Method Development and validation for related...

68 of 305

CHAPTER 4

Method Development and validation for related substances

of Omeprazole in API and formulations by UPLC.

Introduction

Omeprazole

Formula : C17H19N3O3S

CAS Number : 73590-58-6

Molecular Weight : 345.42

Synonyms : 1H-Benzimidazole,5-methoxy-2-[[(4-methoxy- 3,5-dimethyl-

2- pyridinyl)methyl]sulfinyl]; 5-Methoxy-2-(((4-

methoxy- 3,5- dimethyl- 2-

pyridyl)methyl)sulfinyl)benzimidazole; 2- (((3,5-

Dimethyl-4- methoxy-2-pyridyl)methyl)sulfinyl)-5-

methoxy- 1H-benzimidazole

Melting point : 156 °C

Omeprazole having chemical nomenclature of (RS)-5-methoxy-2-((4-methoxy-3,5-

dimethylpyridin-2-yl) methylsulfinyl)-1H-benzo[d]imidazole belongs to a of class

molecule called as proton pump inhibitors. Omeprazole was first approved in Sweden

in 1988 for treatment of duodenal ulcer. It was approved in Canada and in the United

States in 1989, for treatment of duodenal ulcer, gastric ulcers, reflux esophagitis, and

Zollinger-Ellison syndrome1 .The mechanism of action is by means of suppression of

hydrochloric acid secretion by specific inhibition of the H+/K+-ATPase in the gastric

parietal cell thereby reduces the acidity.

of 305

Omeprazole is one of the most widely prescribed drugs internationally even if many

advanced drugs in the same category are available thus large number of batches are

manufactured perennially in a lot of pharmaceutical companies.

So far very few methods have been reported for its determination of Assay by

Chemical methods2,3

. Some methods are also available in the literature for

determinations of Omeprazole by HPLC4-11

. The approximate runtime of the

pharmacopeial methods12-14

ranges from 40-70 min. The time taken for the analysis

significantly delays the batch to be released into the market. Hence there is a need for

the development of fast and reliable method for its routine analysis

So far, to the best of our knowledge, no validated, stability indicating related

substances method by UPLC has been reported for Omeprazole. Hence this method

was developed to quantitatively estimate Omeprazole and five of its impurities

namely Impurity A, B, C, D and E by UPLC .Moreover, this method can be

considered as first choice for analysis of Omeprazole related substances owing to its

extremely short run time providing separation of 5 impurities without compromising

on the selectivity of the method. The resolution between the closest peaks in the

method is 2.1 with a clear base to base separation.

The proposed method has been validated as per the International Conference on

Harmonization (ICH) guidelines15

and United State Pharmacopoeia (USP). The

parameters considered for method validation are specificity, precision, linearity, limit

of detection, limit of quantification, forced degradation, and ruggedness.

Active pharmaceutical ingredient standards and samples were procured as gift

samples from Interlabs India pvt ltd, Hyderabad, India. Omez capsules manufactured

by Dr. Reddy‘s Laboratories Limited, Hyderabad, India were procured commercially

to use them as dosage form for the analysis purpose. The HPLC grade Acetonitrile

and methanol and GR grade ortho phosphoric acid were purchased from HPLC Grade

- E. Merck (India) Ltd., Mumbai, analytical grade Sodium dihydrogen phosphate was

obtained from Spectrochem Pvt. Ltd., Mumbai (India),water was obtained from

Millipore Milli.Q Plus water purification system, Bedford, MA, USA.

Analytical grade hydrochloric acid, glacial acetic acid, sodium hydroxide pellets and

30% (v/v) hydrogen peroxide solution were obtained from Ranbaxy Fine Chemicals,

New Delhi (India).

of 305

Omeprazole Structure confirmation:

1. Thermal Analysis

2.56 mg of the sample was weighed into an aluminum crucible of 25µL and placed

into a DSC. The thermogram was recorded from 20ºC to 250ºC which is carried out

under nitrogen atmosphere at 50mL/min, at 10ºC /min. The thermogram exhibited

sharp endotherm at 160.3 ºC with an onset of 158.6 ºC followed by decomposition as

depicted below

Figure 4.1- DSC Thermogram for Omeprazole

2. UV Study:

The Ultraviolet spectrum was recorded from 200 nm to 400 nm, with API

concentration of 0.0015% in methanol. The spectrum showed two λmax at 207 and

301 nm. As seen below.

of 305

Figure 4.2- UV spectrum for Omeprazole

3. FTIR Study

The FTIR of spectrum of Omeprazole was recorded by preparation of pellet with

KBr. The assignments are given in table No 4.1.

Table 4.1- FTIR assignment table for Omeprazole

Wave Number (cm-1)

Assignment Vibration Mode

3242 -N-H Stretching

3059, 3006 Aromatic C-H Stretching

2985, 2904, 2802 Aliphatic C-H Stretching

1627, 1587, 1568 -C=C Stretching

1461, 1408, Aliphatic C-H Stretching

1204, 1016 Aryl alkyl ether C-O-C Stretching

1076 -S=O Stretching

822, 810 Aromatic C-H Stretching

of 305

Figure 4.3-FTIR spectrum for Omeprazole

4. NMR Study

The 1H and

13C NMR spectra of Omeprazole were recorded in DMSO d6 at 400

MHz and 100 MHz respectively. The chemical shift values are reported on δ scale in

ppm with respect to TMS (0.00ppm) and DMSO d6 (39.5ppm) as internal standard.

of 305

Table 4.2- NMR Assignments of Omeprazole

Position 1H δ(ppm) J(Hz)

2 13C

1 NH 13.41 S -

2 - - - 152.6

4 1H 7.09 Br 94.5

5 - - - 156.8

6 1H 6.92 d, 6.8 112.9

7 1H 7.54 Br 120.5

8 - - - 135.0

9 - - - 137.5

10 Ha 4.68 d, 11.2 60.0

Hb 4.76 d, 11.2 -

11 - - - 120.5

13 1H 8.19 S 149.6

14 - - - 126.4

15 - - - 163.5

16 - - - 125.5

17 3H 2.16 S 11.1

18 3H 3.68 S 59.7

19 3H 2.19 S 12.9

20 3H 3.81 S 55.5

of 305

Figure 4.4- H1NMR spectrum for Omeprazole

Figure 4.5- C13

NMR spectrum for Omeprazole

of 305

5. Mass Spectral study

The mass spectrum of Omeprazole was recorded on 4000-Q trap LCMSMS system.

The sample is introduced into the system through HPLC by bypassing the column.

The ESI +ve ionization spectrum of Omeprazole displayed a protonated molecular ion

at m/z= 346 which corresponds to the molecular formula C17H17N3O3S. The

fragmentation pattern was observed with product ion scan. The possible

fragmentation pattern is shown below (Figure 4.6).

Figure 4.6 Fragmentation pattern for Omeprazole

of 305

Figure 4.7 Mass spectrum for Omeprazole

of 305

Impurities in Omeprazole:

A. Following are the potential impurities in the manufacturing process of

Omeprazole:

1. Impurity –A:

Chemical Name : 5-Methoxy-1H- benzimidazole-2-thiol

Structure :

3. Impurity B

Omeprazole Sulphone Impurity

Chemical name :5-Methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2-yl)

methyl]sulphonyl]-1H-benzimidazole

Structure:

of 305

2. Impurity – C:

Omeprazole N-oxide Impurity

Chemical name : 2-[(RS) [(3,5-dimethylpyridin-2-yl)methyl]sulfinyl]-5-

methoxy-1H-benzimidazole.

Structure:

4. Impurity D

Desmethoxy Omeprazole Impurity

Chemical name : 5-Methoxy-2-[[(3,5-dimethyl-2-pyridinyl)methyl]

sulfinyl]-1H-benzimidazole.

Structure :

of 305

5. Impurity E

Omeprazole Sulphide Impurity

Chemical name : 5-Methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2-

yl)

ethyl]sulphanyl]-1H-benzimidazole

Structure :

of 305

Method Development by UPLC

Objective:

To develop an analytical method for determination of related substances in a drug

product i.e. capsules containing Omeprazole By UPLC with the shortest possible run

time. The method should be capable of

Scope:

This method can be used for routine analysis in Quality control laboratories. This

method can also be applicable in Stability studies for determination of related

substance and also degradation products in API and capsules containing Omeprazole.

Chemicals and reagents:

All the solvents used i.e. Acetonitrile, Methanol, Water were of HPLC grade.

Selection of Mobile phase:

Omeprazole is known to degrade in acidic pH. That is the reason why the

formulations are enteric coated to be released in intestine where the pH is

comparatively basic. Due to this reason, the mobile phases particularly the aqueous

buffers need to be in basic side. The choice of buffers was made based on this reason.

The trials were initiated with borax, where the splitting in peaks was observed;

Ammonium acetate also had the same issue. However disodium hydrogen phosphate

showed improvement in peak shapes. The peak shapes and the separations could not

be achieved to the best of requirement, thus sodium dihydrogen orthophosphate was

selected with ph adjusted towards basic side. Mobile phase modifier, Triethylamine

was added in order to improve the peak shapes. The organic phase was also selected

to achieve better peak shapes and separations. Pure Acetonitrile did not separate all

the peaks. Hence a mixture of Acetonitrile and methanol was selected.

of 305

Selection of Column:

The selection of the column was comparatively a short process. The first column used

was Kinetex 1.7µ XB-C-18 100A 50 x 2.1 mm. By using this column, all the

impurities did not elute within the run time and merging of peaks was observed, thus a

second column i.e. HSS T3, 100*2.1mm, 1.8µm was selected where the separations

improved significantly because of the better aqueous mobile phase compatibility.

Selection of Diluent:

On the basis of solubility of all the impurities as well as Omeprazole, the diluent was

selected to be 75:25% v/v :: Buffer : Acetonitrile .

Selection of wavelength:

Based on the UV spectra of Omeprazole and all the impurities, 280nm was selected

as the wavelength for this method.

Selection of column Oven temperature:

The aim of the experiment was to elute all the peaks as early as possible and to

achieve base to base separation for all the peaks thus the column oven temperature

was also increased to activate the column performance and achieve the separation.

Experiment 1:

Buffer: Disodium hydrogen phosphate buffer Ph 7.50

Mobile phase A: Buffer

Mobile phase B: Acetonitrile

Diluent: 0.01 M Borax: Acetonitrile (3:1)

of 305

Chromatographic conditions:

Flow rate 0.3 ml/min

Wavelength 280 nm

Sample temperature NA

Column temperature 25°C

Injection volume 5 µl

Column : Waters BEH C18, 100X2.1 mm, 1.7µm

Figure 4.8- Chromatogram for experiment No 1

Gradient table

Time % A % B

0.01 80 20

5.00 80 20

of 305

Observation:

Splitting of peak was observed for Omeprazole which may be due to diluent

incompatibility

Way forward:

As Omeprazole degrades even in weakly acidic media the scope of diluent selection is

very restricted, selection of buffer with higher pH in the range (7.0 to 8.0) should

help.

Experiment 2:

Buffer: Disodium hydrogen phosphate buffer Ph 7.50



Diluent: Buffer: Acetonitrile (75:25)



Wavelength 280 nm




Column : Waters BEH C18, 100X2.1 mm, 1.7µm

Gradient table

Time % A % B

0.01 80 20

5.00 80 20

of 305


Observation:

There is improvement in the peak shape because the buffer used in mobile phase and

diluent is same hence there is no incompatibility issue.

Way forward:

The better peak shapes and improvement in separations should be in focus. In order

to achieve this, other mobile phase buffers which reduce the stationary phase

interactions should be tried.

Selection of mobile phase buffer:

The mobile phase buffer pH was selected based on the pKa value of drugs which is 1

unit away from the pH value of mobile phase buffer in order to avoid the problems of

RT shifts. The mobile phase was further optimized based on the following

experiments and the observations are as follows.

5 of 305

Experiment 3:

Buffer: Ammonium Acetate buffer pH 6.0

Mobile phase A : Buffer





Wavelength 280 nm




Column Waters BEH C18, 100X2.1 mm, 1.7µm

Gradient table

Time % A % B

0.01 80 20

0.50 80 20

1.50 50 50

2.50 40 60

2.7 80 20

3.0 80 20

of 305


Observation:

Peak shapes are distorted when ammonium acetate buffer is used in mobile phase.

Way forward:

Switching to more suitable buffer with higher pH should help improve the peak

shapes. The second issue of solution stability with Omeprazole can also be addressed

by this change.

Experiment 4:

Buffer: Disodium Hydrogen Phosphate Buffer pH 7.5




of 305



Wavelength 280 nm



Column : HSS T3, 100 x 2.1mm, 1.8µm


Gradient table

Time % A % B

0.01 75 25

0.50 70 30

1.50 65 35

2.00 55 45

2.30 10 90

2.60 10 90

2.70 75 25

3.00 75 25

of 305

Observation:

Peak shape and resolution between peaks was found to be satisfactory, however

the peaks sharpness still needs to be improved.

Result:

Switching to phosphate buffer with higher pH helped in achieving good peak

shape and better resolution between the API and its related impurities.

Way forward:

The wavelength of detection is 280 nm as this is the λmax of Omeprazole. The

response of the impurities needs to be improved for better detection.

Optimization of injection volume:

Injection volume need to be optimized keeping in mind the peak shapes, resolution

and response of related substances at the same time care should be taken to avoid

column overloading.

Experiment 5:







Wavelength 280 nm



Injection volume 6µl


of 305


Observation:

There is low response in impurity peaks. This may not be suitable as at low

concentrations, the peak areas cannot be quantified with precision and accuracy.

Way forward:

Further higher injection volumes where impurities can be quantified with

precision and accuracy without impacting column performance should help.

Gradient table

Time % A % B

0.01 75 25

0.50 70 30

1.50 65 35

2.00 55 45

2.30 10 90

2.60 10 90

2.70 75 25

3.00 75 25

of 305

Experiment 6:






Flow rate 0.3mL/min

Wavelength 280 nm



Column temperature 30, 40,50°C


Gradient table

Time % A % B

0.01 75 25

0.50 70 30

1.50 65 35

2.00 55 45

2.30 10 90

2.60 10 90

2.70 75 25

3.00 75 25

of 305

Figure 4.13- Chromatogram for experiment No 6 with flow rate of 0.3 ml/min

Figure 4.14- Chromatogram for experiment No 6 with column temperature of 30°C

of 305



Observation:

Acceptable response observed from impurities which the peaks can quantified

with precision and accuracy.

Way forward:

In both the cases i.e. 0.3mL/min as well as 0.6mL/min, the peak shapes and

resolutions were found to be good thus flow rate of 0.6ml/min can be finalized.

Optimization of flow rate:

of 305

Mobile phase flow rate has been optimized keeping in mind the resolution, peak

shape and system pressure as 0.6mL/min as the

Optimization of Column Oven Temperature:

Temperature has been optimized with optimal resolution, peak shape and system

pressure.

Experiment 7







Wavelength 280 nm




Column : Zorbax SB C8 4.6x50 mm, 1.8µ

of 305


Observation:

As seen from Figure 4.17, reduction of resolution between few peaks is observed.

Way forward:

The experimentation with C8 column to bring the nonpolar impurities closer has

resulted in reduced resolution between few peaks.

Gradient table

Time % A % B

0.01 75 25

0.50 70 30

1.50 65 35

2.00 55 45

2.30 10 90

2.60 10 90

2.70 75 25

3.00 75 25

of 305

Experiment 8







Wavelength 280 nm




Column : Kinetex 1.7µ XB-C-18 100A 50 x 2.1 mm

Gradient table

Time % A % B

0.01 75 25

0.50 70 30

1.50 65 35

2.00 55 45

2.30 10 90

2.60 10 90

2.70 75 25

3.00 75 25

of 305


Observation:

Further reduction resolution between few peaks was observed which may be due to

change in non polarity and dimensions of the stationary phase

Way forward:

The experiments with respect to stationary phase e.g. Kinetex 1.7µ XB-C-18 100A 50

x 2.1 mm used in experiment No 8 did not improve the method performance. The use

of ―HSS T3, 100 x 2.1mm, 1.8µm‖ has produced the best resolutions so far owing to

its capability to separate the polar and nonpolar compounds because of the T3

bonding.

of 305

Optimized chromatographic conditions

Buffer preparation:

Weigh and transfer 1.40 gms of sodium dihydrogen phosphate in 1000 ml of water ,

add 1ml of triethylamine, adjust pH to 7.5 with dilute ortho phosphoric acid, filter

through 0.22µm membrane filter.

Mobile phase A : Buffer

Mobile phase B : Mixed Acetonitrile and Methanol in the ratio 900 : 100 and sonicate

for 5 mins.


Column : HSS T3 100 x 2.1 mm, 1.8 µ

Column temperature : 50˚C

Wavelength : 280 nm

Flow rate : 0.6 ml/m

Sample comp temperature : NA

Injection volume : 8 µl

Gradient Program

Time % A % B

0.0 75 25

0.50 70 30

1.50 65 35

2.00 55 45

2.30 10 90

2.60 10 90

Table 4.3- Relative retention time table for all the impurities with respect to

Omeprazole

IMP name RRT

BENZIMIDAZOLE 0.42

DES METHOXY 0.93

NOXIDE 0.57

SULFONE 0.82

SULFIDE 1.34

OMEPRAZOLE 1.00

of 305

Analytical method validation

Analytical method validation is a process that demonstrates the suitability of the

proposed procedures for the intended purpose. More specifically, it is a matter of

establishing documented evidence providing a high degree of assurance with respect

to the consistency of the method and results. It evaluates the product against defined

specifications. The validation parameters viz., specificity, accuracy, precision,

linearity, limit of detection, limit of quantitation, robustness, system suitability have

to be evaluated as per the ICH guidelines for all analytical methods developed by

HPLC.

Validation Characteristics

The following validation characteristics were verified as per the ICH guidelines.

System suitability

Specificity

Linearity

Accuracy

Precision

LOD & LOQ

System suitability

This is an integral part of development of a chromatographic method to verify

that the resolution and reproducibility of the system are adequate enough for the

analysis to be performed. It is based on the concept that the equipment, electronics,

analytical operations and samples constituting an integral system could be evaluated

as a whole. Parameters such as plate number (N), asymmetry or tailing factors (As),

relative retention time (RRT), resolution (Rs) and reproducibility (% R.S.D), retention

time were determined. These parameters were determined during the analysis of a

"sample" containing the main components and related substances. System suitability

terms were determined and compared with the recommended limits (1≥As ≤2 and

Rs>1.5).

of 305

Specificity

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

presence of its process related impurities. The specificity of the developed HPLC

method was performed by injecting blank solution and standard solution spiked with

process-related impurities separately The chromatogram of drug with impurities was

compared with the blank chromatogram, to verify the blank interference. No peak was

observed at the retention time of Omeprazole and its impurities. Hence the method is

specific for the determination of Omeprazole and its combination product.

Precision of Test method

System precision of the method was evaluated by injecting the standard solution six

times and percent relative standard deviation (% R.S.D) for area of none of the peaks

was more than 0.5%. This proves the system precision of the test method. The

precision of the method for the determination of impurities related to Omeprazole

peaks was studied for repeatability at 100 % level. Repeatability was demonstrated by

analyzing the standard solution spiked with impurities for six times. The % R.S.D for

peak area of each impurity was calculated. Repeatability for Omeprazole and its

impurities were found to be optimum Thus proves that this method is precise. The

results are given in Table No 4.4.

Table 4.4- Precision results for Omeprazole and its impurities

S.No. RRF AVG SD %RSD

Benimidazole 0.72 0.188 0.0007 0.36

n-oxide 1.41 0.232 0.0006 0.25

Sulfone 0.85 0.205 0.0005 0.25

desmethoxy 1.18 0.258 0.0014 0.55

Sulfide 0.78 0.239 0.0012 0.50

TOTAL IMP NA 1.122 0.0023 0.21

of 305

Figure 4.19- Chromatogram obtained with optimized chromatographic conditions

Linearity

Standard solutions at different concentration levels ranging from 50% of the spec

level to 300% of the specification limit were prepared and analyzed. In order to

demonstrate the linearity of detector response for Omeprazole and its impurities, the

linearity plot was drawn taking the concentration on X-axis and the mean peak area

on Y-axis. The data were subjected to statistical analysis using a linear-regression

model. The regression equations and correlation coefficients (r2) are given in Tables

below.

of 305

Figure 4.20-Linearity graph for Benzimidazole impurity

Figure 4.21-Linearity graph for N-Oxide impurity

Figure 4.22-Linearity graph for Sulfone impurity

y = 12306x - 110.5R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

40000

0 0.5 1 1.5 2 2.5 3 3.5

Are

abenzimidazole

y = 28986x + 204.2R² = 1

0

20000

40000

60000

80000

100000

0 0.5 1 1.5 2 2.5 3 3.5

N-OXIDE

y = 15532x + 85.69R² = 1

0

10000

20000

30000

40000

50000

0 1 2 3 4

Are

a

Concentration in ppm

SULFONE

of 305

Figure 4.23-Linearity graph for Desmethoxy impurity

Figure 4.24-Linearity graph for Omeprazole

Figure 4.25-Linearity graph for Sulfide impurity

y = 27419x + 317.0R² = 1

0

20000

40000

60000

80000

100000

0 1 2 3 4

Are

a


DESMETHOXY

y = 22287x + 292.7R² = 0.999

0

10000

20000

30000

40000

50000

60000

70000

80000

0 0.5 1 1.5 2 2.5 3 3.5

Are

a


OMEPRAZOLE

y = 15828x - 70.71R² = 0.999

0

10000

20000

30000

40000

50000

0 1 2 3 4

Are

a


SULFIDE

of 305

Accuracy of test method

Accuracy of the test method was determined by analyzing Omeprazole substance

spiked with impurities at five different concentration levels of 50 %, 75%, 100

%,150%, 200% and 300 % of each at the specified limit. The mean recoveries of all

the impurities were calculated individually and are represented in the table 4.5 for

Omeprazole and all the impurities.

Table 4.5- Accuracy results of Omeprazole and its impurities

IMP name ACCURACY

50 % 75 % 100 % 150 % 200 % 300 %

BENZIMIDAZOLE 101.17 98.26 99.86 98.67 98.02 98.20

DES METHOXY 103.31 98.74 102.32 98.38 98.28 97.57

NOXIDE 101.48 97.67 101.44 97.43 97.37 96.85

SULFONE 102.68 99.41 102.18 98.91 98.65 98.23

SULFIDE 102.40 98.32 102.47 98.34 99/.81 98.70

OMEPRAZOLE 99.01 101.30 98.32 97.74 98.32 99.03

Limit of detection (LOD) and limit of quantitation (LOQ)

Limit of detection or LOD is the lowest level at which the impurity or API peak can

be observed or in other words can be distinguished from that of the system noise.

Limit of quantitation or LOQ is the lowest level at which the impurity or API can be

quantitatively estimated with an acceptable accuracy. This estimation was performed

by means of the slope method. The calculation was carried by means of the following

formula.

SLOD 3.3

Where = standard deviation of intercept

S = slope of the calibration curve

SLOQ 10

Where = standard deviation of intercept

S = slope of the calibration curve

of 305

The high level of sensitivity of the method can be observed by means of low levels of

the LOD and LOQ values.

Figure 4.26-LOQ Chromatogram for Omeprazole and its impurities

Table 4.6-LOQ precision for Omeprazole and its impurities

IMP name Avg %of imp % Stdev %RSD

BENZIMIDAZOLE 0.03 0.00 0.088

DES METHOXY 0.01 0.00 1.15

NOXIDE 0.01 0.00 1.68

SULFONE 0.02 0.00 1.19

SULFIDE 0.02 0.00 1.50

OMEPRAZOLE 0.02 0.00 0.89

of 305

Table 4.7-LOQ Accuracy for Omeprazole and its impurities

S.No. RRT LOQ Accuracy

Benimidazole 0.42 95.02

n-oxide 0.57 94.90

sulfone 0.82 94.68

Desmethoxy 0.93 104.91

Omeprazole 1.00 99.89

Sulfide 1.34 94.68

of 305

FORCED DEGRADATION STUDY

The forced degradation of a drug product is performed as a part of method

development or method validation in order to check the degradation product peaks

that are appearing in the chromatogram when the drug product is exposed to extreme

conditions. This is essentially to test the capability of the test method to check if the

method is able to separate any peak thus formed in any of the degradation conditions.

Stability testing of an active substance or finished product provide evidence on how

the quality of a drug substance or drug product varies with time influenced by a

variety of environmental conditions like temperature, humidity and light etc,.

Knowledge from stability studies enables understanding of the long-term effects of

the environment on the drugs. Stability testing provides information about

degradation mechanisms, potential degradation products, possible degradation path

ways of drug as well as interaction between the drug and the excepients in drug

product.

Forced degradation study was carried out by treating the sample under the

following conditions

Acid degradation

Capsule samples containing approx 50 mg of Omeprazole was weighed and

transferred into 100 ml volumetric flask and 5 ml of 1N HCl was added to it. The

solution was warmed on a water bath at 80 °C for 2 hr and then neutralized with 5 ml

of 1N NaOH. The neutralized solution was made up to the volume with diluent.

Alkali degradation


transferred into 100 ml volumetric flask and 5 ml of 1N NaOH was added to it. The

solution was warmed on a water bath at 80 °C for 2 hr and then neutralized with 5 ml

of 1N HCl. The neutralized solution was made up to the volume with diluent.

Oxidative degradation


transferred into 100 ml volumetric flask and 5 ml of 1 % Hydrogen peroxide solution

was added to it. The solution was warmed on water bath at 80 °C for 1 hr. Then the

above mixture was kept aside for few minutes, and the volume was made up with

diluent.

of 305

The above stressed samples were analyzed as per the test procedure using Photodiode

Array detector. The results are summarized in table No 4.7

Table 4.8-Forced degradation results including the %degradation

Spl condition Degradation

conditions

%

Degradation

Mass

Balance

Acid

degradation spl 1 N HCl for 5 min 3.326 99.71

Base

degradation spl

1N NaoH heat fo

30min 60°C 0.249 99.02

Peroxide

degradation spl 3% H2O2 30 min 60°C 68.31 102.99

Thermal

degradation spl 105°C 12 Hrs 1.952 100.80

Uv light

exposed spl

Exposed 254 nm for

1.2 Mn lux hrs 1.849 99.80

Sunlight

exposed spl

Exposed to direct

sunlight for 55 Hrs 0.378 100.55

Table 4.9 Forced degradation results with peak purity plot values

Spl Condition Purity angle Purity threshold Purity Flag

Spl as such 0.818 1.005 No

Thermal spl 0.813 1.434 No

Acid spl 0.567 2.397 No

Base spl 0.462 1.037 No

Peroxide spl 0.205 1.904 No

Uv spl 0.466 1..186 No

Visible spl 0.404 1.054 No

of 305

Figure 4.27-Purity plot for Omeprazole

Figure 4.28-Chromatogram of acid degradation sample

of 305

Figure 4.29-Chromatogram of base degradation sample

Figure 4.30-Chromatogram of peroxide degradation sample

of 305

Figure 4.31-Chromatogram of Thermal degradation sample

Conclusion:

A method for determination of Omeprazole and its related substances has been

successfully developed by UPLC. This method is having lot of advantages owing to

its extremely short run time. This method has also been validated as per ICH

guidelines. The method has demonstrated the stability indicating capability as it has

complied the acceptance criteria of separating all the unknown degradation products

arising from various stress studies, namely acid, base and peroxide.

The method is found to be specific, precise, linear and accurate in the range of its

intended application. This method is suitable for use in routine analysis in any quality

control laboratory and if applied will prove to be extremely beneficial for the

organization and the end user i.e. the patient.

of 305

References:

1. D Israel, M.; E Hassall.; Journal of Pediatric Gastroenterology & Nutrition, 27 - 5 -

pp 568-579 (November 1998)

2.S Pinzauti.; P Gratteri.; S Furlanetto.; P Mura.; E Dreassi.; R. Phan-Tan-Luu,

Journal of Pharmaceutical and Biomedical Analysis, 14, 8–10, pp 881–889 (June

1996)

3.. A. Radi, Journal of Pharmaceutical and Biomedical Analysis, 31,5, pp1007-1012

(2003)

4. H K. Trivedi1.; M C. Patel.; International Journal of ChemTech Research, 2, 3, pp

1355-1367, ( 2010)

5. M Mathew, V. Das Gupta and R E. Bailey, Drug Development and Industrial

Pharmacy, 21, 8 , pp 965-971 (1995)

6. W-K Kang, D-S Kim, K-I Kwon, Archives of Pharmacal Research, 22, 1, pp 86-88

(1999).

7. G W Sluggett.; J D Stong.; J H Adams.; Z Zhao, Journal of Pharmaceutical and

Biomedical Analysis‘ 25, 3–4, , pp 357–361 (June 2001)

8. P K F Yeung.; R Little.; YQ Jiang.; S J Buckley.; P.T Pollak.;H Kapoor.; S.J.O

Veldhuyzen van Zantenb, Journal of Pharmaceutical and Biomedical Analysis

17, 8, pp 1393–1398(1998)

9. L Sivasubramanian.; V Anilkumar, Indian J Pharm Sci, 69,5, pp 674-676 (2007)

10. P. V Rao.; Ch. K. Sanjeeva Reddy.; M. Ravi Kumar.; & Dantu Durga Raoa.;

Journal of Liquid Chromatography & Related Technologies, 35,16 (2012)

11. N L Rezk.; K C Brown, A D M Kashub, Journal of Chromatography B

844, 2, 5, pp 314–321(December 2006)

12. The British Pharmacopoeia, Her Majesty‘s Stationery Office, London, (2000).

13. The Indian Pharmacopoeia, Government of India Ministry of Health and

Family Welfare, the Controller of Publications, Delhi, (1996).

14. United States Pharmacopoeia and National Formulary

15. ICH, Q2(R1): Analytical Validation-Methodology November (1996).

Method Development and validation for related...

Documents

Transcript of Method Development and validation for related...