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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
contains a tricoordinated sulfinyl sulfur in a pyramidal structure and therefore can exist in equal
and (R)-enantiomers. In the acidic conditions of the canaliculi of
parietal cells, both are converted to achiral products (sulfenic
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
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. The stability of omeprazole is a
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 inhibitor used in the treatment of dyspepsia, peptic ulcer disease
(PUD), gastro esophageal reflux disease (GORD/GERD), laryngo pharyngeal reflux (LPR) and
Ellison syndrome and is one of the most widely prescribed drugs internationally.
ppresses 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 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
contains a tricoordinated sulfinyl sulfur in a pyramidal structure and therefore can exist in equal
acidic conditions of the canaliculi of
parietal cells, both are converted to achiral products (sulfenic
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
about 155°C. It is a weak base, freely soluble in ethanol and methanol, and slightly soluble in
e stability of omeprazole is a
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.
bitor used in the treatment of dyspepsia, peptic ulcer disease
(PUD), gastro esophageal reflux disease (GORD/GERD), laryngo pharyngeal reflux (LPR) and
Ellison syndrome and is one of the most widely prescribed drugs internationally.
ppresses gastric acid secretion. By acting specifically on the proton pump,
omeprazole blocks the final step in acid production, thus reducing gastric acidity [1].
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:
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: 5 µL
Data acquisition time: 10 minutes
Detection mode: Ultra violet detection at 302 nm.
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:
Column: Extend C18, Agilent (50mm length, 4.6mm internal diameter and 1.8µm particle
size). A bidentate organosilane combined with double end capping.
Buffe: 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 60: 40 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: 5µL
Data acquisition time: 10 minutes
Detection mode: Ultra violet detection at 302 nm.
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:
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 60: 40 v/v, and
adjusted pH to 7.4 with 1M potassium hydroxide solution, filtered through 0.22 µm
membrane filter.
Flow rate: 1.0 mL minute-1
Injection volume: 5µL
Data acquisition time: 5 minutes
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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