part-[a] - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/46826/8/08... · 2015-12-04 ·...

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


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


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.


53

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].


54

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


55

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].


56

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].


57

[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


58

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.


59

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.


60

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


61

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


62

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-


63

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].


64

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.


65

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)


66

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.


67

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.


68

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


69

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


70

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.


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.


72

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


73

Figure 18: Omeprazole degradation profile in alkali stress condition

Figure 19: Omeprazole degradation profile in oxidative stress condition


74

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


75

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).


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


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

.


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.


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


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.


81

[7] References

[1] E. J. Ariëns: Stereochemistry, a basis for sophisticated pharmacokinetics and

clinical pharmacology European Journal of Clinical Pharmacology, 26; 663-668

(1984).

[2] Development of New Stereoisomeric Drugs, Guidance, Compliance & Regulatory

Information, U.S. Food and Drug Administration.

[3] Angela Abelo, Tommy B. Andersson, Drug. Metab. Dispos August; 28; Issue 8,

966-972 (2000).

[4] M. Mathew, V. Das Gupta, R.E. Bailey, Drug Dev. Ind. Pharm. 21, 965–971

(1995).

[5] S. Agatonovic-Kustrin, D. Williams, N. Ibrahim, B.D. Glass, Curr. Drug Discov.

Technol. 4; 192–197 (2007).

[6] M. Erickson, L. Josefsson, Pharmaceutical Formulation for Omeprazole,

AstraZeneca. US Patent Number 6,090,827 Astra Zeneca AB (2000).

[7] C. Scarpignato, I. Pelosini, Aliment. Pharmacol. Ther. 23, 23–34 (2006).

[8] Sverker von Unge, Vratislav Langer and Lennart Sjolin tetrahedron: Asymmetry

Vol. 8, No. 12, 1967-1970 (1997).

[9] Stedman CA, Barclay ML. Review article: comparison of the pharmacokinetics,

acid suppression and efficacy of proton pump inhibitors. Aliment Pharmacol

Ther.14(8); 963–978 (2000).

[10] Lau WC, Gurbel PA. The drug-drug interaction between proton pump inhibitors

and clopidogrel. CMAJ. 180(7); 699–700 (2009)

[11] Norgard NB, Mathews KD, Wall GC (July 2009). Drug-drug interaction between

clopidogrel and the proton pump inhibitors". Ann Pharmacother. 43(7); 1266–

1274 (2009).


82

[12] Pauli-Magnus C, Rekersbrink S, Klotz U, Fromm MF. Interaction of omeprazole,

lansoprazole and pantoprazole with P-glycoprotein. Naunyn Schmiedebergs Arch

Pharmacol. 364(6); 551–557 (2001).

[13] Prilosec Side Effects & Drug Interactions". RxList.com. Retrieved 2007-06-16

(2007).

[14] Madanick RD. "Proton pump inhibitor side effects and drug interactions: much

ado about nothing?” Cleve Clin J Med. 78(1); 39–49 (2011)

[15] M. Mathew, V. Das Gupta, R.E. Bailey, Drug Dev. Ind. Pharm. 21, 965–971

(1995).

[16] S. Agatonovic-Kustrin, D. Williams, N. Ibrahim, B.D. Glass, Curr. Drug Discov.

Technol. 4, 192–197 (2007).

[17] M. Erickson, L. Josefsson, Pharmaceutical Formulation for Omeprazole,

AstraZeneca. US Patent Number 6,090,827, Astra Zeneca AB (2000).

[18] C. Scarpignato, I. Pelosini, Aliment. Pharmacol. Ther. 23, 23–34 (2006).

[19] Q.B. Cass, F. Batigalhia, J. Chromatogr. A. 987; 445-452 (2003).

[20] Q.B. Cass, A.L.G. Degani, N.M. Cassiano, J. Liq. Chromatogr. Relat. Technol.

26; 2083-2101 (2003).

[21] M. Kato, T. Fukushima, N. Shimba, I. Shimada, Y. Kawakami, K. Imai, Biomed.

Chromatogr. 15; 227-237 (2001).

[22] Jeanette Olsson, Filip Stegander, Nicola Marlin, Hong Wan, Lars G. Blomberg.

Enantiomeric separation of omeprazole and its metabolite 5-hydroxyomeprazole

using non-aqueous capillary electrophoresis. J. Chromatogr. A.1129; 291-295

(2006).

[23] Bonato PS, Paias FO. Enantioselective analysis of omeprazole in pharmaceutical

formulations by chiral high-performance liquid chromatography and capillary

electrophoresis. J Braz Chem (15); 318-23 (2004).


83

[24] Q.B. Cass, V.V. Lima, R.V. Oliveira, N.M. Cassiano, A.L.G. Degani, J.

Pedrazzoli Jr. Enantiomeric determination of the plasma levels of omeprazole by

direct plasma injection using high-performance liquid chromatography with

achiral-chiral column switching. J. Chromatogr. B. (798); 275-281 (2003).

[25] Katia R.A.B., Mariana C., J.C. Barreiro, C.A. Montanari, Q.B. Cass. milligram

enantioresolution of sulfoxide proton pump inhibitors by liquid chromatography

on polysaccharide-based chiral stationary phase. Journal of Pharmaceutical and

Biomedical Analysis. (47); 81-87 (2008).

[26] ICH – international conference on harmonization of technical requirements for

registration of pharmaceuticals for human use. Validation of analytical Procedure:

Text and Methodology ICH Secretariat, Geneva, Switzerland (1996).

[27] Wainer IW, Stiffin RM, Shibata T. Resolution of enantiomeric aromatic alcohols

on a cellulose tribenzoate high-performance liquid chromatography chiral

stationary phase: A proposed chiral recognition mechanism. J Chromatogr A.,

441:139 (1987).

[28] Cirilli R, Ferretti R, Gallinella B, De Santis E, Zanitti L, La Torre F. High-

performance liquid chromatography enantioseparation of proton, pump inhibitors

using the immobilized amylose-based Chiralpak IA chiral stationary phase in

normal-phase, polar organic and reversed-phase conditions. J Chromatogr A.,

1177:105-13 (2008).

[29] Belaz KR, Coimbra M, Barreiro JC, Montanari CA, Cass QB. Multimilligram

enantioresolution of sulfoxide proton pump inhibitors by liquid chromatography

on polysaccharide-based chiral stationary phase. J Pharm Biomed Anal, 47:81-7

(2008).

part-[a] - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/46826/8/08... · 2015-12-04 ·...

Documents