3.1 introduction - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/20011/9/09... ·...

3.1 Introduction

Omeprazole is a substituted benzimidazole proton pump inhibitor. It is a

contains a tricoordinated sulfinyl sulfur in a pyramidal structure and therefore can exist in equal

amounts of both the (S)- and (

parietal cells, both are converted to achiral products (sulfenic

acid and sulfenamideconfigurations) which react with a cysteine group in

(H+/K+) ATPase, thereby inhibiting the ability of the

Omeprazole is chemically designated as “1

dimethyl-2-pyridinyl) methyl] sulfinyl] benzimidazole” with an empirical formula C

and its molecular weight is 345.42 and i

The chemical structure of omeprazole is as shown in Figure 3.1.

Figure 3.1: Chemical structure of

Omeprazole is a white to off

about 155°C. It is a weak base, freely soluble in ethanol and methanol, and slightly soluble in

acetone and isopropanol and very slightly soluble in water. Th

function of pH; it is rapidly degraded in acid media, but has acceptable stability under alkaline

conditions. Proton pump inhibitors are among the world's most widely used therapeutic classes.

Omeprazole is a proton pump inhi

(PUD), gastro esophageal reflux disease (GORD/GERD), laryngo pharyngeal reflux (LPR) and

Zollinger–Ellison syndrome and is one of the most widely prescribed drugs internationally.

Omeprazole suppresses gastric acid secretion. By acting specifically on the proton pump,

omeprazole blocks the final step in acid production, thus reducing gastric acidity [1].

Omeprazole is a competitive inhibitor of the enzymes

There is anevidence that omeprazole administration results in significant decrease in the

clearance of diazepam, phenytoin, and possibly carbamazepine and S

Omeprazole is a substituted benzimidazole proton pump inhibitor. It is a


and (R)-enantiomers. In the acidic conditions of the canaliculi of


sulfenamideconfigurations) which react with a cysteine group in hydrogen potassium

ATPase, thereby inhibiting the ability of the parietal cells to produce

Omeprazole is chemically designated as “1H-Benzimidazole, 5-methoxy-2-[[(4

pyridinyl) methyl] sulfinyl] benzimidazole” with an empirical formula C

and its molecular weight is 345.42 and it is freely soluble in 0.1N sodium hydroxide solution.

The chemical structure of omeprazole is as shown in Figure 3.1.

Figure 3.1: Chemical structure of omeprazole

Omeprazole is a white to off-white crystalline powder that melts with decomposition at


acetone and isopropanol and very slightly soluble in water. The stability of omeprazole is a



Omeprazole is a proton pump inhibitor used in the treatment of dyspepsia, peptic ulcer disease


Ellison syndrome and is one of the most widely prescribed drugs internationally.

ppresses gastric acid secretion. By acting specifically on the proton pump,


Omeprazole is a competitive inhibitor of the enzymes cytochrome P450 (CYPs) 2C9 and 2C19.

ere is anevidence that omeprazole administration results in significant decrease in the

clearance of diazepam, phenytoin, and possibly carbamazepine and S-warfarin [2

Omeprazole is a substituted benzimidazole proton pump inhibitor. It is a racemate and


acidic conditions of the canaliculi of


hydrogen potassium

to produce gastric acid.

[[(4-methoxy-3, 5-

pyridinyl) methyl] sulfinyl] benzimidazole” with an empirical formula C17H19N3O3S

t is freely soluble in 0.1N sodium hydroxide solution.

white crystalline powder that melts with decomposition at


e stability of omeprazole is a



bitor used in the treatment of dyspepsia, peptic ulcer disease


Ellison syndrome and is one of the most widely prescribed drugs internationally.

ppresses gastric acid secretion. By acting specifically on the proton pump,


CYPs) 2C9 and 2C19.

ere is anevidence that omeprazole administration results in significant decrease in the

warfarin [2, 3].

The absorption of omeprazole takes place in the small intestine and is usually completed

within 3–6 hours. The systemic bioavailability of omeprazole after repeated dose is about 60%.

Omeprazole bioavailability is significantly impaired by the presence of food and, therefore,

patients should be advised to take omeprazole with a glass of water on an empty stomach (fast

for at least 60 minutes before taking omeprazole). Additionally, most sources recommend that

after taking omeprazole at least 30 minutes should be allowed to elapse before eating [4, 5].

Omeprazole therapy in an adult with congenital chloridorrhea results in control of his diarrhea

and hypokalemia, by reducing gastric chloride secretion [6]. Omeprazole improves the

antiobesity and antidiabetic activity of Exendin-4 in db/db mice [7]. Omeprazole attenuates

hyperoxic injury in H441 cells via the aryl hydrocarbon receptor [8]. Omeprazole attenuates

hyperoxic lung injury in mice via aryl hydrocarbon receptor activation and is associated with

increased expression of cytochrome P4501A enzymes [9]. Omeprazole inhibits proliferation and

modulates autophagy in pancreatic cancer cells [10]. Omeprazole decreases paracellular cation

permeability and increases the activation energy for passive Mg+2 transport of CaCo-2

monolayers that led to the suppression of passive Mg+2 absorption [11]. The association of

omeprazole with Ca(OH)2 favored a superior repair of rat periapical lesions and seemed to

display different selective activity over endodontic microbiota, in comparison with the

conventional Ca(OH)2 dressing [12]. Omeprazole is also a competitive inhibitor of p-

glycoprotein, as are other proton pump inhibitors [13]. Omeprazole is a specific gastric secretion

inhibitor on oxynticopeptic cells, reduces gizzard erosion in broiler chicks fed with toxic fish

meals [14]. Omeprazole is more effective than a histamine H2receptor blocker for maintaining a

persistent elevation of gastric pH after colon resection for cancer [15]. Omeprazole degradation

in acid medium was mainly dependent on microcrystalline cellulose concentration. A 90-day

accelerated stability test in brown glass bottles with a desiccant showed that all prototype

formulations would result in an acceptable stability profile for both remaining omeprazole, and

also for the increase of impurity concentrations [16]. Omeprazole is beneficial in basal ulcer

healing and it reversed the adverse action of indometacin on ulcer repair under acid-independent

conditions. These actions are likely to be mediated through the promotion of gastric epithelial

cell migration but not cell proliferation [17].

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. 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.

Several analytical methods for the determination of omeprazole in biological samples,

bulk material or pharmaceutical formulations, have been reported in literature. Older methods

were extensively reviewed by Bosch et al [18]. Wei Zhang et al reported a method for

simultaneous determination of tolbutamide, omeprazole, midazolam and dextromethorphan in

human plasma by LC–MS/MS [19]. Harshal K. Trivedi et al developed a high performance

liquid chromatography (HPLC) method for determination of omeprazole and its related

compounds in pharmaceutical formulations [20]. The degradation of lansoprazole and

omeprazole in the aquatic environment was studied and reported by M. DellaGreca et al [21]. A

spectrophotometric method was reported for the determination of omeprazole, lansoprazole and

pantoprazole in pharmaceutical formulations by Abdel-Aziz M. Wahbi et al [22]. The alternating

current polarographic behavioral studies and determination of lansoprazole and omeprazole in

dosage forms and biological fluids was reported by N EL-Enany et al [23]. Zeinab Abdelaziz El-

Sherif et al reported a method for the determination of lansoprazole, omeprazole and

pantoprazole sodium sesquihydrate in the presence of their acid induced degradation products by

using reversed-phase high performance liquid chromatography [24]. F Salama et al reported

avalidated spectrophotometric method for the determination of omeprazole and pantoprazole

sodium via their metal chelates [25]. Statistical assurance of process validation by analytical

method development and validation for omeprazole capsules and blend was reported by

D.Kumaraswamy et al [26]. A column switching high-performance liquid chromatographic

method was reported for the determination of omeprazole and its two main metabolites in human

plasma by Mikiko Shimizu et al [27]. Quantification of omeprazole and its metabolites in human

plasma by liquid chromatography–mass spectrometry method was reported by different authors

[28, 29]. A method for the determination of omeprazole in human plasma by protein

precipitation and liquid chromatography–tandem mass spectrometry was reported by J. Macek et

al [30]. A spectrofluorimetric method for the determination of omeprazole based on its

degradation reaction catalyzed by ultraviolet (UV) light is proposed by Peralta CM et al [31]. Jha

P et al [32] reported a stability indicating high performance thin layer chromatographic (HPTLC)

method for quantitative determination of omeprazole in capsule dosage form.

The determination of four proton pump inhibitors, omeprazole, pantoprazole, lansoprazole and

rabeprazole in human plasma by high performance liquid chromatography was reported by

different authors [33, 34]. A kinetic spectrophotometric method for the determination of

omeprazole in dosage forms was reported by Mahmoud AM [35].A bio-analytical hydrophilic

interaction LC-MS/MS method for the simultaneous quantification of omeprazole and

lansoprazole in human plasma was reported by De Smet J et al [36]. A

spectrophotometric and chromatographic determination of omeprazole in pharmaceutical

formulations was reported by Gallardo V et al [37]. A Validated HPTLC method for

determination of ondansetron in combination with omeprazole in solid dosage form was reported

by Raval PB et al [38]. A bio analytical assay for simultaneous determination of omeprazole and

its three major metabolites in human blood plasma using reverse phased high performance liquid

chromatography (RP-HPLC) with liquid-liquid extraction procedure was reported byRezk NL et

al [39]. HPLC determination of omeprazole in human plasma by using a monolithic column was

reported by Zarghi A et al [40]. Simultaneous determination of omeprazole, hydroxyomeprazole

and omeprazole sulphone in human plasma by isocratic high performance liquid chromatography

with diode array detection (HPLC-DAD) method was reported by Linden, R et al [41]. Chiral

HPLC atmospheric pressure photo ionization tandem mass spectrometry method was reported

for enantioselective quantification of omeprazole and its metabolites in human serum by Jens

Martens-Lobenhoffer et al [42].Podilsky, G. et aldeveloped and validated an HPLC method for

the simultaneous monitoring of bromazepam and omeprazole [43]. Omeprazole and its main

metabolites were analyzed by liquid chromatography with hybrid micellar mobile phases [44].

Vittal S et al reported a method for the determination of omeprazole in human plasma by liquid

chromatography-electrospray ionization tandem mass spectrometry [45]. Simultaneous

determination of omeprazole and domperidone in dog plasma by liquid chromatography with

mass spectrophotometer (LC-MS) methodwas reported byZhan Li et al [46]. Determination of S-

omeprazole, R-omeprazole and racemic omeprazole were reported by M. Hassan-Alinet al [47].

As per the literature survey review on the above reported methodologies, most of the

methods were developed to determine the omeprazole in plasma. As the extraction process of

omeprazole from its formulation and separation of omeprazole from its formulation is becoming

a critical activity, less number of methods was reported for the determination of omeprazole

present in pharmaceutical formulations. For the analysis of omeprazole in biological matrices

and pharmaceutical formulations, spectrophotometry was frequently employed. Compared to

spectrophotometric methods, the HPLC methods are more sensitive, accurate, precise and

specific. Multi component formulations are difficult to be analyzed by spectrphotometric method

as they might have similar ƛmax values which can be separated and determined by high

performance chromatography through retention times. The other techniques reported for the

determination omeprazole are less precise and accurate when compared with chromatographic

techniques. Although these methods are quite suitable for the determination of omeprazole in

biological matrices and pharmaceutical formulations, all of them are characterized by relatively

long analysis times. The reported liquid chromatography-mass spectrometry (LC-MS) methods

are also quite suitable for determination of omeparazole in biological matrices and

pharmaceutical formulations, but this technique involves huge cost for analysis which may be a

limitation for routine quality control applications. But as such there is no validated method

available, which is having short analysis time to estimate the assay of omeprazole with more

precise and accurate as part of routine testing which is more useful in commercial aspect.

Therefore, it is very imperative to develop a suitable analytical method for omeprazole such that

the methods could be easily adapted for routine and in-process quality control analysis or similar

studies.

The aim of this study was to develop a rapid, simple, precise and accurate ultra

performance liquid chromatographic (UPLC) method for the determination of omeprazole in

pharmaceutical formulations. The developed method has to be validated as per the regulatory

requirement to use for routine quality control applications. The proposed method is validated

according to International Conference on Harmonization (ICH) guidelines [48] in terms of

specificity, precision, accuracy, linearity, range, ruggedness and robustness including with

stability of mobile phase, standard and sample solutions.

3.2. Experimental

3.2.1. Reference substances, chemicals, reagents and samples

The entire experiment was performed using “class A” volumetric glassware,

pharmaceutical grade omeprazole active pharmaceutical ingredient (API) and capsules procured

from Dr. Reddy`s laboratories limited. Analytical grade potassium dihydrogen phosphate,

dipotassium hydrogen phosphate purchased from Merck, Germany, potassium hydroxide,

sodium hydroxide pellets purchased from Ranbaxy laboratories limited, HPLC grade acetonitrile

purchased from Merck, highly pure HPLC grade Milli Q water collected from Millipore,

Bedford, MA, USA, 0.22µm membrane filter purchased from millipore, Barcelona were

employed in the studies.

3.2.2. Instrumentation

Omeprazole assay analysis was performed by using Waters UPLC (Milford, MA, USA)

PDA system consisting of a quaternary solvent manager, a sample manager, column-heating

compartment, and photodiode array detector. This system was controlled and out put signal was

monitored by Waters Empower software. Zorbax SB C18, Agilent, 50mm length, 4.6mm

internal diameter column with particle size 1.8µm employed as stationary phase for

chromatographic separation. Sartorius semi micro balance with model ME235S was used for all

weighing and Thermo Orion pH meter was used for buffer pH adjustment. Sonication carried out

with Bandelin sonicator and rotary shaker was adopted for shaking of samples during

preparation. All samples were centrifuged by Hermle centrifuge machine.

3.2.3 Blank, standard and sample solution preparation

Omeprazole API, capsules and the corresponding placebo (without API) were used

throughout the development and validation. All the samples were treated according to test

solution preparation.

3.2.3.1 Standard solution preparation

The standard stock solution of omeprazole was prepared by dissolving an accurately

weighed amount of omeprazole working standard in 0.1N sodium hydroxide solution, resulting

in a concentration of 0.4 mg mL-1. The above solution was further diluted with diluent (mobile

phase) to get a final solution of 0.04 mg mL-1.

3.2.3.2Blank solution preparation

Balnk solution was prepared by following the same procedure as mentioned in standard

solution preparation by omitting omeprazole working standard.

3.2.3.3 Sample solution preparation

The test solution was prepared by dissolving an accurately weighed portion of the

omeprazole enteric coated pellets, equivalent to 100 mg of omeprazole in 150mL of 0.1N sodium

hydroxide. After sonicating for around 15 minutes, the solution was shaken for 20 minutes and

the volume was made up to 250 ml with 0.1N sodium hydroxide solution. A portion of above

solution was centrifuged at 3000 rpm for 15 minutes in order to eliminate insoluble excepients.

2mL of the above supernatant solution was further diluted to 20mL volume with the diluent

(mobile phase) and the same was used for chromatographic analysis.

3.2.4 Chromatographic conditions

The analysis was carried out by using advanced ultra performance liquid chromatography

(UPLC). The omeprazole was separated on an extend C18, Agilent column with 50mm length,

4.6mm internal diameter and 1.8µm particle size at ambient column oven temperature with an

isocratic run program at a flow rate of 1.0mL minute−1. The separation was achieved by isocratic

elution with run time of 2 minutes. The mobile phase was filtered through a 0.45µm Millipore

filter, before use. Ultraviolet (UV) detection was performed at 302 nm. The sample injection

volume was 5µL. The buffer was prepared by dissolving 2.72gm of potassium dihydrogen

orthophosphate (KH2PO4) and 0.525gm of dipotassium hydrogen phosphate (K2HPO4) in

1000mL of water. The degassed composition of buffer and acetonitrile in 60:40 v/v ratio with pH

7.4 (adjusted with 0.1 N potassium hydroxide solution) was used as the mobile phase.

3.2.5 Evaluation of blank

Injected 5µL of blank solution into ultra performance liquid chromatograph and recorded

the chromatogram.

3.2.6 Evaluation of system suitability

From the chromatogram obtained for the standard preparation, the column efficiency was

determined for the analyte peak and found to be not less than 1500 theoretical plates. The tailing

factor was not more than 2.0, and the relative standard deviation of replicate injections was not

more than 2.0 %.

3.2.7 Procedure

The standard preparation and the sample preparation were separately injected into an

ultra performance liquid chromatograph and areas of the major peaks were recorded. The diluent

chromatogram was examined for any extraneous peaks, and the corresponding peaks observed

in the sample chromatogram were ignored. The retention time of omeprazole peak under the

present chromatographic conditions was about 0.9 minutes.

3.2.8 Quantitation

Omeprazole peak areas were recorded for standard and sample injections. Respective

peak areas were taken into account to quantitate the amount of omeprazole present in the sample

as follows:

At Cs P % of omeprazole = ------ x ------- x ------ x 100

As Ct 100

Where, At = Omeprazole peak area obtained from the sample preparation;

As = Omeprazole peak area obtained from the standard preparation;

Cs = Concentration of omeprazole in standard solution;

Ct = Concentration of omeprazole in sample solution and

P = Omeprazole standard purity in percentage.

3.3 Result and Discussion

3.3.1 Method development and optimization

The objective of this work is to develop a rapid, simple and precise method for the

determination of omeprazole (Active Pharmaceutical Ingredient) present in omeprazole

formulation (drug product) by using an ultra performance liquid chromatograph (UPLC).

Method development was initiated by the review of literature survey and studies on omeprazole

physical and chemical characteristics. The solubility of omeprazole was tested in different

solvents and identified that 0.1N sodium hydroxide solution was suitable for extraction of

omeprazole from its formulation. Based on spectral profile and absorption characteristics of

omeprazole, UV detector at 302nm wavelength was selected to detect omeprazole. The degassed

composition of phosphate buffer (Dissolved 2.72 gm of potassium dihydrogen orthophosphate

and 0.525 gm of dipotassium hydrogen phosphate in 1000 ml of water) was found suitable to

cope up for the injection load on the column. Acetonitrile was found to be suitable as an organic

modifier as it is a weak hydrogen acceptor. The participation of residual silanol groups in the

retention process is more pronounced in acetonitrile. Preliminary experiments carried out under

various chromatographic conditions are as follows.

3.3.1.1Optimization of chromatographic parameters

� First trial:

Column: Extend C18, Agilent (50mm length, 4.6mm internal diameter and 1.8µm particle

size). A bidentate organosilane combined with double end capping.

Buffer: Dissolved 2.72 gm of potassium dihydrogen orthophosphate and 0.525 gm of

dipotassium hydrogen phosphate in 1000 ml of water (pH = 7.4).

Mobile phase: Prepared a mixture of buffer and acetonitrile in the ratio of 80: 20 v/v, and

adjusted pH to 7.4 with 1M potassium hydroxide solution, filtered through 0.22 µm

membrane filter.

Flow rate: 0.5 mL minute-1

Injection volume: 2 µL

Data acquisition time: 15 minutes

Detection mode: Ultra violet detection at 302 nm.

In this trial, omeprazole was well separated from related impurities. The peak response

observed for omeprazole and theoretical plates was found very less as peaks were found broad in

shape. The elution time of omprazole was found to be about 5 minutes which need to be

reduced.

� Second trial:







membrane filter.


Injection volume: 5 µL



In this trial, peak response was found satisfactory and omeprazole was well separated

from its related impurities. The omeprazole peak was eluted at about 4 minutes.

� Third trial:



Buffe: Dissolved 2.72 gm of potassium dihydrogen orthophosphate and 0.525 gm of




membrane filter.


Injection volume: 5µL



In this trial, omeprazole peak was eluted in about 2 minutes and well separated from its

related impurities and also the peak was found to be symmetric.

� Fourth trial:







membrane filter.


Injection volume: 5µL


Detection mode: Ultra Violet detection at 302 nm.

In this trial, omeprazole peak was eluted in about 1 minute and was well separated from

its related impurities also found to be a symmetric peak.

From the results of all the above trials, it was finally concluded that the best optimal

conditions for the quantitative separation and elution are a C18 column with 50mm length,

4.6mm internal diameter and 1.8µm particle size as stationary phase, composition of buffer and

acetonitrile (60:40) as mobile phase with 1.0mL minute-1 flow rate, 5µL injection volume and

detection at 302nm with an UV detector.

3.3.2. Method Validation

The proposed test method was validated to include requirements of International

conference on Harmonization (ICH) guidelines [48], in terms of specificity, linearity, precision

(intermediate precision, method precision), accuracy, range, robustness and ruggedness. The

stability study of mobile phase, standard, sample solutions and system suitability were also

examined.

3.3.2.1 Specificity

As part of specificity study, the interference of placebo with omeprazole peak in

duplicate preparation of placebo equivalent present in test preparation was studied as per the

proposed test procedure. The placebo sample solutions were prepared at various concentrations

in the same manner as described in the sample preparation by taking placebo without omeprazole

and were injected into chromatographic system and recorded the chromatograms. There were no

interferences due to placebo and sample diluents at the retention times of omeprazole.

5µL of blank solution, placebo solution, standard and sample solutions were injected

separately into ultra performance liquid chromatograph and the chromatograms were recorded

under optimal conditions as shown in Figure 3.2, Figure 3.3, Figure 3.4, and Figure 3.5

respectively.

To check the impurities interference with omeprazole, impurities blend solution was

prepared and injected within the stability study limit (0.3%) as per test preparation into the

chromatographic system. The obtained results from the chromatograms are tabulated in Table 3.1

and the purity plots are shown in Figure 3.6 & 3.7.

Table 3.1: Results of impurities interference with omeprazole peak

Impurity Name % Stability limit

Omeprazole

Purity

Angle

Purity

threshold

Impurity-1 0.3

0.064 0.246

Impurity-2 0.3

Impurity-3 0.3

Impurity-4 0.3

Impurity-5 0.3

Imp A 0.3

3.3.2.2 Forced degradation studies

The stability indicating nature of the method was evaluated by performing the forced

decomposition (physical and chemical) studies to verify the interference of omeprazole peak

from its degrade impurities. Stress testing is carried out to identify the likely degradation

products or to elucidate the inherent stability characteristics of the active drug product [49]. In

this study, omeprazole enteric coated pellets ware subjected to the following physical and

chemical stress conditions.

3.3.2.2.1 Physical degradation studies

Physical degradation was carried out by exposing the drug product to heat, humidity, sun

light and UV light.

� Heat stress study

Omeprazole enteric coated pellets are heated at 105°C for 6 hours. The test solution was

prepared by dissolving an accurately weighed portion of heat stressed omeprazole enteric coated

pellets, equivalent to 100 mg of omeprazole in 150mL of 0.1N sodium hydroxide. After

sonicating for around 15 minutes, the solution was shaken for 20 minutes and the volume was

made up to 250 ml with 0.1N sodium hydroxide solution. A portion of this solution was

subjected for centrifugation and the supernatant solution was used for chromatographic analysis.

� Humidity stress study

Omeprazole enteric coated pellets were subjected to humidity stress at 90% relative

humidity and at 25°C temperature for 7 days. The humidity stressed omeprazole enteric coated

pellets test solution was prepared by dissolving an accurately weighed portion, equivalent to 100

mg of omeprazole in 150mL of 0.1N sodium hydroxide. The resultant solution was sonicated for

15 minutes and shaken for 20 minutes. The volume of the solution was finally made up to 250 ml

with 0.1N sodium hydroxide solution. A portion of this solution was subjected for centrifugation

and the supernatant solution was used for chromatographic analysis.

� Sunlight stress study

Omeprazole enteric coated pellets were sufficiently spread on Petri plates (1mm thick

layer) and exposed to sunlight for about 55 hours. 100 mg of sun light exposed omeprazole were

dissolved in 150mL of 0.1N sodium hydroxide, sonicated for 15 minutes, shaken well for 20

minutes and the volume was finally made up to 250 ml with 0.1N sodium hydroxide solution. A

portion of this solution was centrifuged and used for chromatographic analysis.

� Ultraviolet light stress study

The drug sample was exposed to ultraviolet light (1.2 million lux hours) at ambient

conditions for 7 days. The UV light exposed pellets of omeprazole drug 100mg were dissolved in

0.1N sodium hydroxide, sonicated for 15 minutes, shaken for 20 minutes and finally the volume

was made up to 250mL with 0.1N sodium hydroxide solution. The resultant solution was

centrifuged and used for chromatographic analysis.

3.3.2.2.2 Chemical degradation studies

Chemical degradation was carried out by exposing the drug product to acid hydrolysis,

base hydrolysis, peroxide oxidation and water stress.

� Acid hydrolysis stress study

The drug product was subjected to acid hydrolysis using 0.1N hydrochloric acid at 60°C

for 25 minutes. An accurately weighed acid hydrolyzed sample (100mg) of omeprazole was

dissolved in 0.1N sodium hydroxide. After sonicating for around15 minutes, and shaking the

solution for 20 minutes the solution was diluted to 250 ml with 0.1N sodium hydroxide solution.

A portion of this solution was centrifuged and used for chromatographic analysis.

� Base hydrolysis stress study

A sodium hydroxide solution of omeprazole drug (100mg in 250mL) was heated at 60°C

and its chromatograms were recorded under optimal conditions developed.

� Peroxide oxidation stress study

Omeprazole pellets were subjected to peroxide oxidation using 1% hydrogen peroxide

solution at a reflection temperature of 60°C for the period of 25 minutes. Accurately weighed

peroxide oxidation stressed sample equivalent to 100mg of omeprazole and dissolved in 0.1N

sodium hydroxide. After sonicating for around 15 minutes, and shaking the solution for 20

minutes, the volume was made up to 250mL with 0.1N sodium hydroxide solution. A portion of

this solution was centrifuged and the supernatant solution was used for chromatographic

analysis.

� Water stress study

The drug was subjected to water stress using purified water at a reflection temperature of

60°C for 60 minutes. 100mg of stressed sample was dissolved in 0.1N NaOH, sonicated and

centrifuged. The resultant solution was made up to 250mL with 0.1N sodium hydroxide and a

portion of the solution was subjected to chromatographic analysis.

All the stressed samples were injected into the ultra performance liquid chromatographic

system with PDA detector as per proposed test method conditions. The corresponding purity

plots were presented in Figure 3.8 (unstress), Figure 3.9 (heat stress), Figure 3.10 (humidity

stress), Figure 3.11 (sunlight stress), Figure 3.12 (UV light stress), Figure 3.13 (acid hydrolysis

stress), Figure 3.14 (base hydrolysis stress), Figure 3.15 (peroxide oxidation stress), and Figure

3.16 (water stress). The obtained results from the chromatographic system were tabulated in

Table 3.2(a) and Table 3.2(b).

Table 3.2(a): Physical degradation study results

Heat stress

Humidity stress

Sunlight stress UV light

stress

Temperature/ intensity/ humidity

105°C 90 % RH,25°C 1.2 million lux hrs UV light

Time 6 Hrs 7 Days 55 Hrs 7 Days

Purity angle 0.277 0.049 0.050 0.049

Purity threshold 0.636 0.245 0.244 0.245

Table 3.2(b): Chemical degradation study results

Acid

hydrolysis Base hydrolysis

Peroxide oxidation

Water stress

Concentration 0.1N HCl 0.1N NaOH 1 % Peroxide Water

Reflection temp 60°C 60°C 60°C 60°C

Reflection time 25 Min 25 Min 25 Min 1Hr

Purity angle 0.736 0.664 0.122 0.376

Purity threshold 1.119 1.100 0.318 0.669

Homogeneity of all the impurities, omeprazole and degradants was established using a

photo diode array (PDA) detector. All known impurities and unknown degradants were well

separated and for all the compounds, purity angle was found to be less than purity threshold.

Apart from the peaks homogeneity, the spectra of all the impurities and omeprazole were

compared against their standard spectra. The degradation studies under various specific

conditions have shown that no impurity which interferes with the peak of omeprazole was

noticed. Based on the forced degradation studies, it was observed that the omeprazole degraded

under acid hydrolysis and hence it is unstable in acid. This shows that the method can be used for

the determination of omeprazole with the same accuracy under all the degradation conditions as

mentioned above.

3.3.2.3. Precision

3.3.2.3.1. System precision

To evaluate system precision, five replicate injection of omeprazole standard solution

were injected into an ultra performance liquid chromatographic system and the chromatograms

were recorded. The relative standard deviation, tailing factor and theoretical plates of omeprazole

peak were calculated. The observed % of relative standard deviation, tailing factor and

theoretical plates, 0.3%, 1.1 and 7206 respectively, which are found to be satisfactory against the

prescribed limits of not more than 2.0%, 2.0 and not less than 1500 respectively.

3.3.2.3.2 Intermediateprecision

As part of intermediate precision for omeprazole, the study was conducted at two

different placebo concentrations (4.5% and 10.0%) of omeprazole by two different analysts using

different UPLC instruments and different columns.

Table 3.3(a): Intermediate precision results of omeprazole at lower and higher strengths.

Sample No: % Assay (4.5%) % Assay (10.0%)

Analyst-1 Analyst-2 Analyst-1 Analyst-2

1 100.47 100.55 99.68 100.51

2 101.21 100.41 99.67 98.78

3 100.73 99.12 99.46 99.16

4 101.46 101.23 99.77 98.59

5 101.92 101.30 99.80 99.06

6 101.56 100.26 99.74 98.91

Average 101.23 100.48 99.69 99.17

% RSD 0.53 0.79 0.12 0.69

The relative standard deviations of the observed results at various conditions are found to

be less than 2.0%. The obtained results are tabulated in Table 3.3(a).

3.3.2.3.3. Methodprecision (repeatability)

The method precision of the test method was determined by assaying six samples

prepared from omeprazole enteric coated pellets as per the proposed test procedure, and

calculated the relative standard deviation of the obtained assay results. The precision at lower

(4.5%) and higher (10.0%) strengths was evaluated and the obtained results were tabulated in

Table 3.3(b).

Table 3.3(b): Method precision results of omeprazole at lower and higher strengths.

S.No.

Assay of omeprazole (%)

4.5% strength

10.0% strength

Precision-1 100.47 99.68

Precision-2 101.21 99.67

Precision-3 100.73 99.46

Precision-4 101.46 99.77

Precision-5 101.92 99.80

Precision-6 101.56 99.74

Average 101.23 99.69

% RSD 0.53 0.12

3.3.2.4 Accuracy

To confirm the accuracy of the proposed method, recovery studies were carried out by

standard addition technique. Samples were prepared six times at lower (50%) and higher (150%)

level concentrations and in triplicate at 75%, 100%, 125% (A nominal concentration of about

0.02 mg mL-1 to 0.06 mg mL-1) of the test concentration. The samples were prepared as per the

proposed test method at various concentration levels, injected into the chromatographic system

and recorded the chromatograms and recoveries were calculated and tabulated in the Table 3.4.

The results of accuracy as determined by both the calculation methods revealed that, the

average recovery at each level was between 97.0% to 103.0% with RSD at each level was ≤ 5%.

No significant difference was seen between the two methods.

Table 3.4: Accuracy results of omeprazole.

Sample No.

Level (%)

Amount added (mg)

Amount found (mg)

Recovery (%)

Statistical Analysis

1 50 50.83 51.96 102.22 Mean* 102.5

2 50 50.79 51.96 102.31

3 50 50.71 52.19 102.90 SD* 0.42

4 50 50.76 52.26 102.97

5 50 50.64 51.62 101.92 RSD* (%) 0.4

6 50 50.70 52.11 102.76

1 75 75.98 76.61 100.83 Mean^ 100.8

2 75 76.06 77.03 101.28 SD^ 0.48

3 75 76.02 76.26 100.32 RSD^ (%) 0.5

1 100 101.46 102.58 101.10 Mean^ 101.6

2 100 101.35 103.39 102.02 SD^ 0.49

3 100 101.43 103.29 101.84 % RSD^

(%) 0.5

1 125 126.56 126.89 100.26 Mean^ 101.6

2 125 126.67 129.33 102.10 SD^ 1.21

3 125 126.55 129.77 102.54 RSD^ (%) 1.2

1 150 152.07 152.93 100.57 Mean* 101.0

2 150 152.09 153.67 101.04

3 150 151.97 154.89 101.92 SD* 0.53

4 150 151.92 153.84 101.26

5 150 152.01 152.76 100.49 RSD* (%) 0.5

6 150 152.03 153.28 100.82

Overall statistical analysis

Mean$ 101.6

SD$ 0.87

RSD$ (%) 0.9

* : For six replicates ^ : For three replicates $ : For twenty one replicates

3.3.2.5. Linearity

To demonstrate the linearity of detector response for omeprazole assay method, six

standard solutions with concentration ranging from 25% to 150% of omeprazole (40µg/ml) were

prepared to cover the concentration of omeprazole from 10 ppm to 60 ppm. The chromatograms

were recorded for these solutions under optimal conditions and the peak areas were measured. A

graph was plotted between concentration (µg mL-1) and average peak area (absorbing units). The

data was used for statistical analysis using a linear regression model. The statistical parameters

of linear curve (Ca), slope, intercept and coefficient of determination values were calculated and

shown in table 3.5.

Table 3.5: Statistical data of omeprazole linearity study

S. No. Concentration (µg mL-1) Peak area (AU) Coefficient of

determination (r2)

01

10.0075 84616 0.9993

02

20.0150 179364

03

30.0225 251867

04

40.0300 344565

05

50.0375 429747

06 60.0450 516881

Figure 3.17: The linearity graph of omeprazole

The linearity curve was established by plotting the values of concentration(µg mL-1) on

X-axis and obtained areas from chromatographic system (absorbing units) on Y-axis as

determined from linearity test (Figure 3.17). The obtained coefficient of determination (r2 is

0.9993) shows that the calibration curve is very much linear in the concentration range

mentioned.

3.3.2.6 Range

To demonstrate the range of the analytical method, data from six values of lower and

higher concentration solutions of accuracy preparation was considered. The obtained mean

recovery at lowest level and highest level was found between 97.0% and 103.0% with the

coefficient of determination (r2) above 0.999 and relative standard deviation for six preparations

at each level was found below 2.0% which shows that the analytical method is more accurate and

precise throughout its range of 10 µg mL-1 to 60 µg mL-1 of omeprazole.

3.3.2.7 Ruggedness

R² = 0.9993

0

100000

200000

300000

400000

500000

600000

0 10 20 30 40 50 60 70Concentration (µg mL-1)

Pe

ak

are

a (

AU

)

Linearity plot of omeprazole

3.3.2.7.1 Bench top stability of omeprazole test preparation and standard preparation:

To demonstrate the bench top stability of omeprazole test and standard preparations, the

assay of omeprazole was performed in duplicate, kept the standard and test preparations on

bench top and analyzed on day 1 and day 2 against a freshly prepared standard each time. The

results are tabulated in Table 3.6(a).

Table 3.6(a) Bench top stability study results of omeprazole standard and sample solutions

Time in days

Standard (Omeprazole )

Similarity factor

Assay of test preparation (%)

Difference

T1 T2 T1 T2

Initial

NA NA 100.47 101.21 NA NA

1

1.013 1.007 100.94 100.69 0.47 0.52

2 0.981 1.001 100.80 100.33 0.33 0.88

The observed omeprazole assay of test solutions were not differed more than 3.0% from

the initial assay value and similarity factors of standard solutions were obtained within 0.98 to

1.02. The obtained results evidenced that omeprazole test solution and standard solutions were

stable for 2 days on bench top.

3.3.2.7.2 Refrigerator stability of omeprazole standard and test preparation:

To demonstrate the refrigerator stability of omeprazole standard and test solutions, the

solutions were analyzed for omeprazole in duplicate. The standard and test preparations were

kept in refrigerator and analyzed twice, on each day. The observed omeprazole assay of test

solutions were not differed more than 3.0% from the initial assay value and similarity factor of

standard solutions were obtained within 1.00 to 1.02. The obtained results evidenced that

omeprazole test solution and standard solutions were stable for 2 days in refrigerator. The results

are shown in Table 3.6(b).

Table 3.6(b): Refrigerator stability study results of omeprazole standard and sample solution

Time in days

Standard Similarity factor

Assay of test preparation (%)

Difference

T1 T2 T1 T2 T1 T2

Initial NA NA 100.47 101.21 NA NA

1 1.012 1.007 100.46 102.05 0.01 0.84

2 1.019 1.000 99.54 100.38 0.93 0.83

3.3.2.7.3 Bench top stability of mobile phase:

To demonstrate the bench top stability of mobile phase, performed the assay of

omeprazole was carriedout by preparing as per test procedure using same lot of mobile phase

stored on bench top at initial, after 1 day to 4 days with one day interval. The chromatograms

were recorded and the results of system suitability parameters and assay of omeprazole are

presented in Table 3.7(a) and Table 3.7(b) respectively. The observed results of system

suitability at each interval were meeting with the system suitability requirement and obtained

assay values at each interval were not differed more than 3.0% from the initial interval results

which evidenced that the mobile phase is stable for 5 days on bench top storage.

Table 3.7(a): System suitability results of mobile phase bench top stability study.

System Suitability Parameters

(obtained from omeprazole peak in standard)

Observed value

Acceptance Criteria

At Initial

(1st day) Day 2 Day 3 Day 4

Day 5

Tailing factor 1.1 1.2 1.2 1.2 1.2 NMT 2.0

Theoretical plates 7206 7366 7060 7165 7178 NLT 1500

RSD for peak area 0.3% 0.1% 0.4% 0.2% 0.1% NMT 2.0

Table 3.7(b): Assay results of mobile phase bench top stability study.

% Assay

Assay of omeprazole (%)

At Initial

(1st day) Day 2 Day 3 Day 4

Day 5

Average Assay

100.84 103.00 100.53 99.05 100.03

Difference from initial

NA 2.16 0.31 1.79 0.81

3.3.2.8. Robustness

3.3.2.8.1 Study on variable conditions

The robustness of the proposed method was demonstrated by the results obtained in the

study of system suitability parameter by injecting the standard preparation with variable mobile

phase composition (90% to 110% acetonitrile), variable flow rates (0.8, 1.0 and 1.2 mL minute-

1), under variable pH conditions (pH 7.2 and 7.5), and at variable column temperatures (25°C

and 30°C), chromatograms were recorded under variable conditions as mentioned above and

plate count and tailing factor were evaluated for each chromatogram which are presented in

Table 3.8.

The observed results of system suitability parameters from the above robustness study

were found well within the acceptance criteria (RSD ≤ 0.3%) and which shows that the method

is robust for the intended purpose.

Table 3.8: Summary of robustness results

Robustness Condition

Variation USP Tailing USP Plate

count RSD (%)

As per test Method

- 1.2 7345 0.3

Mobile phase - 10% 1.1 7501 0.1

+ 10% 1.3 6542 0.2

Flow Rate (1 mL minute-1)

- 0.2 mL minute-1 1.2 7785 0.2

+0.2 mL minute-1 1.2 6808 0.2

pH (7.4) - 0.2 1.2 7371 0.2

+ 0.1 1.2 7111 0.3

Oven temperature (Ambient)

+ 5°C 1.2 7307 0.1

3.3.2.8.2 Filter Validation

The robustness of the assay method was further established from the results obtained in

the validation using three different filters namely, Durapore hydrophilic membrane filter (PVDF),

Nylon 66 membrane filter and nylon 66 syringe SY25NH of 0.45 µm pore size. The test

preparation solutions was filtered through these different filters and the resultant solution was

subjected to chromatographic analysis. The results are presented in Table 3.9. The similarity

factor varied between 0.997 and 1.016 indicating that the robustness of the proposed method.

Table 3.9: Results of filter validation study.

Filter description Preparation Similarity factor

PVDF

(Make: Millipore, Lot No: BM64M5059)

1 1.001

2 1.016

Nylon 66

(Make: Pall Pharma Lab, Lot No: 2003DC052)

1 0.997

2 1.002

Nylon 66SY25NH

(Make: Advanced Microdevices, Lot No: SN1607)

1 1.005

2 1.016

3.3.3 Comparison of the results

A number of methods were reported for the determination of omeprazole in plasma and

limited methods were reported for the determination of omeprazole in pharmaceutical

formulations as it involves critical extraction process as part of sample preparation. Majority of

the reported methods are based on spectrophotometry and high performance liquid

chromatography (HPLC). In general, HPLC methods are having significant importance with

respect to precision and accuracy when compared to spectrophotometric methods for quality

control applications. Harshal K Trivedi et al [20] have reported a precise single HPLC method

for the determination of omeprazole and its related compounds in pharmaceutical formulations.

This method is found to be superior over other reported methods. The results of proposed method

are compared with those reported by Trivedi et al and tabulated the observations in Table 3.10.

Table 3.10: Comparison between HPLC (reported) method and UPLC (present) method.

Parameter HPLC Method [20] UPLC Method

(present method)

Observations

Column Zorbax XDB C8, 150mm

length, 4.6mm internal

diameter and particle size

5µm.

Extend C18, Agilent, 50mm

length, 4.6mm internal

diameter and particle size

1.8µm.

C18 gives good

resolution due to its

hydrophobic nature.

Mobile

phase

Glacine (3gm in 1000mL

of water) buffer as

mobile phase A (pH 8.8)

and mixture of

aceronitrile : methanol

(83:17) as a mobile phase

B.

Mixture of phosphate buffer

(pH 7.4) and aceronitrile in

the ratio of 60:40 as a

mobile phase.

In reported method high

pH buffer was used as

mobile phase when

compared with present

method, High pH mobile

phase damages silica

based columns [50].

Extraction Dimethyl formamide was 0.1N NaOH was used to Aqueous extraction

solvent used to extract the

omeprazole from its

formulation.

extract the omeprazole from

its formulation.

solution mixes easily

with diluent (aqueous).

Diluent Mixture of pH 8.0

phosphate buffer and

acetonitrile in the ratio of

90:10.

Mobile phase was used as

diluent.

In reported method, the

mobile phase and

diluents are of different

composition and hence

there complete

miscibility is doubtful. In

the present reported

method the mobile phase

and diluents are one and

the same and hence no

problem of miscibility.

Flow rate &

Pump mode

1.2 mL minute-1 with

gradient mode.

1.0 mL minute-1 with

isocratic mode.

Isocratic mode is simple,

reliable & gives constant

baseline response.

Data

Acquisition

time

25 minutes per injection 2 minutes per injection Less run time reduces

solvent consumption, and

saves analysis time

Linearity

range

Linearity of the method

covered from 10 µg mL-1

to 40 µg mL-1 of

omeprazole

Linearity of the method

covered from

10 µg mL-1 to 60 µg mL-1 of

omeprazole

Applications will

increase with increased

range.

3.4. Conclusion

This paper reports for the first time a novel method on omeprazole to quantitate assay in

finished dosage form by RP-UPLC. The analytical method was validated according to the ICH

guidelines which reveal that the method is selective, precise and accurate. The proposed UPLC

method has the ability to separate omeprazole from its degradation products, impurities;

excipients found in the tablet dosage form and therefore can be applied to the analysis of samples

at quality control. The method is rapid, direct, specific, accurate, precise, and can be used for the

routine analysis of omeprazole drug in the finished dosage form. The method may also be

extended to evaluate the active drug substance.

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