological action and meta-
bolic process. It is not uncommon for one enantiomer to
be active while other is toxic in biological systems. The
pharmaceutical industry has raised its emphasis on the
generation of enantiomerically pure compounds before
undertaking phamarmacokinetic, metabolic, physiologi-
cal, and toxicological evaluation in the search for drugs
with greater therapeutic benefits and lower toxicity [1,2].
Nowadays, chiral separations are playing a more and
more important role for the analysis of single enanti-
omers in the field of pharmaceutical industry [3]. How-
ever, the development of the methods for the quantitative
analysis of Chiral compounds and for the assessment of
enantiomeric purity is extremely challenging, because the
same physical and chemical properties of the two enan-
tiomers make discriminating and separating them very
difficult [4].
Ezetimibe, a selective inhibitor of intestinal cholesterol
and related phytosterol absorption, is designated as
1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydro-
xypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone. Ezetimi-
be selectively prevents the absorption of cholesterol from
dietary and capillary sources by blocking the transport of
*
Corresponding author.
overall delivery of cholesterol to the liver, thereby pro-
moting the synthesis of LDL receptors and the subse-
quent reduction in serum LDL-C [5,6]. Few HPLC
methods for the determination of Ezetimibe were re-
ported in literatures [7,8]. The (R)-enantiomer is the un-
desired enantiomer, which can be present as a chiral im-
purity without any pharmacological and toxicological
reports by now. So it is essential to find an effective way
to analyze the enantiomers of Ezetimibe, the chemical
structure of Ezetimibe is shown in Figure 1 , the chemi-
cal structures of (R)-enantiomer is shown in Figure 2
and (R)-enantiomer Ezetimibe may be at low level for
little (R)-Ezetimibe exists in starting material or racemi-
zation in synthesis.
Here, the direct enantioseparation of the undesired
enantiomer from an active pharmaceutical ingredient,
Ezetimibe, is isolated by Supercritical fluid chromatog-
raphy (SFC) using modified cellulose as chiral stationery
phases. The aim of this work was to isolate, and charac-
terize and optimized the chromatographic conditions in
terms of mobile phase composition in order to separate
and identify the enantiomers of Ezetimibe. The deve-
loped SFC method was used for isolation of (R)-enan-
tiomer in Ezetimibe.
Supercritical Fluid Chromatography (SFC) is a form of
normal phase chromatography that is used for the analy

Copyright 2013 SciRes. AJAC

 K. CHIMALAKONDA
Figure 1. (3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluoroph-enyl)-3-
hydroxypropyl]-4-(4-hydroxyphenyl) azetidin-2-one.
Figure 2. (3R,4S)-1-(4-fluorophenyl)-3-((3R)-3-(4-fluorophenyl)-3-
hydroxypropyl)-4-(4-hydroxyphenyl)azetidin-2-one.
sis and purification of moderate molecular weight, ther-
mally labile molecules. It can also be used for the separa-
tion of chiral compounds. The principles are similar to
those of high performance liquid chromatography
(HPLC) [9]; however, SFC typically utilizes carbon di-
oxide as the mobile phase; therefore, the entire chroma-
tographic flow path must be pressurized.
Supercritical Fluid Chromatography (SFC) is found to
be useful in industry primarily for the separation of chiral
molecules, and the same columns are used as standard
HPLC systems. SFC is now commonly used for achiral
separations and purifications in the pharmaceutical in-
dustry.
The mobile phase is composed of high pressure liquid
or supercritical carbon dioxide, however, modifiers are
added which can be used to change the chromatography,
these are typically alcohols like methanol, ethanol or
isopropyl alcohol. Other solvents such as Acetonitrile
and chloroform can be used as modifiers. The solvent
limitations are system- and column-based.
Here, the direct enantioseparation of the undesired
enantiomer from an active pharmaceutical ingredient,
Ezetimibe, is isolated by Supercritical fluid chromatog-
raphy (SFC) using modified cellulose as chiral stationery
phases. The aim of this work was to isolate and charac-
terize. Optimized the chromatographic conditions in
terms of mobile phase composition in order to separate
and identify the enantiomers of Ezetimibe, the developed
SFC method was used for isolation of (R)-enantiomer in
Ezetimibe.
2. Experimental
Chemicals and reagents Ezetimibe racemic mixture ()
was obtained from the R&D department of Inogent La-
boratory (Hyderabad, India). Chemical structure is pre-
sented in Figure 1. HPLC grade 2-Propanal was pur-
Copyright 2013 SciRes.
ET AL. 489
chased from Merck (Mumbai, India) CO2, with a purity
of 99.9% was purchased from MRGenterprises (Hydera-
bad, India). HPLC grade n-Hexane, 2-Propanal, Trifluor-
oaceticacid and Ethanol was purchased from Merck
(Mumbai, India).
2.1. Isolation by Supercritical Fluid
Chromatography (SFC)
The isolation of R-enantiomer in Ezetimibe by using
SFC 200 System in large Scale. The Chromatographic
conditions are the total flow was 100 g/min; the % of
solvent was 15% isopropanol and the Supercritical fluid
is 85%. The Automatic back pressure Regulator main-
tained at 180, the preparative column was Chiral Cel
OD-H (250 30 mm), 5 microns. The output signal was
monitored by using Gilson detector.
2.2. Sample Preparation
Ezetimibe racemic mixture () was prepared with 10 mg/
mL dissolving appropriate amount of the substance in di-
luent and injected. Collected the R-Isomer fraction and di-
stilled and characterize by using spectroscopy techniques.
2.3. Characterization of R-Isomer
2.3.1. UV-Vis Spectroscopy
UV-Visible spectrum was recorded by diluting sample in
methanol in UV-2450 Perkin Elmer spectrophotometer.
2.3.2. FT-IR Spectroscopy
The FT-IR Spectroscopy was recorded in the solid state
as KBr dispersion using Thermo Nicolet 380 FT-IR
Spectrophotometer.
2.3.3. Mass Spectroscopy
Electrospray ionization mass spectroscopy was per-
formed using a triple quadrupole mass spectrometer. The
positive and negative Electrospray MS data was obtained
by switching the capillary voltage between +5000 V and
4500 V, respectively.
2.3.4. NMR Spectroscopy
The NMR experiments were performed on Varian 400
MHz. The 1H and 13C chemical shift values were re-
ported on the K scale in ppm.
2.4. High Performance Liquid Chromatography
A validated LC method was used for Identification and
quantification of R-Isomer of Ezetimibe. Waters e2695
with 2998 PDA detector with Empower-2 software was
used. The chromatographic conditions were Chiral Cel
AS-H (250 4.6 mm, 5 was used. The chromatographic
conditions were Chiral Cel AS-ethanol, 2-Propanol and
AJAC
490 K. CHIMALAKONDA
Trifloroacetic acid (84:12:4:0.1 v/v). Detection was car-
ried out at 230 nm and the flow rate 1.0 mL/min.
3. Results and Discussion
Isolation by Supercritical Fluid Chromatography (SFC)
Racemic mixture solution of Ezetimibe and (R)-enanti-
omer (10e solution of prepared in ethanol was used in the
method development. To develop a rugged and suitable
SFC method for the separation of the two enantiomers,
different stationary phases and mobile phases were em-
ployed. Initial screening of chiral column was carried out
by several chiral column suppliers. Various chiral col-
umns, namely Chiralpak AD-H (250 4.6 mm), chiralcel
IA (250 4.6 mm), chiralcel AS-H (250 4.6 mm) of
Daicel were employed. All these columns failed to pro-
vide selectivity between Ezetimibe peak and the unde-
sired enantiomer peak using different possible mobile
phases. In the following method development activities,
Chiralcel OD-H column (250 4.6 mm, 5 Mm) with
mobile phase consisting of Supercritical Carbon dioxide
and methanol was used, but all that was obtained was a
defective separation of two enantiomers with a very low
resolution.
It was continued to select the best stationary and mo-
bile phases that would give optimum resolution and se-
Figure 3. Recemic mixture of Ezetimibe.
Figure 4. Ezetimibe individually.
Copyright 2013 SciRes.
ET AL.
lectivity for the two enantiomers. There was an indica-
tion of separation on Chiralcel OD-H (250 4.6 mm, 5
tiomers. There was mobile phase consisting of upper
critical carbon dioxide and 2-Propanol. The composition
of the mobile phase was optimized to enhance the chro-
matographic efficiency and resolution between the enan-
tiomers. The results of resolution factor (Rs) and selec-
tivity factor (N) are summarized in Table 1. Based on the
data obtained from the method development and optimi-
zation activities, Chiralcel OD-H (250X4.6mm, 5m)
column. The phase of Supercritical carbon dioxide and
2-Propanol (85:15%) was selected for the method devel-
opment. The phase of Supercritical carbon dioxide and
2-Propanol (85:15%) was selected for the method devel-
opment. The flow rate of the final method was 2.0
mL/min with injection volume 20 devel column tem-
perature was 25C, and the detection wavelength was 230
nm. Under these conditions, the two enantiomers were
separated well and the peak of (R)-enantiomer eluted be-
fore the peak of Ezetimibe. In the optimized method, the
typical retention time of Ezetimibe and (R)-enantiomer
were 5.31, 6.27 min, respectively. Base line separation of
Ezetimibe and (R)-enantiomer was obtained with total
run time of 20 min. The separation of an approximately
1:1 (wt/wt) mixture solution (in ethanol) of the two enan-
AJAC
 K. CHIMALAKONDA ET AL. 491

tiomers is shown in Figure 3. Ezetimibe peak confirmed
with by injecting individually is shown as Figure 4.
Cellulose based chiral stationary phase contained five
chiral centers per unit, and Ezetimibe has only one chiral
centre close to the carbonyl group in the structure. The
stereo electronic interaction between the enantiomers and
the chiral stationary phase generated enantio selectivity,
thus causing significant difference in the migration of the
enantiomers inside the column. Having the right amount
of 2-Propanol in the mobile phase also played an impor-
tant role in affecting the steric environment of the chiral
cavities or channels of the stationary phase and contribu-
tes to enantio selectivity. However, an excessive amount
of 2-Propanol was likely to cut down the resolution by
taking up chiral centers of the chiral stationary phase or
forming hydrogen bonding with enantiomers instead of
the hydrogen bonding between the enantiomers and the
stationary phase. Other important interaction between the
enantiomers and the stationary phase, such as - bond-
ing Vander walls forces, dipole induced dipole attrac-
tions, and steric effects can also achieve better resolution
on chiralcel OD-H column and steric effects can also
achieve better resolution on chiralcel OD-H column.
3.1. Characterization of R-Isomer by UV-Visible
Spectroscopy
The UV-Visible spectrum was recorded by diluting
R-Isomer by using 10 ppm solution in methanol. Solution
was scanned 200 - 400 nm against methanol as blank and
recorded the UV-visible spectrum for R-Isomer spectrum
is shown in Figure 5.
3.1.1. FT-IR Spectroscopy
Dispersed about 2 mg of R-Isomer in 300 - 400 mg of
KBr, grind the mixre, spread uniformly on a die and
compress in to a thick disk by applying pressure about
800 Mpa. Kept the pellet in KBr folder in FT-IR spec-
trophotometer and scanned the spectrum from 4000 cm1
to 450 cm1.
Noted the wavelengths and its the spectrum from
4000 cm1 to 450 cm1 and shown at Figure 6. Func-
tional groups are listed in Table 1.
Table 1. Functional groups from FT-IR spectrum.
S.No Assignment Wave number (cm1)
1 Free-OH(Stretching) 3397.7
2 Beta lactoms C = O (Stretching) 1721.7
3 Aromatic C-F (Stretching) 1224.9
4 Aromatic C = C 1559.8
5 Aromatic CH2 2930.2

Figure 5. UV-Visible spectrum for R-Isomer.

Figure 6. FT-IR spectrum for R-Isomer.

Copyright 2013 SciRes. AJAC

492 K. CHIMALAKONDA
3.1.2. Mass Spectroscopy
Analyzed the R-Isomer by using Mass spectroscopy and
observed the mass number (m/z) for R-Isomer and its
confirming the mass number of R-Isomer is 409 (m/z:
negative mode: 408), as shown in Figure 7.
3.1.3. NMR Spectroscopy
R-Isomer sample analyzed 1H NMR and 13C NMR using
Varian 400 MHz. The no. of protons and no. of carbons
confirms the structure of R-Isomer. The reported 1H
NMR spectrum was shown Figure 6. The reported 13C
NMR spectrum was shown Figure 8. 1H NMR and Fig-
ure 9: 13C NMR. 1H NMR and 13C NMR K values are
listed Tables 2 and 3.
3.2. High Performance Liquid Chromatography
A validated LC method was used for Identification and
Figure 7. Mass spectrum (m/z) for R-Isomer.
Figure 8. 1H NMR spectrum for R-Isomer.
Copyright 2013 SciRes.
ET AL.
quantification of R-Isomer of Ezetimibe. Waters e2695
with 2998 PDA detector with Empower-2 software was
used. The chromatographic conditions were Chiral Cel
AS-H (250 4.6 mm, 5 microns was used. The chroma-
tographic conditions were Chiral Cel AS-ethanol, 2-
Propanol and Trifloroacetic acid (84:12:4:0.1 v/v). De-
tection was carried out at 230 nm and the flow rate 1.0
mL/min.
Both Ezetimibe and Isolated R-Isomer ware prepared
in diluent and injected individually and as co-injection.
R-Isomer Retention time was confirmed by injected re-
cemic mixture.Recemic mixture shown as Figure 10,
Isolated R-Isomer shown as Figure 11 and Ezetimibe
shown as Figure 12.
4. Conclusion
The research paper describes the isolation and charac-
AJAC
 K. CHIMALAKONDA ET AL. 493

Figure 9. 13C NMR spectrum for R-Isomer.

Figure 10. R-Isomer and ezetimibe (1:1).

Figure 11. Chromatogram of Isolated R-Isomer.

Copyright 2013 SciRes. AJAC

494 K. CHIMALAKONDA ET AL.

Figure 12. Ezetimibe.

Table 2. 1H NMR values (ppm). Table 3. 13C NMR values (ppm).

S.NO (PPM) VALUES NO. OF HYDROGENS S.NO (PPM )VALUES

1,1 7.313 - 7.349 2H 1,1 127.532 - 127.608

2,2 115.683 - 115.911

2,2 7.102 - 7.147
3 4.525 - 4.564
4 5.264 - 5.267
5 1.709 - 1.864
6 1.669 - 1.709
2H
3 71.136
1H
5 36.403
1H(OH)
6 24.540
2H
7 59.546
2H 8 59.804

7 3.062 - 3.088 1H 9,9 127.554

8 4.785 - 4.791 1H 10,10 115.706

9, 9 7.197 - 7.218 2H 12,12 118.188 - 118.264

10, 10 6.734 - 6.755 2H 13,13 114.545 - 114.757

14 159.843 - 162.242
11 9.502 1H(Ar-OH)
15 133.983 - 133.999
12, 12 7.188 - 7.223 2H
16 127.843
13, 13 7.099 - 7.144 2H
17 157.430

18 142.075 - 142.105
terization of R-Isomer of Ezetimibe by using Supercriti-
cal Fluid Chromatography (SFC). The R-isomer was 19 156.815 - 156.206
isolated by using Supercritical Fluid Chromatography 20 167.305
(SFC) with Chiralcel OD-H (250 4.6 mm, 5D-H col-
umn using a mobile phase consisting of Supercritical
carbon dioxide and 2-Propanol (85:15). The isolated
5. Acknowledgements
R-Isomer was characterized by using UV-Visible spec- The authors would like to acknowledge the management
troscopy, FT-IR, Mass number (m/z) m, 1 H NMR, 13 C of Inogent Laboratories Private limited, Hyderabad, In-
NMR and by Chiral HPLC analysis. dia.

Copyright 2013 SciRes. AJAC

 K. CHIMALAKONDA
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