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PART-[A]
ENANTIOSELECTIVE METHOD
DEVELOPMENT AND VALIDATION
OF SOME PHARMACEUITCALS
SECTION-1
Enantioselective HPLC Method
Development and Validation
of Omeprazole
Omeprazole Section-1
50
[1] Introduction
Stereoisomers are molecules that are identical in atomic constitution and bonding, but
differ in the three-dimensional arrangement of the atoms. They are often readily
distinguished by biological systems, however, and may have different pharmacokinetic
properties (absorption; distribution, biotransformation and excretion) and quantitatively
or qualitatively different pharmacological or toxicological effects. Most biological
molecules (proteins, sugars, etc.) are present in only one of many chiral forms, so
different enantiomers of a chiral drug molecules bind differently (or not at all) to target
receptors. One enantiomer of a drug may have a desired beneficial effect while the other
may cause serious and undesired side effects, or sometimes even beneficial but entirely
different effects [1].
Under the Chemistry, Manufacturing and Control (CMC) the chemistry section of the
application should contain the requisite information to assure the identity, quality, purity
and strength of the drug substance and drug product. In addition, the following
considerations should be taken into account of the method and specification when dealing
with chiral drug substances and drug products;
Drug Substance: Applications for enantiomeric and racemic drug substances should
include a stereochemically specific identity test and/or a stereochemically selective assay
method.
Drug Product: Applications for drug products that contain an enantiomer or racemic drug
substance should include a stereochemically specific identity test and/or a
stereochemically selective assay method [2].
Omeprazole is one of the most widely prescribed proton pump inhibitor (PPI) and
is available over the counter in some countries. PPIs are a group of drugs whose main
action is a pronounced and long-lasting reduction of gastric acid production. Omeprazole
was first approved as a racemic mixture, but the (S)-enantiomer was later introduced to
the market. The major difference is the (S)-omeprazole is metabolized more slowly and
reproducibly than the (R)-omeprazole and racemic omeprazole, because streoselective
Omeprazole Section-1
51
metabolism by cytochrome P450 enzyme [3]. Therefore lower doses of (S)-omeprazole
can be used to produce equivalent acid suppression than omeprazole doses. Omeprazole
is unstable in acidic environment [4]. In aqueous media, the degradation rate proceeds
with a half-life of less than 10 min at pH values lower than 4.3 [5]. Omeprazole is thus
formulated as enteric-coated granules encapsulated in a gelatin shell or as enteric-coated
tablets [6, 7].
1.1 Description
N
NS
O
..
OMe
H
CH
2
N
CH3
CH3
OMe
(S)-(-)-omeprazole
N
NS
O
OMe
H
N CH
2
CH3
CH3
OMe
:
(R)-(+)-omeprazole
Figure 1: Enantiomers of Omeprazole
Omeprazole (Figure 1) is 5-methoxy-2-{[(4-methoxy-3,5-dimethylpyridin-2-yl)
methane]sulfinyl}-1H-1,3-benzodiazole. It has a stereogenic center at the sulphur atom,
and exists as two optically active forms, (S)- and (R)-omeprazole [8]. Its molecular
formula is C17H19N3O3S and the molecular weight is 345.4 g/mol.
1.2 Indication
For the treatment of acid-reflux disorders (GERD), peptic ulcer disease, H. pylori
eradication, and prevention of gastrointestinal bleeds with NSAID use.
1.3 Interaction
Omeprazole is a competitive inhibitor of the enzymes CYP2C19 and CYP2C9,
and may therefore interact with drugs that depend on them for metabolism, such as
Omeprazole Section-1
52
diazepam, escitalopram, and warfarin; the concentrations of these drugs may increase if
they are used concomitantly with omeprazole [9].
Clopidogrel (Plavix) is an inactive prodrug that partially depends on CYP2C19
for conversion to its active form; inhibition of CYP2C19 blocks the activation of
clopidogrel, thus reducing its effects and potentially increasing the risk of stroke or heart
attack in people taking clopidogrel to prevent these events [10, 11]. Omeprazole is also a
competitive inhibitor of p-glycoprotein, as are other PPIs [12].
Drugs that depend on stomach pH for absorption may interact with omeprazole
and drugs that depend on an acidic environment (such as ketoconazole or atazanavir) will
be poorly absorbed. Acid-labile antibiotics (such as erythromycin) will be absorbed to a
greater extent [9].
1.4 Mechanism of Action
Omeprazole is a proton pump inhibitor that suppresses gastric acid secretion by
specific inhibition of the H+/K
+-ATPase in the gastric parietal cell. By acting specifically
on the proton pump, omeprazole blocks the final step in acid production, thus reducing
gastric acidity.
1.5 Pharmacodynamics
Omeprazole is a compound that inhibits gastric acid secretion and is indicated in
the treatment of gastroesophageal reflux disease (GERD), the healing of erosive
esophagitis, and H. pylori eradication to reduce the risk of duodenal ulcer recurrence.
Omeprazole belongs to a new class of antisecretory compounds, the substituted
benzimidazoles, that do not exhibit anticholinergic or H2 histamine antagonistic
properties, but that suppress gastric acid secretion by specific inhibition of the H+/K
+
ATPase at the secretory surface of the gastric parietal cell. As a result, it inhibits acid
secretion into the gastric lumen. This effect is dose-related and leads to inhibition of both
basal and stimulated acid secretion irrespective of the stimulus.
Omeprazole Section-1
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1.6 Side Effects
The occurrence of omeprazole side effect is very less, still headache, diarrhea,
abdominal pain, nausea, dizziness, trouble awakening and sleep deprivation although in
clinical trials the incidence of these effects with omeprazole was mostly comparable to
that found with placebo [13]. Other side effects may include iron and vitamin B12
deficiency, although there is very little evidence to support this [14].
1.7 Dosage
Omeprazole is available as tablets and capsules (containing omeprazole or
omeprazole magnesium) in strengths of 10 mg, 20 mg, 40 mg, and in some markets 80
mg; and as a powder (omeprazole sodium) for intravenous injection. Most oral
omeprazole preparations are enteric-coated, due to the rapid degradation of the drug in
the acidic conditions of the stomach. This is most commonly achieved by formulating
enteric-coated granules within capsules, enteric-coated tablets, and the multiple-unit
pellet system (MUPS).
Omeprazole is also available for use in injectable form (I.V.) in Europe, but not in
the U.S. The injection pack is a combination pack consisting of a vial and a separate
ampule of reconstituting solution. Each 10 ml clear glass vial contains a white to off-
white lyophilized powder consisting of omeprazole sodium 42.6 mg equivalent to 40 mg
of omeprazole
1.8 Chemical Stability
Omeprazole is unstable in acidic environment [15]. In aqueous media, the
degradation rate proceeds with a half-life of less than 10 min at pH values lower than 4.3
[16]. Omeprazole is thus formulated as enteric-coated granules encapsulated in a gelatin
shell or as enteric-coated tablets [17, 18].
Omeprazole Section-1
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1.9 Metabolism
The major metabolites of omeprazole are summarized below;
S.N. Substrate Enzymes Product
1 Omeprazole
Cytochrome P450 2C9
Cytochrome P450 2C19
Cytochrome P450 3A4
5-Hydroxyomeprazole
2 Omeprazole Cytochrome P450 2C19 5’-O-Desmethyl
omeprazole
3 Omeprazole Cytochrome P450 3A4 Omeprazole sulfone
4 Omeprazole Cytochrome P450 3A4 3-Hydroxyomperazole
1.10 Polysaccharide-based Chiral Stationary Phase
The polysaccharide-based chiral stationary phase (CSP) has shown to be effective
on multimodal elution (normal, reverse and organic polar mode). Phenyl carbamate
derivatives are the most successful CSPs as it covers the broad applications for different
compounds [19, 20]. The CSP in Chiracel OD-H is cellulose tris(3,5-dimethylphenyl
carbamate) coated on silica gel (Fig. 2).
Figure 2: Chiral Stationary Phase of Chiracel OD-H
Omeprazole Section-1
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The phenylcarbamates bearing electron-withdrawing substituents, such as
halogens, or electron-donating substituents, such as alkyl groups, exhibit better chiral
recognitions. These substituents appear to affect the polarity of the carbamate group via
an inductive effect and alter the interaction mode between the cellulose derivatives and
the racemates.
The mechanism of separation in direct chiral separation methods is the interaction
of CSP with analyte enantiomers to form short-lived, transient diastereomeric complexes
[21].
The complexes are formed as a result of following mechanism;
1. Hydrogen bonding
2. Dipole-dipole interactions
3. π- π interaction
4. Electrostatic interactions
5. Inclusion complexation
The relative binding strength of the diastereomeric complexes determines
enantioselectivity and rate of elution of enantiomers.
[2] Literature Overview
The literature reviews regarding omeprazole suggest that various analytical
methods were reported for drug substance as well as pharmaceutical formulations. Only
few enantioselective chromatographic separation methods for omeprazole have been
described in the literature.
Jeanette Olsson, Filip Stegander, Nicola Marlin, Hong Wan, Lars G.
Blomberg has reported enantiomeric separation of omeprazole and its metabolite 5-
hydroxyomeprazole using non-aqueous capillary electrophoresis technique. Heptakis-
(2,3-di-O-methyl-6-O-sulfo)-β-cyclodextrin (HDMS-β-CD) was chosen as the chiral
selector. When using UV detection, the value for LOD is very high, therefore MS is
currently being investigated as an alternative detector [22].
Omeprazole Section-1
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Pierina S. Bonato, Fernanda O. Paias have reported comparisons study for
enantioselective analysis of omeprazole in pharmaceutical formulations by chiral high-
performance liquid chromatography and capillary electrophoresis (CE). The enantiomer
separation has achieved by amylose tris(3,5-dimethylphenylcarbamate) coated on silica
gel column. The mixture of hexane and ethanol (40:60, v/v) used as a normal phase
mobile. This linear response has been found over a concentration range from 25 to 150
µg/ml. The results confirm that CE is more versatile and less expensive than HPLC using
chiral columns, since several expensive columns are required to cover a reasonably wide
application range and column lifetime tends to be relatively short. In addition, chiral
HPLC required a large volume of organic solvents. On the other hand, the HPLC is more
sensitive and resulted in a better resolution of omeprazole enantiomer [23].
Q.B. Cass, V.V. Lima, R.V. Oliveira, N.M. Cassiano, A.L.G. Degani, J.
Pedrazzoli Jr. have reported a study on enantioselective determination of the plasma
levels of omeprazole by direct plasma injection using HPLC with achiral-chiral column-
switching. The chiral method has developed using amylose tris (3,5-
dimethylphenylcarbamate) CSP and normal phase mobile phase consisting of hexane and
ethanol (70:30, v/v). The results confirm that the develop method is simple with no time
involved for sample pretreatment. It has proven to be useful for pharmacokinetics studies
of omeprazole enantiomers in plasma samples [24].
Katia R.A.B., Mariana C., J.C. Barreiro, C.A. Montanari, Q.B. Cass have
reported a study for multi milligram enantioresolution of sulfoxide proton pump
inhibitors (omeprazole, lansoprazole, rabeprazole) by liquid chromatography on
polysaccharide-based chiral stationary phase. The enantiomers were isolated using
amylose based different phenyl carbamate derivatives (tris-3,5-dimethylphenylcarbamate
and tris-(S)-1-phenylethylcarbamate). The enantiomers were eluted using normal phase
mobile phase consisting different ratio of hexane and ethanol. Authors have studied the
different injection techniques to achieve the highest production rate of sulfoxide proton
pump inhibitors [25].
Omeprazole Section-1
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[3] Aim of Present Study
Omeprazole is widely prescribed in the form of enteric coated formulations, due
to the rapid degradation of the drug in the acidic condition of the stomach. As per
preceding discussion in the literature review, a few chromatographic methods are
reported for the enantiomeric separation of omeprazole. So far to our present knowledge,
there is no validated, chiral HPLC method reported for the determination of (R)-
omeprazole in enteric-coated formulation. Therefore the major objective of this present
work was to develop short and accurate chiral HPLC method for omeprazole in enteric
coated formulation. As there was no published report, we preferred cellulose based
Chiracle OD-H column to separate omeprazole enantiomers.
Our study deals with the systematic method development studies such as, effect of
stationary phase, effect of organic modifier and effect of column temperature. This work
also deals with systematic validation of developing a stability indicating method as per
the International Conference of Harmonization (ICH) guidelines [26]. This method is
recommended for routine analysis in quality control laboratories.
[4] Experimental
4.1 Chemicals and Drugs
Bulk drug samples and enteric-coated capsule formulation of omeprazole, pure
(R)-omeprazole, (S)-omeprazole and racemic mixture were obtained from local market.
HPLC grade n-hexane, isopropylalcohol (IPA), ethanol and methanol were used as
mobile phase, which is manufactured by Merck and procured from commercial sources.
HPLC grade water was obtained from Milli-Q water purification system.
4.2 Chromatographic conditions
The chiral separation was performed on an Agilent 1200 HPLC system consist of
a quaternary pump, column oven, photo diode array detector and an auto injector. To
check the method’s robustness, the analysis has been also performed on a Shimadzu LC-
2010 HPLC system consist of a quaternary pump, a column oven, a photo diode array
detector and an auto injector. Enantiomeric separation achieved at 40°C column oven
Omeprazole Section-1
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temperatures using Chiralcel OD-H column (250mm × 4.6 mm, 5 µm particle size,
Daicel make). The flow rate was 0.75 ml/min and injection volume was 5 µl. Data
analysis performed at a wavelength of 302 nm.
4.3 Mobile phase preparation
The mobile phase consisted of a mixture of n-hexane, methanol, isopropylalcohol,
ethanol in the ratio of 85:8:6:1 (v/v/v/v). Accurately 850 ml of n-hexane, 80 ml of
methanol, 60 ml of isopropylalcohol and 10 ml of ethanol were transferred to 1000 ml
mobile phase bottle and degassed in an ultrasonic bath (Spincotech Pvt. Ltd., Mumbai)
for 5 mins.
4.4 Diluent Preparation
Mobile phase used as a diluent.
4.5 Sample Preparation
The standard stock solutions of pure (R)-omeprazole, (S)-omeprazole and racemic
sample were prepared by dissolving appropriate amount of the standard samples in
mobile phase. A stock solution concentration was fixed to 800 µg/ml. A working solution
was also prepared in the mobile phase. For formulation sample, 8 capsules (10 mg of (S)-
omeprazole label claim) were opened and the enteric-coated granules were finely ground
using agate mortar and pestle. The ground material, which was equivalent to 80 mg of
(S)-omeprazole was transferred to 50ml volumetric flask containing 45 ml of methanol.
Omeprazole extracted from place in to methanol by ultrasonication for 15 min.
Temperature of ultrasonication bath was maintained at room temperature, i.e. 25 °C. The
volume has made up to 50 ml with methanol and the resultant mixture was filtered
through a 0.45 µm membrane filter. This solution corresponds to analyte concentration of
1600 µg/mL, and further dilutions were prepared in diluent.
Omeprazole Section-1
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4.6 Method Validation
4.6.1 Specificity
The specificity of this method was indicated by the absence of any endogenous
interference at retention times of enantiomeric peaks. The absence of interfering peak
was evaluated by injecting a blank sample consisting of diluent and placebo.
Stability Indicating Method: The drug was subjected to forced degradation under acidic
(0.1M hydrochloric acid), basic (0.1M sodium hydroxide), and oxidative (30% hydrogen
peroxide) stress conditions.
Acidic stress condition:
Acidic stress study was carried by dissolving the drug at 800 ug/mL concentration in
0.1M HCl and kept for 30 mins in water bath at 50°C.
Alkaline stress condition:
An alkaline stress study was carried by dissolving the drug at 800 ug/mL concentration in
0.1M NaOH solution and kept for 30 mins in water bath at 50°C.
Oxidative stress condition:
The study was carried out by dissolving the drug at 800 ug/mL concentrations in 30% v/v
hydrogen peroxide solution and kept for 30 mins in water bath at 50°C.
4.6.2 Precision
The precision of the method is the degree of agreement among the individual test
results when the procedure is applied repeatedly to multiple sampling of a homogenous
sample. The precision of the method was checked by an analyzing nine replicate samples
of (S)-omeprazole (at analyte concentration, i.e. 800.00 µg/mL) spiked with 0.1% (0.8
µg/mL) of (R)-omeprazole on different days and R.S.D. of area under the peaks was
calculated. The intermediate precision was determined at different in another laboratory
by performing nine successive injections.
Omeprazole Section-1
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4.6.3 Linearity of omeprazole enantiomers
Linearity corresponds to the capacity of the method to supply results directly
proportional to the concentration of the substance being determined within a certain
interval of concentration. [9, 10] Detector response linearity was assessed by preparing
12 calibration sample solutions covering from 0.39 µg/mL to 800 µg/mL (0.39, 0.78,
1.56, 3.13, 6.25, 12.50, 25.00, 50.0, 100.00, 200.00, 400.00, and 800.00 µg/mL).
Regression curve was obtained by plotting peak area versus concentration, using the least
squares method. Duplicate injections given for each concentration level.
4.6.4 Sensibility
Lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were
achieved by giving six injections of lowest three concentration levels, prepared for
linearity study. The signal to noise ratio and RSD of the area is considered to evaluate
LLOD and LLOQ.
4.6.5 Recovery Study of (R)-omeprazole in Formulation
The standard addition and recovery experiments were conducted to determine the
accuracy of the present method. The study was carried out in triplicate by spiking placebo
with three concentrations (0.12, 0.15, and 0.18%) of standard (R)-omeprazole and
assaying for the chromatographic method. The recovery for (R)-omeprazole was
calculated from the slope and Y-intercept of the calibration curve, drawn in the
concentration range of 0.39-800 µg/mL.
4.6.6 Ruggedness
To determine the ruggedness, the recovery experiments carried out for (R)-
omeprazole in formulation samples were again carried out in laboratory B using a
different instrument.
4.6.7 Robustness
For the HPLC method, the robustness was determined by the analysis of the
samples under a variety of conditions making small changes in the percentage of
Omeprazole Section-1
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methanol in mobile phase (7 and 9%, v/v), in the flow rate (0.7 and 0.8 ml/min), in the
column temperature (35 and 45°C), and changing the wavelength (299 and 303 nm). The
change in chromatographic resolution between enantiomers was evaluated for the study.
4.6.8 Solution Stability
To check the solution stability of omeprazole and mobile phase, the sample was
analyzed for 24h at room temperature, i.e., at 25°C. Resolution and composition of
omeprazole enantiomers were observed for 3, 6, 9, 12, 18, and 24 h.
[5] Result and Discussion
5.1 Method Development and Optimization
The aim of this study is to separate the enantiomers of omeprazole with optimum
resolution within a short time using polysaccharide based CSP. Omeprazole has no
physiological charge. It has five hydrogen bond acceptor and one hydrogen bond donor
on the structure. The racemic sample solution of 100 ug/ml concentration was used for
the method development and optimization.
Figure 3: UV spectra of Omeprazole
Omeprazole Section-1
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To determine the λmax, the racemic solution was scan between 200 to 400 nm
using UV diode arrays detector and we found two λmax, i.e. 202 and 302 nm (Fig. 3). We
preferred to work on 302 nm, to achieve good detector baseline which is free from mobile
phase interference.
Phenyl carbamate derivatives are the most successful CSPs as it covers the broad
applications for different compounds [19, 20]. Cellulose based phenylcarbamates
derivatives chosen for method development. Chiralcel OJ-H cellulose tris(4-
methylbenzoate)carbamate coated on 5 µm silica gel. Chiracel OJ-H did not show
potential results for enantiomeric separation of omeprazole (Figure 4). Different solvent
combination of n-hexane, isopropylalcohol, methanol and ethanol have been tried to
separate the enantiomers, but there was no sign of separation.
Figure 4: Column: Chiralcel OJ-H, Mobile phase: n-hexane and ethanol in ratio
of (80:20, v/v), Flow: 0.75 ml/min, Column temperature: 25°C
The Chiralcel OD-H column is cellulose tris(3,5-dimethylphenyl carbamate)
coated on silica gel coated of 5 µm particle size. Different solvent combinations of n-
Omeprazole Section-1
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hexane, isopropylalcohol, methanol and ethanol have been tried to separate the
enantiomers. Initial column temperature was kept at 25°C.
The sign of enantiomeric separation was found on Chiracel OD-H column using
mobile phase consisting the mixture of n-hexane and ethanol (Fig. 5). The solutes can
bind to the carbamate groups on the chiral stationary phase forming transient
diastereomers through hydrogen bonding using the C=O and NH groups and also through
dipole–dipole interaction using the C=O moiety. Omeprazole has NH functional group
and this could well be contributing to the interactions with the carbamate groups on CSP,
resulting in separation [27].
Figure 5: Column: Chiralcel OD-H, Mobile phase: n-hexane and ethanol in ratio of
(80:20, v/v), Flow: 0.75 ml/min, Column temperature: 25°C
In presence of IPA, the resolution has improved but the peaks shape was still
broad which was resulting in to the poor resolution. In order to achieve the sharp peak,
the addition of suitable modifier was required. During method development the
methanol was chosen as a polar organic modifier of the mobile phase, because the
methanol has proven good organic modifier for resolution of omeprazole enantiomers
[28, 29].
Omeprazole Section-1
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Figure 6: Column: Chiralcel OD-H, Mobile phase: n-hexane ethanol, IPA in ratio
of (80:10:10, v/v/v), Flow: 0.75 ml/min, Column temperature: 25°C
The percentage of methanol between 6 and 10% had strong effect on separation
and sharpness of the enantiomeric peaks corresponding to omeprazole (Fig. 8, 9 and
10). The increase in certain percentage of methanol content in mobile phase increased
the resolution, and numbers of theoretical plates of the two enantiomers as peaks were
become sharper. But after certain amount of methanol, the chromatographic resolution
and capacity factor were decreased as peaks were started merging. The results are
summarized in Fig. 7.
Omeprazole Section-1
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Figure 7: Effect of methanol content (% v/v) on system suitability parameters
Figure 8: Mobile Phase : n-hexane, methanol, IPA, ethanol in ratio of 85:10:4:1 (v/v/v/v)
Omeprazole Section-1
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Figure 9: Mobile phase: n-hexane, methanol, IPA, ethanol in ratio of 85:8:6:1 (v/v/v/v)
Figure 10: Mobile phase: n-hexane, methanol, IPA, ethanol in ratio of 85:6:8:1 (v/v/v/v)
In order to obtain sharp peaks without compromising on the resolution, the 8 %
(v/v) of methanol content chosen in mobile phase. The chromatographic results are
summarized in Table 1.
Omeprazole Section-1
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Methanol Content
(%, v/v) Rs α Sym-(S) Sym-(R) N-(S) N-(R)
6 1.56 1.10 0.63 0.65 2631 2524
8 1.87 1.11 0.74 0.74 6162 5747
10 1.83 1.12 0.76 0.76 6503 6220
Table 1: Effect of methanol on system suitability results.
(Rs: Resolution, α: selectivity factor, Sym-(S); Symmetry value for (S)-enantiomer,
Sym-(R): symmetry value for (R)-enantiomer, N-(S): number of theoretical plates for
S isomer, N-(R): number of theoretical plates for R isomer)
In the final method, the typical retention times of (S)-omeprazole and (R)-
omeprazole were about 14.2 and 15.7 min, respectively (Fig. 11) and the runtime was set
to 20 min.
Figure 11: Enantiomeric resolution of omeprazole on Chiralcel OD-H column.
Mobile phase consisted of n-hexane, methanol, IPA, ethanol in ratio of 85:8:6:1
(v/v/v/v), flow rate: 0.75 mL/min, UV-302 nm, column temperature: 40°C,
Injection volume: 5 µl.
Omeprazole Section-1
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In order to identify the enantiomeric peaks in racemic mixture, pure omeprazole
enantiomer, i.e. (S)-omeprazole injected in developed method (Fig. 12).
Figure 12: Enantiomeric analysis of (S)-omeprazole
.
Figure 13: Enantiomeric analysis of (S)-omeprazole spiked with 10% (w/w)
(R)-omeprazole
Omeprazole Section-1
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5.2 Results of Method Validation
5.2.1 Results of System suitability
The system suitability results summarized in Table 2, which showed the
resolution between both enantiomers was not less than 1.75. The peak purity results were
passed for both the enantiomers and report of peak purity is presented in Fig. 14 and 15.
Omeprazole Rs α Purity Factor Threshold
(S)-enantiomer 999.364 999.110
(R)-enantiomer 1.87 1.11 999.629 999.076
Table 2: System suitability results.
Figure 14: Peak purity report of (S)-omeprazole
Omeprazole Section-1
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Figure 15: Peak purity report of (R)-omeprazole
5.2.2 Results of Specificity
To evaluate the selectivity, the chromatogram obtained by analyzing blank run
consisting of diluent and placebo was compared in order to check the absence of any
peaks likely to interfere at RTs of S- and R- omeprazole. As can be seen in overlay of
omeprazole (LLOQ level) and blank chromatogram (Fig. 16), blank chromatograms is
free from any interference at RTs of zopiclone enantiomers. The peak purity factor was
within the calculated threshold limit for (S)-omeprazole and (R)-omeprazole enantiomers
(Table 3).
Omeprazole Purity Factor Threshold
(S)-enantiomer 999.298 999.097
(R)-enantiomer 999.144 999.103
Table 3: Peak purity results specificity.
Omeprazole Section-1
71
Figure 16: Overlay of blank and omeprazole chromatograms
Stability Indicating Method
The drug shows 30%, 15%, and 21% degradation under acidic, basic, and
oxidative conditions, respectively. The purity factor is within the threshold limit for
omeprazole enantiomers in forced degradation samples (Table 4). The degradation
products were separated from omeprazole enantiomers (Fig. 17, 18, 19), hence the
developed method was found to be stability indicating and results are free from any
interference.
Omeprazole Section-1
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Stress conditions % Degradation Peak purity result
Acid degradation (0.1M HCL) 30 Pass
Alkali degradation (0.1M NaOH) 15 Pass
Oxidative degradation (30% H2O2) 21 Pass
Table 4: Peak purity results for force degradation study.
Figure 17: Omeprazole degradation profile in acidic stress condition
Omeprazole Section-1
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Figure 18: Omeprazole degradation profile in alkali stress condition
Figure 19: Omeprazole degradation profile in oxidative stress condition
Omeprazole Section-1
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5.2.3 Results of Precision
The precision of the method was performed at analyte concentration (i.e. 800
ug/ml) of (S)-omeprazole spiked with 0.1 % (0.8 ug/ml) of (R)-omeprazole. Percentage
relative standard deviation (% RSD) for major enantiomer, i.e. (S)-omeprazole was less
than 0.25 for retention time and 0.35 for peak area. The precision for (R)-omeprazole
was performed at LLOQ level and %RSD was 2.01 for the retention time and 7.07 for
the peak area. Data of precision study are summarized in Table 5.
The intermediate precision was determined in another laboratory by performing
nine successive injections. In intermediate precision study, results showed that %RSD
values were in the same order of magnitude than those obtained for repeatability and data
are presented in Table 6.
Precision Data
Sr. No. (S)-omeprazole (R)-omeprazole
RT Area RT Area
1 14.23 13700 15.91 23
2 14.22 13789 15.35 24
3 14.24 13777 15.78 21
4 14.26 13777 15.92 23
5 14.27 13730 15.22 25
6 14.28 13740 15.34 21
7 14.29 13712 15.32 23
8 14.28 13656 15.87 26
9 14.21 13688 15.18 23
Average 14.25 13725.78 15.54 23.22
SD 0.03 42.12 0.32 1.64
%RSD 0.20 0.31 2.04 7.07
Table 5: Results of precision study
Omeprazole Section-1
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Precision Data
15.82 (S)-omeprazole (R)-omeprazole
RT Area RT Area
1 15.1 13940 16.06 23
2 15.06 13948 16.68 23
3 15.07 13980 16 19
4 15.02 13960 16.69 24
5 15.01 13911 16.88 23
6 15.06 13925 16.07 23
7 14.99 13992 16.4 22
8 15.04 14014 16.67 22
9 15.08 13890 16.83 23
Average 15.05 13951.1 16.48 22.44
SD 0.04 40.17 0.35 1.42
%RSD 0.24 0.29 2.13 6.34
Table 6: Results of intermediate precision study
5.2.4 Results of Linearity
The described method was linear in wide concentration range, covering from 0.39
to 800 µg/mL. The linearity study evaluated by injecting each calibration level in
duplicate. The calibration curve was drawn by plotting the peak area verses its
corresponding concentration. The each solution was injected in duplicate and R.S.D. of
area under the peak for duplicate runs remain < 2% across the study.
The correlation coefficient was more than of 0.999 for (S)-omeprazole peak, which
shows good linear detector response over determined concentration range in developed
method. The equation of the calibration curve for (S)-omeprazole was y = 16.9 x + 31.4
(Fig. 20).
Omeprazole Section-1
76
Figure 20: Linearity of (S)-omeprazole 0.39 to 800 µg/ml
The calibration curve was drawn by plotting the peak area verses its corresponding
concentration with a correlation coefficient was more than of 0.999 for (R)-omeprazole
peak. The equation of the calibration curve for (R)-omeprazole was y = 16.9 x + 32 (Fig.
21).
Figure 21: Linearity of (R)-omeprazole 0.39 to 800 µg/ml
Omeprazole Section-1
77
5.2.5 Results of Sensibility
The LLOD and LLOQ results for (S)- and (R)-omeprazole were satisfactory. The
results are summarized in Table 7.
(S)-omeprazole (R)-omeprazole
LLOD (ng/mL) 390 390
S/N 3.2 3.3
Precision (n=6) 6.45 6.22
LOQ (ng/mL) 780 780
S/N 12 11.5
Precision (n=6) 3.76 2.99
Table 7: Results of Sensibility
5.2.6 Results of Recovery
The recovery experiments were conducted to determine the accuracy of the
present method for the quantification of (R)-omeprazole in formulation samples. (R)-
omeprazole was spiked to the extracted (S)-omeprazole sample (800 µg/ml) in triplicate at
0.12, 0.15 and 0.18% of target analyte concentration.
Recovery was calculated from the slope and Y-intercept of the calibration curve
obtained in linearity study. The same recovery experiments were also conducted using a
different system in laboratory B at the same concentration levels tested in laboratory A
and results were well in agreement. This confirms the ruggedness of the method. The
results are summarized in Table 8
.
Omeprazole Section-1
78
Laboratory A
% Level of
test
concentration
Added
(ng)
Recovered
(ng)
%
Recovery % RSD
(R)-omeprazole
Recovery
80 965 902 93.5 4.5
100 1215 1155 95.1 3.8
120 1455 1513 104 4.0
Laboratory B
% Level of
test
concentration
Added
(ng)
Recovered
(ng)
%
Recovery % RSD
(R)-omeprazole
Recovery
80 955 904 94.7 4.2
100 1211 1192 98.5 3.3
120 1448 1469 101.5 4.2
Table 8: Results of recovery study
5.2.7 Robustness
The chromatographic resolution of the (S)- and (R)-omeprazole enantiomers peaks
was remain more than 1.75 under all modified conditions, which demonstrate the
sufficient robustness of the method. Results are summarized in Table 8. It can be seen
from the data that method is robust for its intended use.
Omeprazole Section-1
79
Parameters Resolution between two enantiomers
Flow rate (mL/min)
0.36 1.89
0.44 1.87
Column temperature (°C)
35 1.88
40 1.87
45 1.85
Methanol content (%, v/v)
7 1.75
8 1.87
9 1.84
Table 8: Results of robustness study
5.2.8 Results of Solution Stability
No significant change was observed in resolution and peak area composition of
omeprazole enantiomers during the solution stability study.
Time interval (h) % area bias
Resolution (S)-omeprazole (R)-omeprazole
Initial - - 1.87
3 0.12 0.18 1.88
6 -0.19 -0.21 1.87
9 -0.21 -0.25 1.84
12 -0.34 -0.30 1.87
18 -0.73 -0.82 1.85
24 -0.91 0.89 1.86
Table 9: Results of solution stability study
Omeprazole Section-1
80
The data are presented in Table 9, It can be seen from the data that % bias of area
for omeprazole enantiomers was less than 1% hence the sample solution and mobile
phase are stable for 24h at room temperature, i.e., at 25°C.
[6] Conclusion
A simple, suitable, linear, precise and fast chiral HPLC method was described for
the enantiomeric separation of omeprazole.
The baseline separation was achieved on a Chiralcel OD-H column, containing
cellulose tris(3,5-dimethylphenyl carbamate) coated on silica gel as CSP. In this we
found the importance of methanol as a polar organic modifier in normal phase chiral
chromatography, which improved the peak shape of omeprazole enantiomers.
The accuracy data proved that the method can be used for the quantitative
determination of undesired enantiomer of omeprazole in the enteric coated
pharmaceutical formulations. This is the first report to describe the stability indicating
chiral HPLC method for the enantioselective analysis of omeprazole enantiomers. The
method was completely validated and shown satisfactory data for all the method
validation parameters tested. The work also deals with sensitive (LLOQ=0.78 µg/mL)
and linear over the thousand fold concentration range. This method can be used for
routine analysis in quality control laboratories.
Omeprazole Section-1
81
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