chapter-7 determination of assay and related substances...

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Overview:

Method development and validation of rapid stability indicating method

for determination of omeprazole (OME) and its related substances in

solid oral dosage form is presented in this chapter.

Chapter-7

Determination of Assay and

Related Substances of Omeprazole in

Pharmaceutical Dosage Form

209

Determination of Assay and Related Substances of

Omeprazole in Pharmaceutical Dosage Form

7.1 LITERATURE REVIEW

Impurity profiling of active pharmaceutical ingredients (API) in both bulk material and finalized

formulations is one of the most challenging tasks of pharmaceutical analytical chemists under

industrial environment [1]. The presence of unwanted or in certain cases unknown chemicals,

even in small amounts, may influence not only the therapeutic efficacy but also the safety of the

pharmaceutical products [2]. For these reasons, all major international pharmacopoeias have

established maximum allowed limits for related compounds for both bulk and formulated APIs.

As per the requirements of various regulatory authorities, the impurity profile study of drug

substances and drug products has to be carried out using a suitable analytical method in the final

product [3, 4].

Literature search revealed that, papers on degradation of omeprazole [5], determination by UV

spectrophotometry method [6], omeprazole (OME) in human plasma and urine by LC-MS-MS

[7], colorimetric method [8], determination of S-omeprazole, R-omeprazole and racemic

omeprazole [9] are available, but as such there is no validated method available, which reports

more known and unknown impurities precisely and significantly for omeprazole (OME), as such

and in drug product. It’s validated analytical performance in terms of major parameters such as

selectivity, accuracy, precision and sensitivity is adequate for the routine quality control of the

purity of omeprazole containing pharmaceutical formulations. The important part of method is

with help of single injection, quantification of omeprazole and its degradable impurities and

process impurities. Common method for assay and related substances, as well as analytical

methods, are validated to ensure they are suitable for their intended use and give accurate and

reliable data.

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7.2 THE SCOPE AND OBJECTIVES OF PRESENT STUDY

There is no stability-indicating HPLC method reported in the literature that can adequately

separate all impurities and accurately quantify omeprazole API and dosage forms. It is, therefore,

felt necessary to develop a new rapid, stability-indicating method for the related substance

determination and quantitative determination of omeprazole. An attempt is made to determine

whether HPLC can reduce analysis times without compromising the resolution and sensitivity.

The objectives of the present work are as follow:

To developed a rapid, stability indicating RP-HPLC method for determination of

omeprazole and its related substances in solid oral dosage form.

Forced degradation study.

To separates omeprazole from its all impurities (Benzamithazone, N-Oxide, Sulphone,

Des-Methoxy and Sulphide) known impurities/ degradation products and any unknown

degradation product generated during forced degradation study.

Perform analytical method validation for the proposed method as per ICH guideline.

7.3 OMEPRAZOLE

Omeprazole is highly effective inhibitor of gastric acid secretion used in the therapy of stomach

ulcers and zollinger-ellison syndrome. The drug inhibits the H(+)-K(+)-ATPase (H(+)-K(+)-

exchanging ATPase) in the proton pump of gastric parietal cells [10, 11]. The chemical IUPAC

name of omeprazole is 6-methoxy-2-[(4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl]-1H-

benzimidazole. Its empirical formula is C17H19N3O3S, and its structural formula is shown in

Figure 7.1 [12]; omeprazole Mg is a white to off-white free-flowing crystalline powder with a

molecular weight of 713.1. The route of synthesis of omeprazole Mg resulted five known

impurities, Benzamidazole, N-Oxide, Sulphone, Des-Methoxy and Sulphide. The presented

method is capable for quantification of all known and unknown impurities of omeprazole Mg

accurately.

Determination of assay and related substances of omeprazole in pharmaceutical dosage form

211

Figure 7.1 3D and 2D Structure of omeprazole

Indications

For the treatment of acid-reflux disorders (GERD), peptic ulcer disease, H. pylori eradication,

and prevention of gastroinetestinal bleeds with NSAID use.

Pharmacodynamics

Omeprazole is a compound that inhibits gastric acid secretion and is indicated in the treatment of

gastroesophageal reflux disease (GERD), the healing of erosive esophagitis, and H.

pylori eradication to reduce the risk of duodenal ulcer recurrence. Omeprazole belongs to a new

class of antisecretory compounds, the substituted benzimidazoles, that do not exhibit

anticholinergic or H2 histamine antagonistic properties, but that suppress gastric acid secretion

by specific inhibition of the H+/K

+ ATPase at the secretory surface of the gastric parietal cell. As

a result, it inhibits acid secretion into the gastric lumen. This effect is dose-related and leads to

inhibition of both basal and stimulated acid secretion irrespective of the stimulus.

Mechanism of action

Omeprazole is a proton pump inhibitor that suppresses gastric acid secretion by specific

inhibition of the H+/K

+-ATPase in the gastric parietal cell. By acting specifically on the proton

pump, omeprazole blocks the final step in acid production, thus reducing gastric acidity.

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212

Absorption

Absorption is rapid, absolute bioavailability (compared to intravenous administration) is about

30-40% at doses of 20-40 mg.

Protein binding

95%

Metabolism

Hepatic

Route of elimination

Urinary excretion is a primary route of excretion of omeprazole metabolites.

Half life

0.5-1 hour

Toxicity

Symptoms of overdose include confusion, drowsiness, blurred vision, tachycardia, nausea,

diaphoresis, flushing, headache, and dry mouth.

7.4 EXPERIMENTAL

7.4.1 Materials and reagents

Omeprazole tablets, placebo, omeprazole working standard (90.2% w/w), benzamidazole

working standard (99.7% w/w), N-oxide working standard (98.4% w/w), sulphone working

standard (98.7% w/w), des-methoxy working standard (95.1% w/w) and sulphide working

standard (99.8% w/w) are provided by Cadila Pharmaceutical Ltd. Dholka, Ahmedabad, India.

HPLC grade methanol and acetonitrile are obtained from J.T. Baker (NJ., USA). GR grade

glycine and sodium hydroxide are obtained from Merck Ltd (Mumbai, India). Nylon membrane


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filters (0.45 µ), nylon syringe filters and PVDF syringe filters are purchased from Pall Life

Science Limited (India). High purity water is generated with Milli-Q Plus water purification

system (Millipore, Milford, MA, USA).

7.4.2 Equipments

Cintex digital water bath (Mumbai, India) is used for specificity study. Photo stability studies are

carried out in a photo-stability chamber (SUNTEST XLS+, ATLAS, Germany). Thermal

stability studies are performed in a dry air oven (Cintex, Mumbai, India).

7.4.3 Preparation of mobile phase

Mobile phase-A:

0.04 M glycine (adjusted pH 8.8 with diluted sodium hydroxide solution) buffer solution is used

as a MP-A.

Mobile Phase-B:

Mixture of acetonitrile and methanol in ratio of (83:17) is used as MP-B.

Table 7.1 Gradient Program

Time in min Mobile-A Mobile-B

0 88 12

20 40 60

21 88 12

25 88 12

7.4.4 Diluent preparation

Dissolve 4.47 gm of Na2HPO4 and 0.73 gm of NaH2PO4 in water. Make the volume up to 1000

mL with water. Dilute 250mL of this solution to 1000 mL with water (pH 8.0, adjusted with

dilute NaOH solution). Mix buffer (pH 8.0) and acetonitrile at a ratio of 90:10.

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214

7.4.5 Standard solution (RS)

An accurately weight and transfer about 5 mg of each impurity and 5 mg of OME standard into

100mL volumetric flask dissolve and dilute with diluent and mix. Dilute 5.0mL of this solution

to 50mL with diluent.

7.4.6 Resolution solution

An accurately weight and transfer about 1 mg of each impurity and 50 mg OME standard into

100mL volumetric flask, dissolve and dilute up to the mark with diluent, mix well.

7.4.7 Standard solution (Assay)

An accurately weight and transfer about 25 mg of OME working standard in to 50mL volumetric

flask add 10mL of dimethylformamide to dissolve it, dilute up to the mark with diluent (500

µg/mL), mix well.

7.4.8 Sample solution preparation

Transfer 5 tablets in to 200mL volumetric flask add about 10mL of dimethylformamide and

sonicate for five minutes and add about 150mL of diluent and sonicate for 20 minutes, dilute to

volume with diluent. Filter this solution with 0.45µ nylon filter.

7.4.9 Placebo solution preparation

Transfer 5 placebo tablets in to 200 mL volumetric flask add about 10mL of dimethylformamide

and sonicate for five minutes and add about 150mL of diluent and sonicate for 20 minutes, dilute

to volume with diluent. Filter this solution with 0.45µ nylon filter.

7.4.10 Chromatographic conditions

The liquid chromatography consisted of an Agilent and waters system, equipped with automatic

sample injector and PDA detector. For data collection and calculation chemstation Software and

Waters EmpowerTM

-2 software is used. The chromatographic condition is optimized using a

Zorbax XDB C8 (150 x 4.6mm, 5µ) column. 0.04M glycine buffer (pH adjusted 8.8) as a MP-A


215

and mixture of acetonitrile: methanol (83:17) as a MP-B. The mobile phase is filtered through a

0.45µ Nylon membrane filter and degassed under vacuum prior to use. The flow rate is 1.2

mL/min with gradient program [Table 7.1]. The monitoring wavelength is 302 nm and the

injection volume is 10 µL with maintaining column oven temperature at 25ºC and sample cooler

temperature is 10˚C.

7.5 METHOD VALIDATION

The method described herein has been validated for simultaneous determination of assay and its

related substances by RP-HPLC.

7.5.1 Specificity

Forced degradation studies are performed to demonstrate selectivity and stability-indicating

capability of the proposed method. The sample are exposed to acid hydrolysis [0.1 N HCl (5

mL), 25°C, 15 min], alkaline hydrolysis [0.1N NaOH (5 mL), 60°C, 3h], oxidative [1 % H2O2 (5

mL), 60°C, 3h], water hydrolysis [at 60°C for 4h], thermal [104°C, 4h], and photolytic

degradation [1.2 million Lux hours]. All exposed samples are than analysed by the proposed

method.

7.5.2 System suitability

Inject diluent, one injection of resolution solution, six replicate of standard solution (RS), five

replicate injection of standard solution (Assay) and check the system suitability as follows.

1) In resolution solution, the resolution between OME and des-methoxy should not less than 2.0.

2) The % RSD of area due to all impurities peaks and OME peaks in six replicate injections of

standard solution (RS) should not more than 5.0.

3) The % RSD of the area due to OME in five replicate injection of standard preparation (Assay)

should not more than 2.0.

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216

7.5.3 Precision

The precision of the system is determined using the sample preparation procedure described

above for six real samples of formulation and analysis using the same proposed method.

Intermediate precision is studied by other scientist, using different columns, different HPLC, and

is performed on different day.

7.5.4 Accuracy

To confirm the accuracy of the proposed method, recovery experiments are carried out by the

standard addition technique for assay and related substances. The accuracy of the assay method

for OME is evaluated in triplicate (n=3) at the three concentrations of 250, 500 and 750 µg/mL

(50, 100 and 150 %) of drug product, and the recovery is calculated for each added (externally

spiked) concentration. The mean of percentage recoveries (n=9) and the relative standard

deviation are calculated. For all impurities, the recovery is determined in triplicate (n=3) for

LOQ, 0.5, 1.0 and 1.5 µg/mL (LOQ, 50, 100, and 150 %) and the recovery of the impurities are

calculated. The mean of percentage recoveries (n=12) and the relative standard deviation are also

calculated for related substances.

7.5.5 Linearity

For related substances test, linearity is demonstrated from 0.1 to 1.5µg/mL of standard

concentration of impurities using a minimum of ten calibration levels (0.10, 0.15, 0.20, 0.25,

0.30, 0.50, 0.75, 1.0, 1.25 and 1.50µg/mL) for benzamidazole, N-oxide, suphone, des-methoxy,

and sulphide impurities. For assay test, linearity is demonstrated from 50% to 150% of standard

concentration using a minimum of five calibration levels (250, 375, 500, 625 and 750µg/mL) for

OME. The method of linear regression is used for data evaluation. The peak areas of the standard

compounds are plotted against the respective OME, benzamidazole, N-oxide, sulphone, des-

methoxy and sulphide concentrations. Linearity is described by the linearity equation, correlation

coefficient and Y-intercept bias is also determined.


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7.5.6 Limit of detection (LOD) and limit of quantification (LOQ)

Limit of detection and quantification concentrations of omeprazole determined based on standard

deviation of response and slope method. The linearity study is performed in the range of 10% to

150.0% of the limit concentration of OME, benzamedazole, N-Oxide, sulphone, des-methoxy

and sulphide, considering 0.2% limit for all known impurities and 0.10% for any unknown

impurities. Duplicate injection of linearity solutions are injected performed. Linearity graph of

concentration in µg/mL (X-axis) versus peak area response (Y-axis) is plotted. LOD and LOQ

concentrations of omeprazole and its impurities are determined on the basis of equation given

below.

Limit of Detection = (3.3 X σ) / S

Limit of Quantification = (10 X σ) / S

Where, σ = Residual standard deviation of regression line

S = Slope of calibration curve

Injected six replicate injections of these LOD and LOQ concentrations and ensured the peak is

detected and responses are measured.

7.5.7 Robustness

The robustness is a measure of the capacity of a method to remain unaffected by small but

deliberate changes, change in wavelength (± 2.0nm), change in column oven temperature (±

5°C), changes in flow rate (± 0.1mL/min), and change in buffer pH (± 0.2). All important

characteristic including resolution between des-methoxy and OME, tailing factor, theoretical

plate number and the retention behaviour of interested compound are evaluated.

7.5.8 Solution stability

The stability of the sample solution (for related substances) is established by storage of the

sample solution at 10°C temperature for 25h. The sample solution is re-analyzed after 7h, 16h,

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21h, 25h and the results of the analysis are compared with the results of the fresh sample. The

stability of standard solution and sample solution for assay are established by the storage of them

at ambient temperature for 54h. The standard and sample solution are re-injected after 7h, 16h,

21h, 25h, 32h, 44h and 54h, % difference is calculated for it.

7.5.9 Filter compatibility

Filter compatibility is performed for 0.45 μ nylon syringe filter and 0.45 μ PVDF syringe filter.

To confirm the filter compatibility in proposed analytical method, filtration recovery experiment

is carried out by sample filtration technique. Sample is filtered through both syringe filters and

percentage assay and impurities are calculated and compared against centrifuged sample.

7.6 RESULTS AND DISCUSSION

7.6.1 Method Development and Optimization

The main objective of the RP-HPLC method development is to rapid assay and related

substances determination of omeprazole in pharmaceutical formulation. Developed method

should be able to determine assay (AS) and related substances (RS) in single run and should be

accurate, reproducible, robust, stability indicating, filter compatible, linear, free of interference

from blank/ placebo/ impurities / degradation products and straightforward enough for routine

use in quality control laboratory.

The spiked solution of omeprazole (500 µg/mL), benzamidazole (10 µg/mL), N-oxide (10

µg/mL), sulphone (10 µg/mL), des-methoxy (10 µg/mL) and sulphide (10 µg/mL) is subjected to

separation by RP-HPLC. Initially the separation of all compounds is studied using water as a

mobile phase-A (MP-A) and acetonitrile (ACN) as a mobile phase-B (MP-B) on a HPLC column

Hypersil BDS (C8, 150 x 4.6mm, 5µ) using a Waters (HPLC) system with the linear gradient

program. The flow rate of 1.0 mL/min is selected with regards to the backpressure and analysis


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time as well. Various types of MP-A and MP-B are studied to optimize the method; some of

them are summarized in Table 7.2 with the associated observations.

Table 7.2 Summary of method optimization

Experimental condition Observation

Water (MP-A) and ACN (MP-B), linear gradient;

Hypersil BDS-C8 (150 × 4.6 mm, 5µ); 25°C

No resolution found between

omeprazole and impurities

0.01 M KH2PO4, pH-7.0 (MP-A) and ACN (MP-B),

linear gradient; Hypersil BDS-C8 (150 × 4.6 mm, 5µ);

25°C

Peak shape is not good

0.04 M Glycine pH-9.0 (MP-A) and ACN (MP-B), linear

gradient; Zorbax Eclipse XDB C8 (4.6 x 150 mm) 5 µm;

25°C

Peak separation observed but

peak shape is not good

0.04 M Glycine pH-8.8(MP-A) and ACN : methanol,

(83:17)(MP-B), linear gradient; Zorbax Eclipse XDB-C8

(150 x 4.6 mm, 5µ); 25°C

Satisfactory peak separation

and sharp peak shape

ACN … Acetonitrile

Based on above experimental study, the optimized HPLC parameters are; flow rate 1.2 mL/min;

column oven temperature 25°C; 0.04 M glycine buffer pH-8.8 as MP-A and, a mixture of ACN,

and MeOH in the ratio of 83:17 v/v as MP-B. In order to achieve symmetrical peak shape of all

substances and more resolution between OME and des-Methoxy, different stationary phases are

explored. Finally the desired separation with symmetrical peaks is obtained using Zorbax Eclipse

XDB C8 (150 x 4.6 mm, 5 µ) column. Column oven temperature is also studied and found that

25°C is more appropriate with respect to separation and peak shape. Based on compounds UV

response, 302nm is found to be more appropriate for determination of OME and its impurities

from single run. OME, benzamidazole, N-oxide, sulphone, des-methoxy and sulphide are well

resolved from each other and there is no chromatographic interference observed due to blank and

placebo in a reasonable time of 25.0 minutes, which are presented in Figure 7.2.

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220

Figure 7.2 Expanded overlaid specimen chromatograms of blank, placebo and sample

7.6.2 Analytical Parameters and Validation

After satisfactory development of the method it is subjected to method validation as per ICH

guideline [13]. The method is validated to demonstrate that it is suitable for its intended purpose

by the standard procedure to evaluate adequate validation characteristics (specificity, system

suitability, precision, accuracy, linearity, LOD, LOQ, robustness, solution stability and filter

compatibility).

7.6.2.1 Specificity

Specificity is the ability of the method to measure the analyte response in the presence of its

potential impurities. Forced degradation studies are performed to demonstrate selectivity and

stability indicating capability of the proposed RP-HPLC method. Figure 7.2 is show that there is

no any interferences at the RT (retention time) of OME due to blank, placebo and impurities.

Stress studies are performed at concentration of 500 µg/mL of OME to provide the stability

indicating property and specificity of the proposed method.

The sample is exposed to acid hydrolysis, alkaline hydrolysis, oxidative, water hydrolysis,

thermal, and photolytic degradation to evaluate the ability of the proposed method to separate

OME from its degradation products. Minor degradation products are observed when OME is


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subjected to base, heat, photolytic and hydrolytic conditions. Significant degradation is observed

when the drug product is subjected to acid hydrolysis, and oxidation. The purity of the peaks

obtained from the stressed sample is verified using the PDA detector. The results of forced

degradation study are summarized in Table 7.3. Specimen chromatogram of specificity study

presented in Figure 7.3 to 7.11.

Table 7.3 Summary of forced degradation results

Degradation

condition

Mass

balance#

Purity Observation

Control sample 99.8 Pass NA

Acidic hydrolysis 100.1 Pass Significant degradation observed

Alkaline hydrolysis 98.8 Pass No significant degradation observed

Oxidation 97.5 Pass Significant degradation observed

Water hydrolysis 98.4 Pass No significant degradation observed

Thermal (solid) 98.3 Pass No significant degradation observed

Photolytic 99.1 Pass No significant degradation observed

NA= Not applicable; # = % assay + % known impurities + area % unknown impurities

Figure 7.3 Specimen chromatogram of diluent

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222

Figure 7.4 Specimen chromatogram of placebo

Figure 7.5 Specimen chromatogram of OME

Figure 7.6 Specimen chromatogram of benzamedazole


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Figure 7.7 Specimen chromatogram of N-oxide

Figure 7.8 Specimen chromatogram of sulphone

Figure 7.9 Specimen chromatogram of des-methoxy

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224

Figure 7.10 Specimen chromatogram of sulphide

Figure 7.11 Specimen chromatogram of impurities spiked in OME sample

7.6.2.2 System suitability

The % RSD of area counts of five replicate injections is below 1.0 % in OME standard solution

(Assay). The % RSD of all impurities area counts for six replicate injections are below 1.0% in

standard solution (RS). The resolution between des-methoxy and OME is more than 3.0 in

resolution solution. The all parameters complied with the acceptance criteria and system

suitability is established. The system suitability result for system precision, method precision and

intermediate precision are summarized in Table 7.4.


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Table 7.4 System suitability results (system precision, method precision and intermediate

precision)

Test Parameters % RSD Resolution between OME

and Des-Methoxy

System precision Omeprazole (assay) 0.1 3.1

Benzamedazole 0.4

N-Oxide 0.5

Sulphone 0.3

Des-Methoxy 0.2

Sulphide 0.2

Omeprazole (RS) 0.9

Precision Omeprazole (assay) 0.1 3.0

Benzamedazole 0.3

N-Oxide 0.4

Sulphone 0.2

Des-Methoxy 0.1

Sulphide 0.4

Omeprazole (RS) 0.7

Intermediate

precision

Omeprazole (assay) 0.2 3.1

Benzamedazole 0.1

N-Oxide 0.4

Sulphone 0.3

Des-Methoxy 0.5

Sulphide 0.2

Omeprazole (RS) 0.2

RS … Related substance

7.6.2.3 Precision

In this study, % RSD of sample is observed, by injecting six sets of sample solution. %RSD for

assay and related substances results of six samples are calculated. The result should not deviate

more than 1.0% for assay value and for related substances it’s not deviate more than 0.01% for

all impurities. It is well within the acceptances criteria for all the sets and method found

repeatable and precise for intended purpose. Method precision and intermediate precision results

are summarized in Table 7.5.

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226

Table 7.5 Precision (n=6) and intermediate precision (n=6) results

Substance Precision Intermediate precision

% Difference Mean % % RSD Mean % % RSD

OME (1000µg/mL) 99.1 1.0 98.8 0.8 0.3

Benzamedazole 0.006 7.7 0.008 6.8 0.002

N-Oxide 0.086 0.5 0.086 1.7 0.000

Sulphone 0.227 1.9 0.226 2.6 0.001

Des-Methoxy 0.031 2.7 0.032 3.1 0.001

Sulphide 0.041 1.8 0.040 4.3 0.001

Unknown impurity 0.050 1.0 0.051 2.1 0.001

Total impurities 0.441 3.2 0.443 2.5 0.002

7.6.2.4 Accuracy

Accuracy of a method is defined as the closeness of the measured value to the true value for the

sample. Accuracy has been performed for RS in the range of LOQ to 150.0% (LOQ, 50.0%,

100.0%, and 150.0%) of target concentration of OME considering limit 0.1% (limit of individual

unknown impurity) and 0.2% of (limit of all known impurities). Accuracy has been performed

for assay in the range of 50 to 150 % of target concentration (500 µg/mL).

The % recovery for the range 50.0% to 150.0% of target concentration has found within the

acceptance criteria with acceptable % RSD of NMT 2.0 at each level. The recovery at each level

is within 98.0% to 102.0%. The recovery at each level of each impurity is within 80% to 120%

of target concentration. This indicates that the method is accurate for the analysis of OME assay

and related substances method. Accuracy study results are summarized in Table 7.6. Overlaid

specimen chromatograms of accuracy study (related substance) are presented in Figure 7.12.


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Figure 7.12 Overlaid specimen chromatograms of accuracy study

Table 7.6 Accuracy results

Substance

LOQ (n=3) At 50% (n=3) At 100% (n=3) At 150% (n=3)

%

Rec.$

%

RSD

%

Rec.$

%

RSD

%

Rec.$

%

RSD

%

Rec.$

%

RSD

OME (Assay) NA NA 99.6 0.8 99.2 0.5 101.2 0.7

#Benzamedazole 85.1 2.3 82.8 1.3 85.4 0.3 89.3 4.7

#N-Oxide 87.1 1.2 91.3 1.5 94.7 2.1 96.0 1.7

#Sulphone 98.4 0.8 103.5 2.1 98.9 1.7 93.3 1.0

#Des-Methoxy 81.3 1.1 85.9 2.0 87.4 1.0 85.1 0.3

#Sulphide 81.3 1.6 82.9 0.1 97.3 0.6 92.2 0.2

Unknown impurity 99.9 2.4 100.1 0.2 100.0 0.2 99.7 0.1

$... Recovery, #... For Related Substances, NA… Not applicable

7.6.2.5 Linearity

Concentration levels are 50, 75, 100, 125 and 150% of the claimed analyte concentration of

assay, corresponding to the range of about 250-750 µg/mL. For related substances each

impurities concentration levels are LOQ, 15%, 20%, 25%, 30%, 50%, 75%, 100%, 125% and

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228

150% of the claimed analyte concentration of impurities, corresponding to the range of about

LOQ to1.5 mcg/mL. The calibration curve obtained by plotting the peak area versus the

concentration is linear in the mentioned concentration range of 50% to 150% for assay and LOQ

to 150% for related substances. For assay correlation coefficient of linearity curve is less than

0.999 and Y-intercept bias is within ± 2.0% of 100% linearity response. For RS correlation

coefficient of linearity curve is less than 0.998 and Y-intercept bias is within ± 5.0% of 100%

linearity response.

Linearity regression statistic results are summarized in the Table 7.7. Linear curve of OME

(assay), OME (RS) and all known impurities are presented in Figure 7.13 to 7.19. Obtained

results are indicated that the method is linear up to the specified range of concentrations.

Overlaid specimen chromatograms of linearity (RS) study are presented in Figure 7.20.

Table 7.7 Regression statistics

Compound Linearity

(µg/mL)

Correlation

coefficient (r2)

Linearity (Equation) Y- intercept

bias at 100%

Omeprazole (Assay) 250 to 750 0.999 Y= 21034x + 5521.8 0.6

Benzamidazole 0.1 to 1.5 0.999 y= 47630x-372.9 0.8

N-Oxide 0.1 to 1.5 0.999 y= 13718x + 99.4 0.7

Sulphone 0.1 to 1.5 0.999 y= 14419x + 40.9 0.3

Des-Methoxy 0.1 to 1.5 0.999 y= 18966x + 1374.9 2.7

Sulphide 0.1 to 1.5 0.999 y= 17722x + 595.5 3.2

Omeprazole (RS) 0.1 to 1.5 0.999 y= 59502x-372.9 2.2


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Figure 7.13 Linearity curve of omeprazole (assay)

Figure 7.14 Linearity curve of benzamedazole

Figure 7.15 Linearity curve of N-Oxide

y = 21034x + 5521.8

R² = 0.9997

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

0 100 200 300 400 500 600 700 800

Are

a

Conc. In ppm

Linearity of Omeprazole (Assay)

y = 47630x - 372.9

R² = 0.9998

0.000

10000.000

20000.000

30000.000

40000.000

50000.000

60000.000

70000.000

80000.000

0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600

Are

a

Conc. in ppm

Linearity of Benzamedazone

y = 13718x + 99.4

R² = 0.9998

0.000

5000.000

10000.000

15000.000

20000.000

25000.000

0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800

Are

a

Conc. in ppm

Linearity of N-Oxide

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230

Figure 7.16 Linearity curve of sulphone

Figure 7.17 Linearity curve of Des-Methoxy

Figure 7.18 Linearity curve of sulphide

y = 14419x + 40.9

R² = 0.9999

0.000

5000.000

10000.000

15000.000

20000.000

25000.000

0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800

Are

a

Conc. In ppm

Linearity of Sulphone

y = 18966x + 1374.9

R² = 0.9999

0.000

5000.000

10000.000

15000.000

20000.000

25000.000

30000.000

35000.000

0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600

Are

a

Conc. In ppm

Linearity of Des-Methoxy

y = 17722x + 595.5

R² = 0.9998

0.000

5000.000

10000.000

15000.000

20000.000

25000.000

30000.000

0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800

Are

a

Conc. In ppm

Linearity of Sulphide


231

Figure 7.19 Linearity curve of omeprazole (RS)

Figure 7.20 Overlaid specimen chromatograms of linearity study (RS)

7.6.2.6 Limit of detection (LOD) and limit of quantification (LOQ)

The detection limit of individual analytical procedure is the lowest amount of analyte in a sample

which can be detected but not necessarily quantified as an exact value and quantification limit is

the lowest amount of analyte in a sample which can be quantitatively determined with suitable

precision and accuracy. Limit of detection and quantification concentrations of omeprazole

determined based on standard deviation of response and slope method.

y = 59502x - 372.92

R² = 0.9998

0.00

10000.00

20000.00

30000.00

40000.00

50000.00

60000.00

70000.00

80000.00

0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400

Are

a

Conc. In ppm

Omeprazole (Related Substance)

Chapter-7

232

Duplicate injection of linearity solutions are injected into chromatography system. Linearity

graph of concentration in µg/mL (X-axis) versus peak area response (Y-axis) is plotted. The

correlation coefficient, slope of regression line and RSD of regression line are calculated.

Injected six replicate injections of these LOD and LOQ concentrations and ensured the peak is

detected and responses are measured. Chromatogram of LOD and LOQ are presented in Figure

7.21 and Figure 7.22 respectively. Results for LOD, LOQ and LOQ precision are summarized in

Table 7.8.

Figure 7.21 Specimen chromatogram of LOD

Figure 7.22 Specimen chromatogram of LOQ


233

Table 7.8 Results of LOD, LOQ and LOQ precision (n=6)

7.6.2.7 Robustness

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by

small but deliberate variations in method parameters and provides an indication of its reliability

during normal usage. No significant effect is observed on system suitability parameters such as

RSD, and resolution, when small but deliberate changes are made to chromatographic conditions.

The results are summarized in Table 7.9, along with the system suitability parameters of normal

conditions. Thus, the method is found to be robust with respect to variability in applied

conditions.

Table 7.9 Robustness study results

Condition Name of component Resolution between Des-

Methoxy and OME

% RSD of five

injections

Normal

methodology

Omeprazole (Assay) 3.0 0.13

Benzamidazone 0.34

N-Oxide 0.42

Sulphone 0.23

Des-Methoxy 0.11

Sulphide 0.40

Omeprazole (RS) 0.72

Name of

compound LOD (µg/mL)

LOQ

(µg/mL)

LOQ precision

(% RSD)

OME 0.060 0.180 2.2

Benzamedazole 0.020 0.060 2.4

N-Oxide 0.016 0.048 4.2

Sulphone 0.010 0.030 2.5

Des-Methoxy 0.015 0.045 1.7

Sulphide 0.017 0.051 2.5

Chapter-7

234

At 300 nm

Wavelength


Benzamidazone 0.40

N-Oxide 0.33

Sulphone 0.32

Des-Methoxy 0.57

Sulphide 0.29


At 304 nm

Wavelength


Benzamidazone 0.32

N-Oxide 0.19

Sulphone 0.11

Des-Methoxy 0.95

Sulphide 0.05


Column temperature

35˚C


Benzamidazone 0.50

N-Oxide 0.31

Sulphone 0.03

Des-Methoxy 1.50

Sulphide 0.04


Column temperature

25˚C


Benzamidazone 0.38

N-Oxide 0.11

Sulphone 0.08

Des-Methoxy 0.23

Sulphide 0.04


At 1.3 mL/min flow Omeprazole (Assay) 2.9 0.11

Benzamidazone 0.28

N-Oxide 0.16

Sulphone 0.37

Des-Methoxy 0.11


235

Sulphide 0.10


At 1.1 mL/min flow Omeprazole (Assay) 3.1 0.19

Benzamidazone 1.58

N-Oxide 0.14

Sulphone 0.37

Des-Methoxy 0.37

Sulphide 0.21


Buffer pH 8.6 Omeprazole (Assay) 2.9 0.16

Benzamidazone 0.47

N-Oxide 0.21

Sulphone 0.16

Des-Methoxy 0.59

Sulphide 0.16


Buffer pH 9.0 Omeprazole (Assay) 3.1 0.36

Benzamidazone 0.90

N-Oxide 0.36

Sulphone 0.41

Des-Methoxy 0.58

Sulphide 0.31


7.6.2.8 Solution stability

The solution stability is checked for sample preparation from initial to 25h for related substances

and initial to 54h for assay. For related substances % difference is not more than 0.1. For assay %

area difference is not more than 2.0. The results obtained are well within the acceptance criteria

up to 25h at 10˚C temperature. Solution stability result of sample, standard for assay and related

substance summarized in Table 7.10 and 7.11.

Chapter-7

236

Table 7.10 Solution stability of sample preparation for related substances

Time in

Hours Benzamidazole Sulphone N-Oxide

Uk-

(RRT-

0.36)

Uk-

(RRT-

0.85)

Total

impurity

%

Difference

Initial 0.02 0.25 0.08 0.03 0.05 0.43 NA

7 h 0.01 0.27 0.08 0.03 0.06 0.45 -0.02

16 h 0.01 0.29 0.08 0.03 0.07 0.48 -0.05

21 h 0.01 0.30 0.08 0.03 0.08 0.50 -0.07

25 h 0.02 0.30 0.08 0.03 0.08 0.52 -0.09

NA … Not Applicable; Uk … un known; RRT … related retention time

Table 7.11 Solution stability of standard and sample preparation for assay

Time in

Hours

Area of

standard

solution

% Difference

Area of

sample

solution

% Difference

Initial 10071745 NA 10696650 NA

7 h 10049421 0.22 10715280 0.17

16 h 10045327 0.26 10677310 0.18

21 h 10070728 0.01 10691846 0.04

25 h 10073984 0.02 10702234 0.05

32 h 10059240 0.12 10698153 0.01

44 h 10074238 0.02 10729678 0.31

54 h 10043342 0.28 10677789 0.18

NA… Not Applicable

7.6.2.9 Filter compatibility

The filter compatibility is studied for two different filters 0.45 µm PVDF filter and 0.45 µm

nylon filters. A single set of sample solution is prepared and some of the portion of this solution

is centrifuged, filtered 10mL of sample solution through 0.45μm nylon filter and 0.45µm PVDF

filter. Centrifuged sample solution and filtered solutions are injected in the chromatography

system. For assay, the % difference in assay value is less than 1.0, from centrifuge solution


237

value. For RS, the % difference in the value is less than 0.05, from centrifuge solution value.

Filter compatibility study results for assay and RS are summarized in Table 7.12 and 7.13,

respectively. Based on obtained results PVDF and Nylon both the filters are compatible for the

developed method.

Table 7.12 Filter compatibility study result for Assay

Component Name Centrifuge sample Sample filter with

Nylon filter

Sample filter with

PVDF filter

Omeprazole 100.0% 99.8% 99.5%

% difference Not applicable 0.2% 0.5%

Table 7.13 Filter compatibility study result for related substance

Name of

Impurity

Centrifuged

sample

Sample filter

with Nylon

%Difference

against

centrifuge

Sample

filter with

PVDF

%Difference

against

centrifuge

Un Single Max 0.04% 0.03% 0.01% 0.01% 0.01%

Benzamidazole 0.16% 0.15% 0.01% 0.16% 0.00%

N-Oxide 0.14% 0.14% 0.00% 0.15% 0.01%

Sulphone 0.15% 0.15% 0.00% 0.14% 0.01%

Des-methoxy 0.15% 0.14% 0.01% 0.15% 0.00%

Sulphide 0.16% 0.15% 0.01% 0.15% 0.01%

Total impurities 0.80% 0.76% 0.04% 0.76% 0.04%

Un … unknown; Max … Maximum

7.7 CALCULATION FORMULA

7.7.1 Assay (% w/w)

Calculated the quantity, in mg, of OME in the portion of solid oral pharmaceutical formulation

using the following formula:

Chapter-7

238

Where,

Cstd = Concentration of standard solution in mg/mL

Cs = Concentration of sample solution in mg/mL

Rs = Compound peak response obtained from the sample preparation

Rstd = Compound peak response (mean peak area) obtained from the standard preparation

7.7.2 Impurity (% w/w)

Calculate % impurity present in the finished product formulation using the following formula:

Where,

Cstd = Concentration of standard solution in mg/mL

Cs = Concentration of sample solution in mg/mL

Rs = Compound peak response obtained from the sample preparation

Rstd = Compound peak response (mean peak area) obtained from the standard preparation

7.7.3 Relative standard deviation (% RSD)

It is expressed by the following formula and calculated using Microsoft excel program in a

computer.

Where,

SD= Standard deviation of measurements

= Mean value of measurements


239

7.7.4 Accuracy (% Recovery)

It is calculated using the following equation:

7.8 CONCLUSION

A precise and accurate method is successfully developed and validated for simultaneous

determination of omeprazole and its related substances in omeprazole dosage form, to meet

global regulatory requirements. The methodology is evaluated for specificity, linearity, precision,

accuracy and range in order to establish the suitability of the analytical method. Stability of

analytical solution, filter compatibility, LOD and LOQ is also studied. The total run time is 25.0

min, within which the drug and their degradation products are eventually separated. This method

can be successfully applied for the routine analysis as well as stability study.

Chapter-7

240

7.9 REFERENCES

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quantification of nebivolol and their related substance by HPLC-UV-PDA detection in

its pharmaceutical drug product” Int J of Pharm Tech Resea, 2009; 1(4): 1139-1147.

[2] Van de WA, Xhonneux R, Reneman R and Janssen P, “Cardiovascular effects of dl-

nebivolol and its enantiomers-a comparison with those of atenolol” Eur J Pharma,

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[3] ICH Guideline Q3A (R2), Impurities in New Drug Substances, October 25, 2006.

[4] ICH Guideline Q7A, Good Manufacturing Practice Guide for Active pharmaceutical

Ingredients, November 2005.

[5] Della GM, Iesce MR, Previtera L, Rubino M, Temussi F and Brigante M, “Degradation

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1087-1093.

[6] Abdel-Aziz MW, Omayma AR, Azza AG, Hoda M and Marwa SM,

“Spectrophotometric determination of omeprazole, lansoprazole and pantoprazole in

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[7] Espinosa BM, Ruiz SAJ, Sánchez RF and Bosch OC, “Analytical methodologies for the

determination of omeprazole: An overview” J of Pharma and Biom Anal, 2007; 44(4):

831-844.

[8] el-Kousy NM and Bebawy LI, “Stability-indicating methods for determining

omeprazole and octylonium bromide in the presence of their degradation products”

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[9] Hassan-Alin M, Andersson T, Niazi M, Röhss K, “A pharmacokinetic study comparing

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[10] Tommy A, Johan H, Kerstin R, Anders W, “Pharmacokinetics and effect on caffeine

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