Chapter II Simultaneous determination of Omeprazole …shodhganga.inflibnet.ac.in/bitstream/10603/12745/10/10_chapter 2.pdfMolecular formula of Domperidone is C22H24ClN5O having molecular

Chapter II

Simultaneous determination of Omeprazole and

Domperidone impurities in active pharmaceutical

ingredients by UPLC

Chapter II

26

Introduction

Omeprazole

The chemical IUPAC name of Omeprazole is 6-methoxy-2-[(4-methoxy-3,5

dimethylpyridin-2-yl) methylsulfinyl]-1H-benzimidazole. The chemical structures

Omeprazole and its impurities are shown in Fig: 2.1. Omeprazole is a white to off-white

free-flowing crystalline powder. 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 in the proton pump of gastric parietal cells[5].

Omeprazole is a racemate, it contains a tricoordinated sulfinyl sulfur in a

pyramidal structure and therefore can exist in equal amounts of both the (S)- and (R)-

enantiomers. In the acidic conditions of the canaliculi of parietal cells, both are converted

to achiral products (sulfenic acid and sulfenamide configurations) which reacts with a

cysteine group in H+/K+ ATPase, thereby inhibiting the ability of the parietal cells to

produce gastric acid. Omeprazole undergoes a chiral shift in vivo which converts the

inactive (R)-enantiomer to the active (S)-enantiomer doubling the concentration of the

active form. This chiral shift is accomplished by the CYP2C19 isozyme of cytochrome

P450.

Omeprazole

NH

NOCH3

S N

CH3

CH3

O CH3

O

Chapter II

27

Chemical Formula : C17H19N3O3S

CAS number : 73590-58-6

Molecular weight : 345.416

IUPAC Name : 6-methoxy-2-{[(4-methoxy-3,5-dimethylpyridin-2-

yl)methane]sulfinyl}-1H-1,3-benzodiazole

Benzimidazole Impurity

NH

NOCH3

SH

Chemical Formula : C8H8N2OS


IUPAC Name : 5-methoxy-1H-benzimidazole-2-thiol

N-oxide Impurity

NH

NOCH3

S N+

CH3

CH3

O CH3

O o-



IUPAC Name : 5-Methoxy-2-(((4-methoxy-3,5-dimethyl-2-

yridinyl)methyl)sulfinyl)1H-Benzimidazole.

Chapter II

28

Sulphone Impurity

NH

NOCH3

S N

CH3

CH3

O CH3

O

O



IUPAC Name : 5-methoxy-2-{[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]sulfonyl}-1H-benzimidazole

Desmethoxy Impurity

NH

NS N

CH3

CH3

O CH3

O

Chemical Formula : 7: C16H19N3O2S


IUPAC Name : 2-{[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]sulfinyl}-1H-benzimidazole

Chapter II

29

Sulphide Impurity

NH

NOCH3

S N

CH3

CH3

O CH3



IUPAC Name : 5-methoxy-2-{[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]sulfanyl}-1H-benzimidazole

Fig: 2.1 Structure of Omeprazole and its impurities

Domperidone

Domperidone is chemically 5- chloro- 1- [1- [3- (2- oxo- 2, 3- dihydro-

1Hbenzimidazol-1- yl) propyl]- piperidin- 4- y l ] - 1, 3- dihydro- 2H benzimidazol- 2-

one. Molecular formula of Domperidone is C22H24ClN5O having molecular weight

425.911 g/mol. and its melting point is 244°C – 246°C[2,3,4]. Domperidone is slightly

soluble in water, sparingly soluble in dimethylformamide, slightly soluble in methanol,

Very slightly soluble in alcohol and its pKa value is 7.9.The Chemical structure of

Domperidone and its impurities shown in Fig: 2.2

Domperidone blocks the action of dopamine. It has strong affinities for the D2 and

D3 dopamine receptors, which are found in the chemoreceptor trigger zone, located just

outside the blood brain barrier, which among others regulates nausea and vomiting. Acts

by selectively antagonizing the peripheral dopaminergic D2 receptors in the

gastrointestinal wall, thereby enhancing gastrointestinal peristalsis and motility and

increasing lower esophageal sphincter tone. This increased gastrointestinal motility can

facilitates the movement of acid contents further down in the intestine preventing reflux

esophagitis and thereby controlling nausea and vomiting[4]. Domperidone is used,

together with metoclopramide, cyclizine, and 5HT receptor antagonists (such as

granisetron) in the treatment of nausea and vomiting.

Chapter II

30

Fig: 2.2 Structure of Domperidone and its impuritires

Domperidone

N

NH

Cl

N

N NH

O

O

Chemical Formula : C22H24ClN5O

CAS number : 57808-66-9


IUPAC Name : 5- chloro- 1- [1- [3- (2- oxo- 2, 3- dihydro- 1Hbenzimidazol-1-

yl) propyl]- piperidin- 4- y l ] - 1, 3- dihydro- 2H

benzimidazol- 2-one

Impurity A

N

NH

Cl

NH

O

Chemical Formula : C12H14ClN3O


IUPAC Name : 5-chloro-1-(piperidin-4-yl)-1,3-dihydro-2H-benzimidazol-2-one

Chapter II

31

Impurity B

N

NH

Cl

N

O

O

Chemical Formula : C13H14ClN3O2


IUPAC Name : 4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)piperidine-1-carbaldehyde

Impurity C

N

NH

Cl

N+

N NH

O

Oo

-



IUPAC Name : 5-chloro-1-{trans-1-oxido-1-[3-(2-oxo-2,3-dihydro-1H-

benzimidazol-1-yl)propyl]piperidin-4-yl}-1,3-dihydro- 2H-

benzimidazol-2-one

Chapter II

32

Impurity D

N

NC l

N

N N H

O

O

N N H

O



IUPAC Name : 5-chloro-3-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)propyl]-1-{1-[3-(2- oxo-2,3-dihydro-1H-benzimidazol-1-yl)propyl]piperidin-4-yl}-1,3-dihydro- 2H-benzimidazol-2-one

Impurity F

N

N

N

N N H

O

O

N

N N H

O

C l

C l

Chemical Formula : C37H42Cl2N8O3


IUPAC Name : 1,3-bis{3-[4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol-1-

yl)piperidin-1-yl]propyl}-1,3-dihydro- 2H-benzimidazol-2-one

Chapter II

33

Omeprazole and Domperidone Combination

Combination Proton Pump Inhibitors with prokinetics will improve the effect of

PPIs. PPIs are unstable at a low pH, dysmotility will slow down gastric emptying,

resulting in retention of PPIs. Retention of PPIs inside the stomach for a long time may

result in an impaired acid suppressive effect, so co-administration of prokinetic drugs will

rapidly transit PPIs to the upper intestine. This leads to improvement of lower esophageal

sphincter function, improvement of esophageal motility, and acceleration of gastric

emptying. The combination of Omeprazole and Domperidone is used duodenal ulcers,

gastric ulcers, reflux or ulcerative oesophagitis, etc[5-6].

The primary objective of this study was to implement QbD approach[7,8] to develop and

validate an UPLC method that could separate Omeprazole and Domperidone from

its potential related substances and to establish an in‐depth understanding of

the method and build in the quality during the method development to ensure optimum

method performance over the life time of the product.

Literature review

Several reported methods are available for determinations of assay of both these

drugs individually or simultaneously but have not come across any simultaneous

determination method for determination of all the impurities. This experiment aims to

achieve very short run times which have not yet been reported.

A few methods have been reported for determination of Omeprazole and its

impurities - Naser et al. reported - A simple sensitive bioanalytical assay for simultaneous

determination of omeprazole and its 3 major metabolites in human blood plasma using

RP-HPLC after a simple liquid-liquid extraction procedure[9]. Ribani et al. reported-

Validation of chromatographic methods: evaluation of determination detection and

quantification limits in the determination of impurities in Omeprazole[10]. Danica et al.

reported Densitometric determinaion of Omeprazole, Pantoprazole and their impurities in

Pharmaceuticals[11]. Kobayashi et al. Reported - Simultaneous determination of

Chapter II

34

omeprazole and its metabolites in plasma and urine by reversed-phase high-performance

liquid chromatography with an alkalin resistant polymer coated C18 column[12]. Macek et

al. reported - Determination of omeprazole in human plasma by high-performance liquid

chromatography[13].

A few methods have been reported for determination of domperidone and its

impurities - Seema et al. reported - Quantitative planar chromatographic analysis of

pantoprazole sodium sesquihydrate and domperidone in tablets[14]. Sivakumar et al.

reported - Development and validation of a reversed-phase HPLC method for

simultaneous determination of domperidone and pantoprazole in pharmaceutical dosage

forms[15]. Veronique Metal. An improved HPLC assay with fluorescence detection for the

determination of domperidone and three major metabolites for application to in vitro drug

metabolism studies[16]. Kalirajan et al. reported -, Simultaneous determination of

rabeprazole and domperidone in dosage forms by RP-HPLC[17]. Patel et al reported -

Determination of Pantoprazole, Rabeprazole, Esomeprazole, Domperidone and Itopride

in Pharmaceutical Products by Reversed Phase Liquid Chromatography using single

mobile phase[18]. Patel et al reported - Simultaneous Estimation of Lansoprazole and

Domperidone in Combined Dosage Form by RP-HPLC[19]. Sivasubramanian et al.

reported - Simultaneous HPLC estimation of omeprazole and domperidone from

tablets[20]. Patel et al. reported - HPLC analysis for simultaneous determination of

rabeprazole and domperidone in pharmaceutical formulation[21]. Karthik et al. reported -

Simultaneous estimation of paracetamol and domperidone in tablets by reverse phase

HPLC method[22].

To the best of the author‘s knowledge no method is available in the literature for

simultaneous determination of Omeprazole, domperidone and their impurities by UPLC.

In this study the combination of Omeprazole and Domperidone has to be taken up and

evaluate the impurities with all their degradation products with unique and simple method

using UPLC.

Chapter II

35

MATERIALS AND METHODS

Reagents and Chemicals

HPLC gradient grade acetonitrile and methanol from Merck (Mumbai, India) has

been used. Di sodium hydrogen phosphate (AR grade), Ortho phosphoric acid,

diethylamine and triethylamine solution from Merck have been used. Demineralized

water was further purified in the laboratory by filtering through an ultrapure Milli-Q

(Millipore, Milford, MA, USA). The drug substances, standards and impurities required

for this work were obtained from Dr Reddy‘s laboratories ltd.

Instrumentation and liquid chromatographic conditions

Chromatographic separation was carried out on a Waters Aquity UPLC with

photodiode array detector. The output signal was monitored and processed using

Empower 2 software. The mobile phase buffer consisted of 0.01M of Di sodium

hydrogen phosphate, added 1mL of Triethylamine and pH adjusted to 7.5 with diluted

Ortho phosphoric acid as mobile phase buffer and mobile phase A (buffer: methanol

(900:50)) and mobile phase B (acetonitrile: methanol (830:170)). The chromatographic

separation was performed in gradient mode (min/%B, 0/20, 0.5/25, 5/50, 5.5/72, 6.2/85,

6.8/85, 7.0/20, 8.0/20). The chromatographic separation was carried out in used Zorbax

XDB C 18 (100 mm X 4.6 mm, and 1.8 µm particle size) at flow rate of volume 1.5

ml/min with 6.0 µl injection volume. The column temperature was at 50°C. UV detection

was performed at λmax 285 nm. The standard and sample preparation was made with

methanol, water,diethylamine in the ratio of 800:200:1 as diluents. All the glassware used

for the following experimentation is of class A grade to obtain maximum precision.

Chapter II

36

Standard preparation

Accurately weighed and transferred Omeprazole and Domperidone working

standard in 50ml volumetric flask, dissolved in 30 ml of diluents and sonicated for 5

minutes and made up to volume with diluents and further dilutions are made to get final

concentration of 3µg/mL and filtered through 0.22 µm filter.

Sample preparation

Accurately weighed and transferred Omeprazole and Domperidone active

pharmaceutical ingredient in 100ml volumetric flask, dissolved in 60 ml of diluents and

sonicated for 5 minutes made up to volume with diluents to get final concentration of

1000µg/ mL and filtered through 0.22 µm filter.

Results and Discussion

A new UPLC method for simultaneous determination of related substances for

Omeprazole, Domperidone and its main impurities has been developed and evaluated.

The UPLC method was tested for selectivity, linearity, sensitivity, accuracy, precision

and robustness.

Method Development

A chromatographic condition for initial method was selected based on the

literature review. On the basis of solubility of all the impurities and active pharmaceutical

ingredients (Omeprazole and domperidone), as well as considering its compatibility with

mobile phase, methanol and water in ratio of (80:20) v/v was selected as diluent. But

from the literature review and our practical experience, we have observed that

Omeprazole was unstable in a solution at pH below 6.0. Hence the diluent was designed

considering the stability concern, which made us to add Diethylamine in diluent to

mitigate the problems associated with instability of Omeprazole. The initial method used

for method development is described in Table: 2.1 and the obtained chromatogram for the

analysis of active pharmaceutical sample spiked with impurities is presented in Fig: 2.3.

Chapter II

37

Table: 2.1 A Comparison of Initial UPLC Method and Optimized UPLC Method

Parameter Initial method Optimized method

Column ZorbaxXDB C 18

50*4.6,1.8µ

ZorbaxXDB C 18

100*4.6,1.8µ

Flow rate 1.5 ml/ min

Column temperature 40°C 50°

Injection volume 4µL 6µL

Detection 285 nm

Mobile phase A Buffer : methanol

(900mL:100mL)

Buffer : methanol

(900mL:50mL)

Mobile phase B Acetonitrile : Methanol

(850:150)

Acetonitrile : Methanol

(830:170)

Gradient (min/%B, 0/20, 0.5/25, 5/50,

5.5/70, 6.0/85, 6.8/85, 7.0/20,

8.0/20)

(min/%B, 0/20, 0.5/25,

5/50, 5.5/72, 6.2/85, 6.8/85,

7.0/20, 8.0/20)

Chapter II

38

BE

NZI

MID

AZO

LE

IMP

A

N-O

XID

E

IMP

C IMP

BS

ULP

HO

NE

DE

SM

ETH

OX

YO

ME

PR

AZO

LE

DO

MP

ER

IDO

NE

SU

LFID

E

IMP

D

IMP

F

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.3 Chromatogram obtained from initial method

As could be seen from the chromatograms, resolution of

- Impurity C and impurity B (Rs1),

- Impurity B and sulphone impurity (Rs2),

-desmethoxy impurity and Omeprazole (Rs3),

-sulphide impurity and Domperidone (Rs4),

represented the main problem because of close eluting pattern. In the optimization

experiments, effects of three parameters; column temperature (40, 50, and 55°C), mobile

phase A organic modifier (methanol), mobile phase B organic modifier, (acetonitrile:

methanol) were simultaneously evaluated to assess the effects of these parameters on

each of the four response variables.

Preliminary analyses revealed that the following conclusions are made: 1. Lower

column temperature 40°C led to a poor separation of sulphide impurity and Domperidone

(Rs 4), as well as that of impurity C and impurity B (Rs 1) (Fig: 2.3). In addition, higher

column temperature 55°C, poorer separation of impurity C and impurity B was observed

in Fig: 2.4.

Rs1

Rs3

Rs4

Rs2

Chapter II

39

BENZ

IMID

AZO

LE

IMP

A

N O

XIDE

IMP

CIM

P B

SULF

ONE

DESM

ETHO

XY

OM

EPRA

ZOLE

SULF

IDE

DOM

PERI

DONE

IMP

D

IMP

F

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.4 Chromatogram obtained from modified column temperature (55°C) from initial

method

2. At higher column temperature (55°C), reduction of methanol content in the

mobile phase A from 100ml to 50ml is resulted in a better separation of impurity C and

impurity B (Rs 1), but poor resolution of impurity B and sulphone impurity (Rs 2) was

observed and showed in Fig: 2.5.

BENZ

IMID

AZOL

E

IMP

A

N OX

IDE

IMP

CIM

P B

SULP

HONE

DESM

ETHO

XYOM

EPRA

ZOLE

SULF

IDE

DOM

PERI

DONE

IMP

D

IMP

F

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.5 Chromatogram obtained from modifed column temperature (55°C) with mobile

phase A ratio(900 mL of buffer :50 mL of methanol) from intial method

Chapter II

40

Further decrease of acetonitrile concentration to 0% provided complete co-elution

of impurity B of and sulphone impurity (Rs 2) (Fig: 2.6).

BENZ

IMID

AZO

LE

IMP

A

N O

XIDE

IMP

CSU

LFO

NE

DESM

ETHO

XY

OM

EPRA

ZOLE

DOM

PERI

DONE

SULF

IDE

IMP

D

IMP

F

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.6 Chromatogram obtained from modifed column temperature (55°C) with mobilephase A as only buffer from intial method

At higher column temperature 55°C, mobile phase A (Buffer: methanol, 900 mL:

50 mL), addition of methanol to mobile phase B, resulted in a satisfactory separation of

the impurities (Fig: 2.7).

BENZ

IMID

AZO

LE

IMP

A

N O

XIDE

IMP

CIM

P B

SULF

ONE

DESM

ETHO

XYO

MEP

RAZO

LE

SULF

IDE

DOM

PERI

DONE

IMP

D

IMP

F

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.7 Chromatogram obtained from modifed column temperature (55°C) , mobile

phase A ratio (900 mL of buffer and 50 mL of acetonitrile) and mobile phase B ratio (830

mL of acetonitrile :170 ml of methanol) from intial method

Chapter II

41

But, at column temperature of 50°C, mobile phase A (Buffer: methanol, 900 mL: 50 mL),

mobile phase B (acetonitrile: methanol, 830 mL: 170 mL) at which a better separation of

the impurities was achieved (Fig: 2.8).

BE

NZ

IMID

AZ

OLE

- 1

.401

IMP

-A -

1.7

51

N-O

XID

E -

1.9

51

IMP

C -

2.2

01

IMP

-B -

2.4

91S

ULF

ON

E -

2.6

06

DE

SM

ET

HO

XY

- 3

.020

OM

EP

RA

ZO

LE -

3.2

07

SU

LFID

E -

4.9

77

DO

MP

ER

IDO

NE

- 5

.110

IMP

-D -

6.0

61

IMP

-F -

6.4

26

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.8 Chromatogram obtained from optimized method

Method optimization by Multifactor response surface

Optimization of the analytical method was tested applying multifactor response surface

methodology. The experimental domain of the selected factors is shown in Table: 2.2.

Table: 2.2 Chromatographic conditions and the range investigated during method

optimization

Factors (chromatographicvariables)

Low High

Column temperature 40°C 55°CMobile Phase A Organic

Modifier0 mL 100 mL

Mobile Phase B OrganicModifier

150 mL 200 mL

Resolution between following peaks were chosen as the response parameters.

- impurity C and impurity B (Rs1),

- impurity B and sulphone impurity (Rs2),

- desmethoxy impurity and Omeprazole (Rs3),

- sulphide impurity and Domperidone (Rs4),

The design matrix of multifactor response surface methodology shows 20 treatment

combinations of a low (–) and high (+) level of the factors Table: 2.3.

Chapter II

42

Table: 2.3 Model matrix for multifactor response surface methodology with responsedata

Runs ColumnTemperature

MobilePhase AOrganicModifier

MobilePhase BOrganicModifier

Rs 1 Rs 2 Rs 4 Rs 4

1 55.00 50.00 150.00 1.59 1.7 2.34 1.59

2 50.00 50.00 170.00 2.1 2 2.9 4.17

3 50.00 50.00 170.00 1.99 2.1 2.8 4.2

4 40.00 100.00 170.00 1.01 1.03 2.85 3.64

5 50.00 50.00 150.00 1.97 2 2.84 3.26

6 40.00 100.00 200.00 0 1.2 1.95 1.23

7 40.00 50.00 170.00 1.29 1.7 2.71 1.26

8 50.00 50.00 200.00 1.8 1.9 2.9 3.52

9 50.00 50.00 170.00 1.98 2.06 2.85 4.29

10 50.00 50.00 170.00 2 2 2.87 4.16

11 55.00 50.00 170.00 1.63 1.9 2.38 5.3

12 40.00 0.00 200.00 0 1.5 2.1 1.56

13 55.00 0.00 200.00 0 1.4 2.54 1.1

14 55.00 0.00 150.00 0 1.6 2.32 1.9

15 55.00 100.00 170.00 1.14 2.08 2.46 6.7

16 50.00 50.00 170.00 2.15 2.2 2.86 4.13

17 50.00 100.00 170.00 0 1.1 3.04 6.15

18 50.00 0.00 170.00 1.05 1.27 2.47 1.07

19 50.00 50.00 170.00 2 2.1 2.76 4.05

20 40.00 100.00 150.00 0.99 1.66 2.65 1.3

Chapter II

43

The obtained values for the factor effects indicate that the content of methanol in

the mobile phase A (factor B) and organic modifier composition of the mobile phase B

(factor C) had the greatest impact on the chromatographic behavior of the system. The

factor A temperature of the column also expressed a strong influence. Based on the

results of these experiments, the following statistical parameters and ANOVA equations

with model graph that correlates resolution of impurity B and sulphone impurity (Rs2),

desmethoxy impurity and Omeprazole (Rs3), sulphide impurity and Domperidone (Rs4),

as well as that of impurity C and impurity B (Rs1), was derived using Design Expert 8.0.

Resolution between impurity C and impurity B (Rs1):

Table: 2.4 Analysis of variance table

Source Sum ofSquares

Mean Square F Value p-value Prob> F

Model 11.08 1.11 5.78 0.0072

A-ColumnTemperature

7.779E-003 7.779E-003 0.041 0.8448

B-Mobile PhaseA Organicmodifier

0.098 0.098 0.51 0.4916

C-Mobile PhaseB Organicmodifier

0.056 0.056 0.29 0.6012

AB 5.927E-004 5.927E-004 3.094E-003 0.9569

AC 0.027 0.027 0.14 0.7171

BC 6.824E-003 6.824E-003 0.036 0.8545

A2 0.18 0.18 0.93 0.3612

B2 4.25 4.25 22.20 0.0011

C20.10

0.10 0.52 0.4872

ABC 0.029 0.029 0.15 0.7068

Lack of Fit 1.70 0.45 85.18 <0.0001

Chapter II

44

The Model F-value of 5.78 implies the model is significant.There is only a 0.72%

chance that a "Model F-Value" this large could occur due to noise. Values of "Prob > F"

less than 0.0500 indicate model terms are significant. The "Lack of Fit F-value" of 85.18

implies the Lack of Fit is significant. There is only a 0.01% chance that a “Lack of Fit F-

value” this large could occur due to noise.

ANOVA equation

Rs1 = +2.00 +0.040 * A +0.15 * B -0.14 * C-0.019 * A * B+0.14 * A * C-0.076 * B* C-0.28 * A2-1.21 *B2-0.21 * C2+0.12 * A * B * C

3D Surface Plots

The resolution between impurity C and impurity B (Rs1) predicted from 3D

surface plots also coincide with observed resolution values which is listed in Table 3.

Moreover from the above 3D surface plots, where one parameter kept constant and other

parameter can be changed and we can predict resolution by clicking at any point of the

contour plot. If we extrapolate to other two axis, it will give corresponding values of the

other two variable factors.

Chapter II

45

Factor A (Column temprature) Kept Constant:

Design-Expert® SoftwareFactor Coding: ActualRs1

2.15

0

X1 = B: Mobile Phase A Organic modifierX2 = C: Mobile Phase B Organic modifier

Actual FactorA: Column Temperature = 47.50

150.00155.00

160.00

165.00170.00

175.00

180.00

185.00

190.00

195.00

200.00

0.00

25.00

50.00

75.00

100.00

00.51

1.52

2.5 R

s1

B: Mobile Phase A Organic modifier C: Mobile Phase B Organic modifier

2.025942.02594

Factor B (Mobile Phase A Organic modifier) Kept Constant:


Design points above predicted valueDesign points below predicted value2.15

0

X1 = A: Column TemperatureX2 = C: Mobile Phase B Organic modifier

Actual FactorB: Mobile Phase A Organic modifier = 50.00

150.00

155.00

160.00

165.00

170.00

175.00

180.00

185.00

190.00

195.00

200.00

40.00

43.00

46.00

49.00

52.00

55.00

0

0.5

1

1.5

2

2.5

Rs1

A: Column Temperature

C: Mobile Phase B Organic modifier

2.01257

Chapter II

46

Factor C (Mobile Phase B Organic modifier) Kept Constant:


2.15

0

X1 = A: Column TemperatureX2 = B: Mobile Phase A Organic modifier

Actual FactorC: Mobile Phase B Organic modifier = 175.00

0.00

25.00

50.00

75.00

100.00

40.00

43.00

46.00

49.00

52.00

55.0000.51

1.52

2.5

Rs1

A: Column Temperature B: Mobile Phase A Organic modifier

2.00311

Fig: 2.9 3D Surface plots for resolution between impurity C and impurity B (Rs1)

Chapter II

47

Resolution between impurity B and sulphone impurity (Rs2):

Table: 2.5 Analysis of variance table


Mean Square F Value p-valueProb > F

Model 2.54 0.18 32.00 0.0006

A-ColumnTemperature

6.784E-0003 6.784E-003 1.20 0.3238

B-Mobile Phase AOrganic modifier

3.753E-003 3.753E-003 0.66 0.4528

C-Mobile Phase BOrganic modifier

0.031 0.031 5.45 0.668

AB 0.20 0.20 34.42 0.0020

AC 0.15 0.15 26.79 0.0035

BC 0.17 0.17 29.90 0.0028

A2 0.0026 0.026 4.62 0.0844

B2 0.89 0.89 4.62 0.0844

C2 1.197E-003 1.197E-003 0.21 <0.0001

ABC 0.34 0.34 60.50 0.6651

A2B 0.33 0.33 59.09 0.0006

A2C 0.077 0.077 13.51 0.0144

AB2 0.35 0.35 62.35 0.0005

AC2 0.14 0.14 25.32 0.0040



less than 0.0500 indicate model terms are significant.

Chapter II

48

ANOVA equation

Rs 2 = +2.12-0.080*A-0.051*B+0.22*C-0.88*A*B-0.99*A*C-1.05*B*C-0.17*A2-

1.39*B2+0.025*C2+0.91*A*B*C+0.98*A2*B+0.52*A2*C+1.49*A*B2-

0.44*A*C2

3D Surface Plots

Resolution between impurity B and sulphone impurity (Rs2) predicted from 3D







Design-Expert® SoftwareFactor Coding: ActualRs 2

2.2

1.03



150.00

155.00

160.00

165.00

170.00

175.00

180.00

185.00

190.00

195.00

200.00

0.00

25.00

50.00

75.00

100.00

-4-20

24

6

Rs 2

B: Mobile Phase A Organic modifier


2.445792.445792.44579

Chapter II

49




1.03



150.00

155.00

160.00

165.00

170.00

175.00

180.00

185.00

190.00

195.00

200.00

40.00

43.00

46.00

49.00

52.00

55.00

-4

-2

0

2

4

6

Rs 2



2.17077



2.2

1.03



0.00

25.00

50.00

75.00

100.00

40.00

43.00

46.00

49.00

52.00

55.00

-4

-2

0

2

4

6

Rs

2



2.25963

Fig: 2.10 3D Surface plots for resolution of impurity B and sulphone impurity (Rs2)

Chapter II

50

Resolution between desmethoxy impurity and Omeprazole (Rs3):

Table: 2.6 Analysis of variance



Model 1.38 0.14 4.41 0.0178

A-ColumnTemperature

3.225E-003 3.225E-003 0.10 0.7548


0.045 0.045 1.44 0.2606


4.413E-003 4.413E-003 0.14 0.7163

AB 6.457E-004 6.457E-004 0.021 0.8891

AC 0.066 0.066 2.11 0.1807

BC 1.473E-003 1.473E-003 0.047 0.8333

A2 0.27 0.27 8.50 0.0171

B2 0.028 0.028 0.89 0.3693

C2 0.012 0.012 0.39 0.5498

ABC 0.015 0.015 0.49 0.5013

Lack of Fit 0.27 0.067 25.91 0.0015

The Model F-value of 4.41implies the model is significant.There is only a1.78%


less than 0.0500 indicate model terms are significant. The "Lack of Fit F-value" of 25.91

implies the Lack of Fit is significant. There is only a 0.15% chance that a “Lack of Fit F-

value” this large could occur due to noise.

Chapter II

51

ANOVA equation

Rs 3 = +2.90 +0.026*A+0.10*B-0.038*C-0.019*A*B+0.22*A*C-0.035*B*C-0.35*

A2-0.098*B2-0.073*C2+0.089*A*B*C

3D Surface Plots

The resolution between desmethoxy impurity and Omeprazole (Rs3) predicted

from 3D surface plots also coincide with observed resolution values which is listed in

Table: 2.3. Moreover from the above 3D surface plots, where one parameter kept

constant we can predict resolution at any of the point in contour plot. If we extrapolate to

other two axis, it will give corresponding values of the other two variable factor.



3.04

1.95



150.00

155.00

160.00

165.00

170.00

175.00

180.00

185.00

190.00

195.00

200.00

0.00 25.00

50.00 75.00

100.00

2

2.2

2.4

2.6

2.8

3

Rs 3



2.92665

Chapter II

52




1.95



150.00

155.00

160.00

165.00

170.00

175.00

180.00

185.00

190.00

195.00

200.00

40.00 43.00

46.00 49.00

52.00 55.00

2

2.2

2.4

2.6

2.8

3

Rs 3



2.90397



3.04

1.95



0.00

25.00

50.00

75.00

100.00

40.00 43.00

46.00 49.00

52.00 55.00

2

2.2

2.4

2.6

2.8

3

Rs 3



2.92332

Fig: 2.11 3D Surface plots for resolution between desmethoxy impurity and Omeprazole

(Rs3)

Chapter II

53

Resolution between sulphide impurity and Domperidone (Rs4):

Table 2.7 : Analysis of variance



Model 53.12 5.31 8.96 0.0015

A-ColumnTemperature

13.81 13.81 23.30 0.0009


16.98 16.98 28.63 0.0005


3.43 3.43 5.79 0.0395

AB 0.13 0.13 0.22 0.6466

AC 0.17 0.17 0.28 0.6093

BC 0.11 0.11 0.18 0.6805

A2 0.78 0.78 1.31 0.2822

B2 0.091 0.091 0.15 0.7051

C2 5.38 5.38 9.08 0.0147

ABC 3.55 3.55 6.00 0.0368

Lack of Fit 5.30 1.33 211.63 <0.0001



less than 0.0500 indicate model terms are significant. The "Lack of Fit F-value" of

211.63 implies the Lack of Fit is significant. There is only a 0.01 chance that a “Lack of

Fit F-value” this large could occur due to noise.

Chapter II

54

ANOVA equation

Rs 4 = +3.94 +1.70 * A+1.94* B+1.07 * C +0.28 * A * B-0.35 * A * C-0.30 * B * C-

0.59 * A2-0.18 * B2 -1.53 * C2+1.35 * A * B * C

The Resolution between sulphide impurity and Domperidone (Rs4) predicted from 3D








6.7

1.07



150.00

155.00

160.00

165.00

170.00

175.00

180.00

185.00

190.00

195.00

200.00

0.00

25.00

50.00

75.00

100.00

-2

0

2

4

6

8

Rs 4



4.86877

Chapter II

55




1.07



150.00

155.00

160.00

165.00

170.00

175.00

180.00

185.00

190.00

195.00

200.00

40.00

43.00

46.00

49.00

52.00

55.00

-2

0

2

4

6

8

Rs 4



4.59562



6.7

1.07



0.00

25.00

50.00

75.00

100.00

40.00

43.00

46.00

49.00

52.00

55.00

-2

0

2

4

6

8

Rs 4



4.93414

Fig: 2.12 3D Surface plots for resolution between sulphide impurity of Omeprazole andDomperidone (Rs4)

Chapter II

56

Method Validation

Validation is required for any new or amended method to ensure that it is capable

of giving reproducible and reliable results. Once the chromatographic conditions had

been selected, the method was validated, whereby attention was paid to the selectivity,

linearity, limit of detection, limit of quantification, precision, accuracy and robustness.

Specificity

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

in the presence of all potential sample components. The specificity of the method was

checked by injecting standard solution, sample solution, diluents as blank, and all

impurities individually. To check the performance of the optimized LC method for the

separation of degradation products, the drug was subjected to various stress conditions.

The corresponding chromatograms are shown in Fig: 2.13-2.25. As can be seen from the

figures, the method is capable of separating all the degradation products formed under the

various stress conditions. Further, forced degradation study was performed on

Domperidone alone to find out corresponding unknown degradants.

Table: 2. 8 Degradation behavior of Omeprazole

DegradationType

Degradation Condition Netdegradation

Purity angle Puritythreshold

As suchSample

--- 0.012 0.067 0.253

Acid Exposed for 5min benchwith 0.1N HCl

4.3 0.064 2.254

Base Exposed for 1hr with 2NNaoH at 60°C

0.019 0.064 0.256

Peroxide Exposed for 1hr with 10%H2O2 at 60°C

6.043 0.095 0.285

Thermal Exposed to 48 Hrs at105°C

2.04 0.066 0.255

UVlight Exposed to 200 wats/Hr 0.054 0.062 0.254

Sun light Exposed to 55 Hrs 0.022 0.062 0.254

Humidity Exposed to 90% Humidityfor 7 days

0.045 0.081 0.319

Chapter II

57

Table: 2.9 Degradation Behavior of Domperidone

DegradationType

DegradationCondition

Netdegradation

Purityangle

Puritythreshold

As suchSample

------ 0.1019 0.228 0.269

Acid Exposed for 4hrswith 2N HCl at60°C

0.741 0.217 0.259

Base Exposed for 1hrwith 2N NaoH at60°C

0.343 0.214 0.262

Peroxide Exposed for 1hrwith 10% H2O2 at60°C

35.80 0.274 0.316

Thermal Exposed to 48 Hrs at105°C

0.41 0.226 0.254

UVlight Exposed to 200wats/Hr

0.385 0.223 0.259

Sun light Exposed to 55 Hrs 0.681 0.204 0.262Humidity Exposed to 90%

Humidity for 7 days0.315 0.231 0.295

Degradation was observed in Omeprazole and Domperilone, base under stress

conditions like acid, peroxide hydrolysis, thermal. Peak purity has been verified for all of

the impurities and for both main peaks. Peak purity shows that impurity peaks as well as

main peaks are homogeneous under all the stress conditions. By the above-mentioned

fact we can confirm that the method is a stability-indicating method. The summary of the

forced degradation studies and peak purity details are given in Table: 2.8 and 2.9. The

chromatograms and purity plots of the stressed samples are shown in Fig: 2.21 & Fig:

2.25.

Mass balance is also useful in method validation, in order to demonstrate that

analytical methods are stability-indicating for which unstressed and stressed(reference)

materials are often compared. In mass balance calculations, the loss of parent drug or the

amount of drug remaining is determined from a sample assay, and the increase in

degradation products is determined by a related substances method. The % assay of

Chapter II

58

stressed sample is determined by diluting the stressed sample to the concentration where

it can be precisely quantified. (Absorbance below 1 Au). Mass balance study results are

tabulated below which clearly indicates that assay results of the stressed spl is coinciding

with the % impurities formed in the related substances method (Table: 2.10 & 2.11).

Mass Balance = % assay of stressed sample + % Net degradation

Table: 2.10 Results of Mass balance of Omeprazole

Degradation type % Assay ofstressed sample

% impurities. Mass balance

As such sample 100.12 0.012 100.11

Acid 92.17 4.3 96.47

Base 99.35 0.019 99.37

Peroxide 91.58 6.043 97.62

Thermal 98.32 2.04 100.36

UV 99.15 0.054 99.20

Sunlight 100.15 0.022 100.17

Humidity 100.51 0.045 100.56

Table: 2.11 Results of Mass balance of Domperidone

Degradation type % Assay ofstressed sample

% impurities. Mass balance

As such sample 100.23 0.1019 100.33

Acid 97.23 0.741 97.97

Base 98.13 0.343 98.47

Peroxide 61.24 35.80 97.04

Thermal 100.65 0.41 101.06

UV 100.12 0.385 100.51

Sunlight 99.86 0.681 100.54

Humidity 99.12 0.315 99.435

Chapter II

59

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.13 Chromatogram obtained from Blank sample

IMP-

A - 1

.753

N-O

XIDE

- 1.

945

SULF

ONE

- 2.

597

OM

EPRA

ZOLE

- 3.

202

DOM

PERI

DONE

- 5.

106

IMP-

D - 6

.051

IMP-

F - 6

.425

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.14 Chromatogram obtained from unspiked sample

Pea

k1 -

1.7

75

Pea

k2 -

1.9

39

OM

EP

RA

ZO

LE -

3.1

91

DO

MP

ER

IDO

NE

- 5

.087

Pea

k5 -

6.0

49

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.15 Chromatogram obtained from base degraded sample

Chapter II

60

UNK-

1 - 0.

914

UNK-

2 - 0.

968

UNK-

3 - 1.

116

UNK-

4 - 1.

192

UNK-

5 - 1.

286

BENZ

IMID

AZOL

E - 1

.329

UNK-

6 - 1.

393 UN

K-7 -

1.59

8IM

P-A

- 1.70

4

IMP-

C - 2

.174

UNK-

8 - 2.

382

SULF

ONE

- 2.59

6

OMEP

RAZO

LE -

3.183

DOMP

ERID

ONE

- 5.07

2

UNK-

9 - 5.

940

IMP-

D - 6

.043

UNK-

10 -

6.098

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.16 Chromatogram obtained from peroxide degraded sample

UNK-

1 - 0.

921

UNK-

2 - 1.

124

UNK-

3 - 1.

191

UNK-

4 - 1.

235

UNK-

5 - 1.

289

UNK-

6 - 1.

433

UNK-

7 - 1.

488

IMP-

A - 1

.686

N-OX

IDE

- 2.04

2

SULF

ONE

- 2.78

5UN

K-8 -

2.89

9

OMEP

RAZO

LE -

3.190

UNK-

9 - 3.

758

UNK-

10 -

4.159

UNK-

11 -

4.238

DOMP

ERID

ONE

- 5.09

0

UNK-

12 -

5.965

IMPD

- 6.0

20

UNK-

13 -

6.258

IMPF

- 6.3

78

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.17 Chromatogram obtained from thermal degraded sample

Peak

1 - 1.

777

Peak

2 - 1.

940

OMEP

RAZO

LE -

3.192

DOMP

ERID

ONE

- 5.09

1

Peak

5 - 6.

049

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.18 Chromatogram obtained from UV degraded sample

Chapter II

61

IMP

-A -

1.7

78

N-O

XID

E -

1.9

41

SU

LFO

NE

- 2

.597

OM

EP

RA

ZO

LE -

3.1

93

DO

MP

ER

IDO

NE

- 5

.091

IMP

-D -

6.0

50

IMP

-F -

6.4

22

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.19 Chromatogram obtained from sunlight degraded sample

AS such sample UV light degraded sample

OM

EP

RA

ZO

LE

- 3

.172

PurityAuto Threshold

AU

Degre

es

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes3.10 3.12 3.14 3.16 3.18 3.20 3.22 3.24 3.26 3.28 3.30 3.32 3.34 3.36 3.38

OM

EP

RA

ZO

LE

- 3

.173


AU

Degre

es

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes3.110 3.120 3.130 3.140 3.150 3.160 3.170 3.180 3.190 3.200 3.210 3.220 3.230 3.240 3.250 3.260 3.270

Sunlight degraded sample Base degraded sample

OM

EP

RA

ZO

LE

- 3

.174


AU

Degre

es

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes3.110 3.120 3.130 3.140 3.150 3.160 3.170 3.180 3.190 3.200 3.210 3.220 3.230 3.240 3.250 3.260 3.270 3.280 3.290 3.300 3.310 3.320

OM

EP

RA

ZO

LE

- 3

.173


AU

Degre

es

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes

3.12 3.14 3.16 3.18 3.20 3.22 3.24 3.26 3.28 3.30 3.32 3.34 3.36

Chapter II

62

Thermal degraded sample Peroxide degraded sample

OM

EP

RA

ZO

LE

- 3

.172


AU

Degre

es

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes3.12 3.14 3.16 3.18 3.20 3.22 3.24 3.26 3.28 3.30 3.32 3.34 3.36 3.38 3.40

OM

EP

RA

ZO

LE

- 3

.175


AU

Degrees

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes3.10 3.12 3.14 3.16 3.18 3.20 3.22 3.24 3.26 3.28 3.30 3.32 3.34 3.36 3.38 3.40

Acid degraded sample Humidity degraded sample

OM

EP

RA

ZO

LE

- 3

.171


AU

Degre

es

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes3.12 3.14 3.16 3.18 3.20 3.22 3.24 3.26 3.28 3.30 3.32 3.34

OM

EP

RA

ZO

LE

- 3

.173


AU

Degre

es

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes3.110 3.120 3.130 3.140 3.150 3.160 3.170 3.180 3.190 3.200 3.210 3.220 3.230 3.240 3.250 3.260 3.270

Fig: 2.20 Peak purity plots of Omeprazole

Peak

1 - 0.

404

Peak

2 - 0.

579

Peak

3 - 0.

716

Peak

4 - 1.

850

Peak

5 - 1.

964

Peak

6 - 2.

165

Peak

7 - 2.

379

Peak

8 - 2.

458

Peak

9 - 2.

836

Peak

10 -

3.751

Peak

11 -

4.528

DOMP

ERID

ONE

- 5.06

5

Peak

13 -

6.039

Peak

14 -

6.415

Peak

15 -

7.002

Peak

16 -

7.078

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.21 Chromatogram obtained from Acid degraded Domperidone sample

Chapter II

63

Pea

k1 -

0.58

0

Pea

k2 -

0.71

6

Pea

k3 -

1.15

3

Pea

k4 -

1.35

1

Pea

k5 -

1.84

8P

eak6

- 1.

963

Pea

k7 -

2.16

7

Pea

k8 -

2.38

1

Pea

k9 -

2.83

8

Pea

k10

- 4.5

26

DO

MP

ER

IDO

NE

- 5.

064

Pea

k12

- 6.0

42

Pea

k13

- 7.0

03P

eak1

4 - 7

.080

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.22 Chromatogram obtained from Base degraded Domperidone sample

Pea

k1 -

0.40

1P

eak2

- 0.

510

Pea

k3 -

0.57

8

Pea

k4 -

0.71

1

Pea

k5 -

1.85

4

Pea

k6 -

2.16

5

Pea

k7 -

2.38

0P

eak8

- 2.

456

Pea

k9 -

2.83

0

Pea

k10

- 3.7

51

Pea

k11

- 4.5

18

DO

MP

ER

IDO

NE

- 5.

060

Pea

k13

- 6.0

38

Pea

k14

- 6.4

14

Pea

k15

- 7.0

03P

eak1

6 - 7

.082

AU

0.000

0.010

0.020

0.030

0.040

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.23 Chromatogram obtained from Theraml degraded Domperidone sample

Peak

1 - 0

.737

Peak

2 - 0

.790

Peak

3 - 0

.949 Pe

ak4

- 1.0

14

Peak

5 - 1

.571

Peak

6 - 1

.647

Peak

7 - 1

.700

Peak

8 - 1

.840

Peak

9 - 1

.959

Peak

10 -

2.07

1Pe

ak11

- 2.

167

Peak

12 -

2.37

9

Peak

13 -

2.62

6

Peak

14 -

2.89

5

Peak

15 -

3.31

2

Peak

16 -

3.49

0Pe

ak17

- 3.

554

Peak

18 -

3.77

6Pe

ak19

- 3.

849

Peak

20 -

3.94

8Pe

ak21

- 4.

041

Peak

22 -

4.11

9

Peak

23 -

4.86

6

DOM

PERI

DONE

- 5.

056

Peak

25 -

5.36

8Pe

ak26

- 5.

446

Peak

27 -

5.82

8

Peak

28 -

6.04

5

Peak

29 -

7.00

4Pe

ak30

- 7.

083

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.24 Chromatogram obtained from Peroxide degraded Domperidone sample

Chapter II

64

Acid degraded sample Peroxide degraded sample

DO

MP

ER

IDO

NE

- 5

.048


AU

Deg

rees

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes4.98 5.00 5.02 5.04 5.06 5.08 5.10 5.12 5.14 5.16 5.18 5.20

OM

EP

RA

ZOLE

- 3.

175


AU

Deg

rees

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes3.10 3.12 3.14 3.16 3.18 3.20 3.22 3.24 3.26 3.28 3.30 3.32 3.34 3.36 3.38 3.40

Thermal degraded sample Base degraded sample

DO

MP

ER

IDO

NE

- 5

.047


AU

Deg

rees

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes4.98 5.00 5.02 5.04 5.06 5.08 5.10 5.12 5.14 5.16 5.18 5.20

DO

MP

ER

IDO

NE

- 5.

049


AU

Deg

rees

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes

4.98 5.00 5.02 5.04 5.06 5.08 5.10 5.12 5.14 5.16 5.18 5.20 5.22

Humidity degraded sample Sunlight degraded sample

DO

MP

ER

IDO

NE

- 5

.047


AU

Deg

rees

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes4.98 5.00 5.02 5.04 5.06 5.08 5.10 5.12 5.14 5.16 5.18 5.20

DO

MP

ER

IDO

NE

- 5

.053


AU

Deg

rees

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes5.00 5.02 5.04 5.06 5.08 5.10 5.12 5.14 5.16 5.18 5.20 5.22 5.24

UV degraded sample As such sample

DO

MP

ER

IDO

NE

- 5

.050


AU

Degre

es

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes4.990 5.000 5.010 5.020 5.030 5.040 5.050 5.060 5.070 5.080 5.090 5.100 5.110 5.120 5.130 5.140 5.150 5.160

DO

MP

ER

IDO

NE

- 5

.048


AU

Degre

es

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes4.98 5.00 5.02 5.04 5.06 5.08 5.10 5.12 5.14 5.16 5.18 5.20 5.22

Fig: 2.25 Peak purity plots of Domperidone

Precision

The precision of the method was evaluated by injecting the six individual sample

preparations and spiked with impurities at concentration of 0.2% with respect to test

Chapter II

65

concentration. The %RSD values for all the six impurities found to be less than 10.

Typical precision sample chromatogram was shown in Fig 2.26.

The intermediate precision of the method was investigated by repeating the

precision studies on other days by different analyst on different system using reagents

from different lot. The intermediate precision, expressed as the %RSD was found to be

less than 10. The data obtained suggested that the method exhibited an excellent precision

and intermediate precision. The results are given in Table 2.12 and 2.13.

BE

NZ

IMID

AZ

OLE

- 1

.390

IMP

-A -

1.7

58

N-O

XID

E -

1.9

38

IMP

C -

2.1

86

IMP

-B -

2.4

76

SU

LF

ON

E -

2.5

83

DE

SM

ET

HO

XY

- 3

.006 O

ME

PR

AZ

OLE

- 3

.193

SU

LF

IDE

- 4

.963

DO

MP

ER

IDO

NE

- 5

.099

IMP

-D -

6.0

60

IMP

-F -

6.4

24

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.26 Chromatogram obtained from Precision sample

Table: 2.12 Results of precision and intermediate precision of Omeprazole

Impurities

Name

RRF Precision%RSD Intermediate

Precision %RSD

Benzimidazole

N-oxide

Sulphone

Desmethoxy

Sulfide

1.19

1.06

0.88

1.40

0.98

0.42

0.25

0.32

0.23

0.32

0.36

0.25

0.25

0.55

0.50

Chapter II

66

Table: 2.13 Results of precision and intermediate precision of Domperidone

Impurities

Name

RRF Precision%RSD Intermediate

Precision %RSD

Imp A

Imp B

Imp C

Imp D

Imp F

0.77

0.75

0.80

1.00

0.76

0.45

0.47

0.39

0.36

0.36

0.30

0.36

0.39

0.39

0.48

Limit of Detection and Quantification

Prepared series of dilutions of impurities and analytes in different concentrations

and injected them into the chromatographic system till the signal to noise ratio is between

2 and 3.4 for limit of detection and signal to noise ration ratio is between 9.0-11.4 for

limit of quantification Table: 2.14 and 2.15. Prepared six individual solutions containing

impurities concentration at limit of quantification level Injected each solution once and

calculated the % RSD for the area of impurities. Typical Limit of Detection and

Quantification sample chromatogram was shown in Fig: 2.27 and 2.28.

Table: 2.14 Results of LOD and LOQ of Omeprazole and its impurities

Impurity/ Analyte LOD in ppm LOQ in ppm LOQ Precision(%RSD)

Benzimidazole 0.11 0.29 3.18

N-oxide 0.12 0.29 1.14

Sulphone 0.11 0.29 0.79

Desmethoxy 0.12 0.27 0.96

Sulfide 0.13 0.29 1.99

Omeprazole 0.09 0.32 1.76

Chapter II

67

Table: 2.15 Results of LOD and LOQ of Domperidone and its impurities

Impurity/ Analyte LOD in ppm LOQ in ppm LOQ Precision

(%RSD)

Impurity A 0.12 0.29 5.31

Impurity B 0.11 0.33 1.76

Impurity C 0.10 0.31 1.34

Impurity D 0.13 0.26 1.50

Impurity F 0.10 0.30 1.56

Domperidone 0.12 0.30 2.03

BE

NZ

IMID

AZ

OLE

- 1

.385

IMP

-A -

1.7

75

N-O

XID

E -

1.9

37

IMP

-C -

2.1

78

IMP

-B -

2.4

73S

ULF

ON

E -

2.5

85

DE

SM

ET

HO

XY

- 3

.003

OM

EP

RA

ZO

LE -

3.1

76

SU

LFID

E -

4.9

55D

OM

PE

RID

ON

E -

5.0

79

IMP

-D -

6.0

58

IMP

-F -

6.4

21

AU

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.27 Chromatogram obtained from LOD sample

BE

NZ

IMID

AZ

OLE

- 1

.394

IMP

-A -

1.7

66

N-O

XID

E -

1.9

42

IMP

-C -

2.1

90

IMP

-B -

2.4

80S

ULF

ON

E -

2.5

87

DE

SM

ET

HO

XY

- 3

.009

OM

EP

RA

ZO

LE -

3.1

84

SU

LFID

E -

4.9

62D

OM

PE

RID

ON

E -

5.0

88

IMP

-D -

6.0

61

IMP

-F -

6.4

26

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.28 Chromatogram obtained from LOQ sample

Chapter II

68

Accuracy

The accuracy of the method was studied by recovery studies. The sample solution

was prepared at six different concentration levels i.e. 50%,75%, 100%, 150%, 200% &

300%, specified amounts of impurities had been added to sample solutions and recovery

of these solutions was performed. The added amounts were calculated in terms of

recovery, which were found to be between 90 – 110% (Table: 2.16 and 2.17). Typical

accuracy sample chromatogram at each level was shown in Fig: 2.29 – 2.34.

Table: 2.16 Results of Accuracy of Omeprazole and its impurities

Level Benzimidazole N-oxide Sulphone Desmethoxy Sulfide Omepraz

ole

LOQ 107.96 107.46 109.36 109.21 108.45 98.19

50% 97.19 98.99 102.68 99.62 101.19 99.01

75% 99.24 99.99 100.54 99.39 101.01 101.30

100% 97.24 100.52 103.38 100.06 98.85 98.31

150% 101.13 98.64 100.62 98.17 99.95 97.74

200% 98.73 100.85 103.93 100.56 101.08 98.32

300% 99.97 100.50 100.05 99.72 100.12 99.03

Table: 2.17 Results of Accuracy of Domperidone and its impurities

Level Imp A Imp B Imp C Imp D Imp F Domperidone

LOQ 99.10 92.98 92.43 112.26 101.26 106.57

50% 98.01 104.01 97.89 101.77 99.04 98.22

75% 97.32 99.06 96.98 99.14 100.94 103.31

100% 99.52 103.35 98.78 98.16 92.63 102.74

150% 98.62 103.62 97.52 97.10 98.67 102.47

200% 100.99 105.40 99.8. 98.28 101.82 100.95

300% 101.99 98.56 99.76 97.46 99.70 102.53

Chapter II

69

BE

NZI

MID

AZO

LE -

1.38

8

IMP

-A -

1.76

0

N-O

XID

E -

1.93

5

IMP

-C -

2.18

3

IMP

-B -

2.47

2S

ULF

ON

E -

2.57

8

DE

SM

ETH

OX

Y -

3.00

2

OM

EP

RA

ZOLE

- 3.

178

SU

LFID

E -

4.95

7D

OM

PE

RID

ON

E -

5.08

4

IMP

-D -

6.05

9

IMP

-F -

6.42

3

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.29 Chromatogram obtained from 50 % accuracy sample

BE

NZ

IMID

AZ

OLE

- 1

.390

IMP

-A -

1.7

63

N-O

XID

E -

1.9

38

IMP

-C -

2.1

86

IMP

-B -

2.4

76S

ULF

ON

E -

2.5

83

DE

SM

ET

HO

XY

- 3

.005

OM

EP

RA

ZO

LE -

3.1

81

SU

LFID

E -

4.9

62D

OM

PE

RID

ON

E -

5.0

88

IMP

-D -

6.0

60

IMP

-F -

6.4

24

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00


BE

NZ

IMID

AZ

OLE

- 1

.392

IMP

-A -

1.7

56

N-O

XID

E -

1.9

39

IMP

C -

2.1

88

IMP

-B -

2.4

78S

ULF

ON

E -

2.5

87

DE

SM

ET

HO

XY

- 3

.007

SU

LFID

E -

4.9

64

IMP

-D -

6.0

61

IMP

-F -

6.4

25

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00


Chapter II

70

BENZ

IMID

AZOL

E - 1

.389

IMP-

A - 1

.772 N-

OXID

E - 1

.933

IMP-

C - 2

.174

IMP-

B - 2

.468

SULF

ONE

- 2.5

95

DESM

ETHO

XY -

2.99

9

OMEP

RAZO

LE -

3.17

5

SULF

IDE

- 4.9

52DO

MPE

RIDO

NE -

5.07

1

IMP-

D - 6

.056

IMP-

F - 6

.424

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00


BENZ

IMID

AZO

LE -

1.38

2

IMP-

A - 1

.772

N-O

XIDE

- 1.

927

IMP-

C - 2

.168

IMP-

B - 2

.460

SULF

ONE

- 2.

583

DESM

ETHO

XY -

2.99

3

OM

EPRA

ZOLE

- 3.

169

SULF

IDE

- 4.9

48DO

MPE

RIDO

NE -

5.06

8

IMP-

D - 6

.055

IMP-

F - 6

.422

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00


BE

NZI

MID

AZO

LE -

1.38

1

IMP

-A -

1.77

1

N-O

XID

E -

1.92

5

IMP

-C -

2.16

6

IMP

-B -

2.45

8S

ULF

ON

E -

2.58

1

DE

SM

ETH

OX

Y -

2.99

1

OM

EP

RA

ZOLE

- 3.

167

SU

LFID

E -

4.94

5D

OM

PE

RID

ON

E -

5.06

6

IMP

-D -

6.05

5

IMP

-F -

6.42

2

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00


Chapter II

71

Linearity

Linearity was demonstrated by injecting impurities at limit of quantification level,

50%, 75%, 100%, 150%, 200% and 300% with respect to the specification level. Plotted

the calibration curve by taking concentration on X-axis and peak area on Y-axis,

calculated the correlation coefficient and % y-intercept at 100% specification level Fig:

2.35 and 2.36. The linearity study reveals that the method is linear from LOQ to 150%

Table: 2.18 and 2.19.

Table: 2.18 Results of Linearity statistical parameter of Omeprazole and its impurities

Impurity name/

Statistical parameter

Correlation

coefficient

Slope Intercept Bias at 100%

Benzimidazole 0.9995 7824 706.9 3.1055

N-oxide 0.9999 7118 203.2 0.954

Sulphone 1.0000 6055 0.423 0.0023

Desmethoxy 0.9999 9033 105.3 0.33880

Sulfide 0.9999 6609 6.932 0.0355

Omeprazole 0.9994 6850 900.5 4.5441

Table: 2.19 Results of Linearity statistical parameter of Domperidone and its impurities

Impurity name/

Statistical parameter

Correlation

coefficient

Slope Intercept Bias at 100%

Imp A 0.9989 5314 650 4.1798

Imp B 0.9983 5126 802 4.812

Imp C 0.9999 5318 339.5 2.1626

Imp D 0.9999 6711 29.49 0.1453

Imp F 0.9993 5176 363.9 2.5523

Domperidone 0.9998 6850 800.5 3.7867

Chapter II

72

Fig: 2.35 Linearity graph of Omeprazole and its impurities

Chapter II

73

Fig: 2.36 Linearity graph of Domperidone and its impurities

Chapter II

74

Conclusion

A method for determination of Omeprazole, Domperidone, and their 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. Forced degradation studies are carried out by stressing at variety

of conditions. All the degradant peaks are well separated from both the principle peaks

and the impurity peaks. The method is validated with respect to precision and found to be

precise. The accuracy is carried out on 7 levels from LOQ % to 300% of the specification

limit and the recoveries of all the peaks are within acceptable limits. The linearity is

carried out on 7 levels from LOQ to 300% of the specification limit. The correlation

coefficient is found to be more than 0.998. Limit of detection (LOD) and Limit of

quantification (LOQ) results demonstrated the extremely high sensitivity of the method.

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

intended application. The QbD based method optimization helped in generating a design

space and operating space with knowledge of all method performance characteristics and

limitations and successful method robustness within the operating space. Hence, it 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.

The above validated related substances method further scrutinized for

determination of related substance of Rabeprazole and Domperidone in active

pharmaceutical ingredient. The method showed similar kind of elution pattern (Fig: 2.37)

which can be attributed to close resemblance of structure and its degradation pathway.

This method can be potentially explored to study the related substances of PPIs in

combination with Domperidone.

Chapter II

75

D-I

MP

-A -

1.7

97

R-I

MP

-A -

2.0

83D

-IM

P-C

- 2

.161

D-I

MP

-B -

2.4

52R

-IM

P-B

- 2

.557

R-I

MP

-D -

2.7

01

R-I

MP

-C -

3.2

25R

AB

EP

RA

ZO

LE -

3.3

49

DO

MP

ER

IDO

NE

- 5

.052

R-I

MP

-E -

5.2

81

D-I

MP

-D -

6.0

40

D-I

MP

-E -

6.4

11

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Fig: 2.37 Chromatogram of Rabeprazole, Domperidone and its impurities

Chapter II

76

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Documents

Chapter II Simultaneous determination of Omeprazole …shodhganga.inflibnet.ac.in/bitstream/10603/12745/10/10_chapter 2.pdfMolecular formula of Domperidone is C22H24ClN5O having molecular