Method Development and validation for Related substances...

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CHAPTER 5

Method Development and validation for Related substances

of Montelukast & Levocetirizine in combination by HPLC

Introduction

Montelukast

Formula : C35H35ClNO3S·Na

CAS Number : 151767-02-1

Molecular Weight : 608.17

Synonyms : Cyclopropaneacetic acid,1-[[[(1R)-1-[3-[(1E)-2-(7-chloro-2-

quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy- 1-

methylethyl)phenyl]propyl]thio]methyl]-,monosodium

salt;Singulair (TN);Singulair;sodium 2-[1-[[(1R)-1-[3-[2-(7-

chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-

yl)phenyl]propyl]sulfanylmethyl]cyclopropyl]acetate;

Melting point : 135.5°C

Montelukast is chemically belongs to leukotriene receptor antagonist (LTRA) used

for the maintenance treatment of asthma and to relieve symptoms of seasonal

allergies.1,2

It is usually administered orally in the form of tablets and oral granules

etc. Montelukast is a CysLT1 antagonist; it blocks the action of leukotriene D4 (and

secondary ligands LTC4 and LTE4) on the cysteinyl leukotriene receptor CysLT1 in

the lungs and bronchial tubes by binding to it. Montelukast is a once-daily leukotriene

receptor antagonist, in asthma and allergic rhinitis in both adults and children3

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Cetrizine Dihydrochloride

Molecular Formula : C21H25ClN2O3·2HCl

CAS No. : 83881-51-0 (Base)

83881-52-1 (Dihydrochloride)

130018-87-0 (Levocetrizine)


Synonyms :(1S.2S)-2-Methylamino-1-phenyl-1-propanol dichloride;

(2-(4-((4-Chlorophenyl)phenylmethyl)-1-

piperazinyl)ethoxy)acetic acid dichloride;

Melting point : 110 to 115°C

215-220 ºC (Levocetrizine)

Cetrizine is chemically (±)-[2-[4-[(4-chlorophenyl)phenylmethyl]-1-

piperazinyl]ethoxy] acetic acid. It is a second-generation4 antihistamine, is a major

metabolite of hydroxyzine, and a racemic selective H1 receptor inverse agonist used

in the treatment of allergies, hay fever, angioedema, and urticaria. The most

commonly it is used in reducing the severity of common cold. Levocetirizine (as

levocetirizine dihydrochloride) is a third-generation non-sedative antihistamine,

developed from Cetirizine. Chemically, levocetirizine is the active enantiomer of

cetirizine5. It is the R-enantiomer of the Cetirizine which is a racemate. Levocetirizine

works by blocking histamine receptors. It does not prevent the actual release of

histamine from mast cells, but prevents it binding to its receptors. This in turn

prevents the release of other allergy chemicals and increased blood supply to the area,

and provides relief from the typical symptoms of hay fever

Levo isomer (levocetrizine)

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Montelukast and Cetirizine/levocetirizine combination therapy

Allergic rhinitis is the most common allergic disease worldwide and affects about

18% to 40% of the general population. Combination therapy (Montelukast plus

levocetirizine) is a more effective strategy than monotherapy in the treatment of

persistent allergic rhinitis6. Montelukast sodium is a selective and orally active

leukotriene receptor antagonist that inhibits the cysteinyl leukotriene (CysLT 1),

receptor. Levocetirizine is the R-enantiomer of Cetirizine. Levocetirizine is an orally

active, potent, selective and long acting H 1 -histamine receptor antagonist with no

anticholinergic activity.

Montelukast sodium is alkaline, stable and levocetirizine Dihydrochloride is acid

stable, when we prepare a matrix tablet, both the drugs would be in contact and make

it unstable during the shelf life of the formulation thus it becomes very important to

develop a method to determine the impurities in a combination product and check for

its stability during the shelf life.

Literature study shows many methods estimation methods for Cetirizine- A.M.Y.

Jaber et al described ―Determination of Cetirizine Dihydrochloride, related impurities

and preservatives in oral solution and tablet dosage forms using HPLC‖7. Paw B et al

published Development and validation of a HPLC method for the determination of

Cetirizine in pharmaceutical dosage forms8.

Estimation methods are also available for Montelukast- Ibrahim A. Alsarra,

“development of a stability-indicating hplc method for the determination of

Montelukast in tablets and human plasma and its applications to pharmacokinetic and

stability studies‖9. R. M. Singh et al ―Development and Validation of a RP-HPLC

Method for Estimation of Montelukast Sodium in Bulk and in Tablet Dosage Form‖10

.

Many articles are available for simultaneous assay determinations of both these drugs

Atul S. Rathore et al Development of Validated HPLC and HPTLC Methods for

Simultaneous ―Determination of Levocetirizine Dihydrochloride and Montelukast

Sodium in Bulk Drug and Pharmaceutical Dosage Form‖11

, Arindam Basu et al,

―Simultaneous RP-HPLC Estimation of Levocetirizine Hydrochloride and

Montelukast

Sodium in Tablet Dosage Form‖12

, Laskhmana Rao et al ―development and validation

of a reversed phase hplc method for simultaneous determination of levocetirizine and

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montelukast sodium in tablet dosage form‖13

. A few more articles are published for

simultaneous determination of Levocetirizine and Montelukast by HPLC14-17

No article could be traced containing simultaneous determination method for

determination of all the impurities of both the drugs.

In this chapter a related substances method for determining the impurities in such

combination products was developed. For this purpose, the drug substances, standard

and impurities were gifted by Dr Reddy‘s laboratories ltd. The drug product used for

this exercise was obtained commercially from the market. The brand called Alerfix

from Eris.

Alerfix tablets contain levocetirizine hydrochloride 5 mg and Montelukast 10 mg.

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Montelukast Structure confirmation:

The following physicochemical techniques were used to confirm the structure of

Montelukast Sodium. These are given below

Thermal study

UV study

FTIR

NMR spectrophotometry

Mass spectrophotometry

1. Thermal Analysis

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

into DSC. The thermogram was recorded from 30ºC to 200ºC which is carried out

under nitrogen atmosphere at 50mL/min, at 5ºC /min. The thermogram exhibited two

endotherms. The 1st endotherm was at 54.7 ºC, which may be due to the loss of

solvent or water. The second endotherm at 135.5 ºC

2. UV Study

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

concentration of 0.0007% in methanol. The spectrum showed five λ maxima at 212,

283, 327, 344 and 358 nm.

3. FTIR Study

The FTIR of spectrum of Montelukast Sodium was recorded by preparation of pellet

with KBr. The assignments are given in the below table.

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Table 5.1 FTIR assignments for Montelukast Sodium

Wave number (cm-1

) Assignment Mode of vibration

3392 O-H Stretching

3058 Aromatic -C-H Stretching

2975, 2928 Aliphatic -C-H Stretching

1637, 1607, 1594, 1497 -C=C Stretching

1563, 1408 -C=O Stretching

1440, 1341 Aliphatic -C-H Bending

1144 -C=O Stretching

1068 -C-Cl Stretching

963, 837, 761 -C-H Bending

4. NMR Study

The 1H and

13C NMR (Fig 6&7) data of Montelukast Sodium were recorded In

DMSO-d6 at 400 MHz and 100MHz respectively on 400MHz spectrometer. The

chemical shift values are reported on 3 scale in ppm with respect to TMS (δ 0.00ppm)

and DMSO-d6 (δ 39.5ppm) as internal standard respectively. The exchangeable

proton was observed from M exchange spectrum The NMR assignment are given in

the Table below.

NMR assignments of Montelukast Sodium.

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Table 5.2 NMR assignments for Montelukast Sodium

Position1 1

H δ (ppm) J (Hz)2 13

C

2 - - - 156.8

3 1H 7.95 d,8.2 120.3

4 1H 8.40 d,8.2 136.5

5 1H 8.00 d,8.8 129.7

6 1H 7.58 dd,1.8,8.8 125.7

7 - - - 125.6

8 1H 8.03 d,1.8 127.2

9 - - - 148.0

10 1H - - 134.3

11 1H 7.89 d,16.8 135.1

12 1H 7.50 d,16.8 128.3

13 - - - 136.0

14 1H 7.73 s 126.6

15 - - - 144.1

16 1H 7.04 -7.63 - 128.9

17 1H 7.04 -7.63 - 131.0

18 1H 7.04 -7.63 - 126.3

19 1H 4.02 D,7.2 49.4

20 Ha 2.12 m 39.0

Hb 2.21 m -

21 Ha 2.75 dd,4.0,12.6 32

Hb 3.06 m -

22 - - - 146.8

23 1H 7.04 -7.63 - 126.6

24 1H 7.04 -7.63 - 125.1

25 1H 7.04 -7.63 - 128.4

26 1H 7.04 -7.63 - 125.3

27 - - - 139.9

28 - - - 71.6

28 OH* 5.15 br -

29 3H 1.44 s 31.7

29 3H 1.44 s 31.6

30 Ha 2.00 d,14.6 43.7

Hb 2.13 d,14.6 -

31 - - - 18.0

32 Ha 0.16- 0.28 m 12.4

Hb 0.34- 0.45 m -

33 Ha 0.16- 0.28 m 12.0

Hb 2.54 m -

34 Ha 2.69 d,12.6 39.9

Hb d,12.6 -

35 - - - 176.0

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5. Mass spectral study

The ESI mass spectrum of Montelukast sodium was studied on 400Q trap LCMSMS

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

The ESI +ve mass spectrum of Montelukast sodium displayed the protonated

molecular ion at m/z =586 which corresponds to the molecular formula

C35H36ClNO3S. The possible fragmentation pattern is shown below.

Figure 5.1- Mass fragmentation pattern for Montelukast Sodium

m/z=568 m/z=440

m/z=442

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Impurity Details of Montelukast

Impurity 1

Chemical Name : 1-[[[(1R)-1-[3-[2-(7-Chloro-quinolinyl) ethenyl] phenyl-3-

[2- (1- hydroxyl-1-methylethyl) phenyl] propyl]thio]

methyl] cyclopropane acetic acid

Molecular Formula : C35H38ClNO3S


Molecular Structure :

Impurity-2

Chemical Name : 1-[[[(1R)-1-[3-[(1E)-2-(7-Chloro-quinolinyl) Ethenyl] phenyl-

3- [2- (1- (1-methyl) ethenyl)] phenyl] propyl]thio]

methyl] cyclopropane acetic acid

[Or]

1-[[[(1R)-1-[3-[(1E)-2-(7-Chloro-quinolin-2-yl) Ethenyl]

phenyl-3- [2- (1-methylenyl)phenyl] propyl]sulfanyl]

methyl] cycloprpyl]acetic acid

Molecular Formula : C35H34 ClNO2S



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Impurity 3

Chemical Name : 1-[[[(1R)-3-(2-acetylphenyl)-1-[3-[(E)-2-(7-Chloroquin-2-yl)

Ethenyl] phenyl]propyl]sulfanyl] methyl] cyclopropyl]

acetic acid

[Or]

2-[1-(3-(2- acetylphenyl)-1-{3-[(E)-2-(7-Chloro-2-quinolyl)-1-

Ethyl] phenyl] propyl]sulfanyl] methyl]

cycloprpyl]acetic acid




Impurity 5:

Chemical Name : 2-[2-[3-(S)-[3-[2-[7-Chloro-2-quinolinyl] Ethyl] phenyl]-3-

hydroxy propyl]phenyl]-2-propanol

Molecular Formula : C29H28 ClNO2



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Impurity-6

Chemical Name :2-(1-{(1r)-1-{3- [(E)-2-(7-chloro-quinolyl)1-1ethrnyl]phrnyl}-

3- [2-(1-hydroxy-1-methyl ethyl ) phenyl]prpyl sufinyl

methyl}cycloprpyl] acetic acid.

[OR]

1-[[[(1-3-[(E)-2-(7-Chloroquinolin-2-yl)Ethenyl] phenyl]-3-[2-

(1- hydroxy-1-methylethyl)phenyl]propyl]sulfanyl] methyl]

cyclopropyl] acetic acid




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Cetirizine Structure confirmation:

The following physicochemical techniques were used to confirm the structure of

Cetirizine Dihydrochloride. These are given below

Thermal study

UV study

FTIR

NMR spectrophotometry

Mass spectrophotometry

1. Thermal Analysis

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

into the DSC. The thermogram was recorded from 30ºC to 300ºC which is carried out

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

endotherm at 205 and 214 ºC followed by decomposition. This was confirmed by

melting point apparatus which showed melting between 200 and 210 ºC.

2. UV Study

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

concentration of 0.0007% in methanol. The spectrum showed two λmax at 204 and

231 nm.

3. FTIR Study

The FTIR of spectrum of Cetirizine Dihydrochloride was recorded by preparation of

pellet with KBr. The assignments are given in the below table.

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Table 5.3 FTIR assignments for Cetirizine Dihydrochloride

Wave number (cm-1

) Assignment Mode of vibration

3461 -N-H, O-H Stretching

2981,2949 Aliphatic -C-H Stretching

2629, 2358 N-H+

Stretching

1743 Acid C=O Aromatic -C=C Stretching

1601 Aromatic -C=C Stretching

1496, 1382, 1357 Aliphatic -C-H Bending

1319 -C-N Stretching

1135 Ether C-O Stretching

1092 Aromatic C-Cl Stretching

805, 757, 699 Aromatic C-H Stretching

4. NMR study

The 1H and

13C NMR (Fig 6&7) data of Cetirizine Dihydrochloride were recorded In

DMSO-d6 at 400 MHz and 100MHz respectively on 400MHz spectrometer. The

chemical shift values are reported on 3 scale in ppm with respect to TMS (δ 0.00ppm)

and CD3COOD (δ 39.5ppm) as internal standard respectively. The exchangeable

proton was observed from M exchange spectrum The NMR assignment are given in

the Table below.

NMR assignments of Cetirizine Dihydrochloride

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Table 5.4 NMR assignments for Cetirizine Dihydrochloride

Position1 1

H δ (ppm) J (Hz)2 13

C

2&6 Ha

Hb

2H

2H

4.20

4.06

m 50.02

2&6 Ha

Hb

2H

2H

3.96

3.69

m 50.02

7 1H 5.72 s 76.34

8 - - - 133.52

9 1H 7.48 m 130.77

10 1H 7.95 m 131.51

11 - - 136.65

12 1H 7.95 m 131.51

13 1H 7.48 m 130.77

14 - - 134.49

15 1H 7.48 m 130.77

16 1H 7.95 m 129.68

17 1H 7.42 m 130.77

18 1H 7.95 m 129.68

19 1H 7.48 m 130.77

20 2H 3.59 m 57.44

21 2H 4.06 m 65.90

22 2H 4.24 s 68.35

23 - - - 178.11

5. Mass spectral study

The ESI mass spectrum of Cetirizine Dihydrochloride was studied on 400Q trap

LCMSMS system. The sample is introduced through HPLC system by bypassing the

column. The chemical ionization was performed by using isobutene gas to enhance

ionization. The CI mass spectrum showed base peak at m/z =389. The possible mass

fragmentation is shown below.

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Figure 5.2- Mass Fragmentation pattern of Cetirizine dihydrochloride

C21H25ClN2O3 C19H22ClN2

Exact Mass 388 Exact Mass 313

C18H20ClN2 C13H10Cl+

Exact Mass 299 Exact Mass 201

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Impurities of Cetirizine

1. Impurity A

Chemical Name: 1-[(4-Chlorophenyl) phenylmethyl] pipeazine

Molecular Formula: C17H19ClN

Molecular Weight: 286.80

Chemical Structure:

1. Impurity B

Chemical Name: 2-[4-[(4-Chlorophenyl) phenyl methyl] pipeazin-1-yl] acetic

acid

Molecular Formula: C19H21N2O2Cl


Chemical Structure:

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2. Impurity C

Chemical Name: 2-[2-[4-[(4-Chlorophenyl) phenylmethyl] pipeazin-1-yl]

ethoxy] acetic acid

Molecular Formula: C21H25ClN2O3


Chemical Structure:

3. Impurity D

Chemical Name: Bis-[(4-Chlorophenyl) phenyl methyl] pipeazine

Molecular Formula: C30H28N2Cl2


Chemical Structure:

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4. Impurity E

Chemical Name: 2-[2-[2-[4-[(4-Chlorophenyl) phenylmethyl] pipeazin-1-yl]

ethoxy] acetic acid

Molecular Formula: C23H23N2O4Cl


Chemical Structure:

5. Impurity F

Chemical Name: 2-[2-[4-(diphenylmethyl) pipeazin-1-yl] ethoxy]acetic acid

Molecular Formula: C21H26N2O3


Chemical Structure:

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Method Development By HPLC

Objective:

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

combination drug product i.e. tablets containing Montelukast and Cetirizine.

Scope:

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

method will also be checked for its applicability during the Stability studies for

determination of related substance and also degradation products in a combination

product of Montelukast and Cetirizine.

Chemicals and reagents:

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

Selection of Mobile phase:

Mobile phase was selected on the basis of chemical properties of Cetirizine and

Montelukast. Cetirizine and its impurity showed different polarity as Imp A,B,C,E

were eluting faster and Imp D was eluting slower, whereas for Montelukast and its

impurities , elution is dependent on high ratio of organic modifiers, thus 0.1% of

OPA buffer was selected. The selection of buffers was made by taking into account,

the solubility of the buffers in the organic phase. In order to ensure that difference in

readings of different pH meters does not affect the method performance, buffers were

avoided initially. Organic modifiers used in the beginning was a combination

Acetonitrile and Water in ratio 95:5, but in this combination, Cetirizine and all its

impurity except Imp D eluted at around 2 minutes, hence instead of Water, Methanol

was used to provide optimum polarity so that the Cetirizine and its impurity retentions

time increase. This change however increased the retention of Montelukast also but it

turned out to be beneficial as it provided sufficient space for Cetirizine and its

impurity to elute.

Selection of Column: Column study was done initially using Inertsil ODS 3V,

250X4.6mm, 5µm, but as the organic modifier combination was changed from

Acetonitrile: Methanol: 90:10% v/v to 90:15:Acetonitrile: Methanol, Impurity 2 and

Impurity 5 of Montelukast resolution decreased significantly, hence column study was

done on 5 different column of almost same chemical property,

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1. Inertsil C8, 250X4.6 mm, 5µm

2. Waters symmetry shield RP18, 250X4.6mm, 5µm

3. Xterra RP-18, 250X4.6mm, 5µm

4. Unison US-C18, 250X4.6mm, 5µm

5. YMC pack ODS, 250X4.6mm, 5µm

Among these set of columns Waters symmetry shield RP-18, 250X4.6mm, 5µm

showed enhanced resolution and peak shape to its High carbon load of 17%, whereas

for Inertsil ODS 3V is 15%, Xterra RP-18 is 15%, Unison US-C18 is 15% and YMC

ODS-AQ is 14%.

Selection of Diluent: On the basis of solubility of both the compounds diluents was

selected to be 70:30 :: Methanol : Water.

Selection of wavelength: Absorption maxima of Cetirizine is around 243 and 229

and that of Montelukast is 266 and 283 wavelength was selected to be 225nm since 10

nm below this the response of Montelukast and its impurities were significantly

reduced along with the irregular baseline due to proximity of cut off wavelength of

Acetonitrile, 10 nm above this response of Cetirizine Imp F was significantly

reduced and 15 nm above this wavelength, the peak responses of all the impurities

decreased drastically.

Experiment 1:

Buffer: Pipetted out 1mL of 85%v/v Orthophosphoric Acid in to a 1000mL

volumetric flask, 500mL water added, shaken for 10min, subjected to ultrasonication,

cooled to room temperature and made up to the volume with water

Mobile Phase A: Buffer

Mobile Phase B: Acetonitrile::Water :: 95:5% v/v

Diluent: Methanol: Water :: 70:30% v/v

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Chromatographic Conditions:

Flow rate 1.5 ml/min

Wavelength 225 nm

Sample temperature Ambient

Column temperature 25°C

Column Inertsil ODS 3V, 250X4.6 mm, 5µm

Gradient Program

Figure 5.3 Chromatogram for experiment No 1showing Montelukast and related

impurities

Time %A %B

0.01 40 60

10 30 70

15 10 90

20 0 100

30 0 100

32 40 60

40 40 60

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Figure 5.4 Chromatogram for experiment No 1showing Cetirizine and related

impurities

Observation:

Montelukast and all its Impurities eluted within 25 minutes. Cetirizine and all its

impurity were eluted within 2 minutes.

Way forward:

Introduction of methanol in mobile phase B with replacement of water with methanol

should help in retention Cetirizine and its impurity. It should also extend the retention

time of Montelukast and its impurities. This change would also ensure proper

separation of all impurities.

Experiment 2:





Mobile Phase B: Acetonitrile::Water :: 90:10% v/v


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Chromatographic Condition:


Wavelength 225 nm




Gradient Program

Time %A %B

0.01 70 30

10 70 30

15 65 35

20 50 50

30 30 40

40 10 90

50 0 100

60 0 100

62 70 30

70 70 30

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Figure 5.5 Chromatogram for experiment No 2 with all the peaks

Observation:

This change has achieved the required results up to a certain extent. Montelukast and

all its Impurities were well separated. The decrease in Acetonitrile content in mobile

phase B impacted on Montelukast and its impurities which seem to be relatively

nonpolar and the runtime was extended upto 45 min. Cetirizine and all its impurities

eluted within 20 minutes but with very less resolution.

Way forward:

Slight increment in the percentage of methanol and corresponding reduction in the

percentage of Acetonitrile should improve the resolution of Cetirizine and its

impurities.

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Experiment 3:





Mobile Phase B: Acetonitrile:: Methanol :: 85:15% v/v




Wavelength 225 nm




Gradient Program

Time %A %B

0.01 70 30

10 70 30

15 65 35

20 50 50

30 60 40

40 10 90

50 0 100

55 0 100

58 70 30

65 70 30

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Figure 5.6 Chromatogram for experiment No 3 showing all peaks

Observation:

Changing the percentage of Acetonitrile and Methanol in mobile phase B worked as

expected. The resolution was improved for Cetirizine and all its impurity whereas in

case of Montelukast resolution between Imp 2 and Imp 5 was reduced. This indicates

that it is necessary to keep the percentage of Acetonitrile to 90% for optimum

separation of Montelukast and its impurity and 15% of methanol for improved

separation of Cetirizine and its impurity.

Way forward:

Increasing the percentage of Acetonitrile to 90% and maintaining the same

percentage of Methanol should workout logically to separate all the impurities.

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Experiment 4:









Wavelength 225 nm




Gradient Program

Time %A %B

0.01 70 30

10 70 30

15 65 35

20 50 50

30 60 40

40 10 90

50 0 100

55 0 100

58 70 30

65 70 30

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Observation:

Changing the percentage of Acetonitrile and Methanol in mobile phase B didn‘t work

as expected, resolution was not improved between Imp 2 and Imp 5 of Montelukast.

This shows that the impurity 2 and impurity 5 peaks are not organic phase sensitive.

Way forward:

Need to study the impact of change in stationary phase parameters thus perform

column study on equivalent columns keeping all the other chromatographic conditions

same as that of previous Experiment.

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Experiment 5:









Wavelength 225 nm



Column Inertsil C8, 250X4.6 mm, 5µm

Gradient Program

Time %A %B

0.01 70 30

10 70 30

15 65 35

20 50 50

30 60 40

40 10 90

50 0 100

55 0 100

58 70 30

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Observation:

The separation between the impurities did not improve significantly. Needs to

improve more in order to finalize the method

Way forward:

Need to check for similar columns for solutions

Experiment 6:







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Wavelength 225 nm



Column Unison US-C18, 250X4.6mm, 5µm

Gradient Program

Time %A %B

0.01 70 30

10 70 30

15 65 35

20 50 50

30 60 40

40 10 90

50 0 100

55 0 100

58 70 30

65 70 30

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Observation:



Way forward:


Experiment 7:







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Wavelength 225 nm



Column Xterra RP-18, 250X4.6mm, 5µm

Gradient Program

Time %A %B

0.01 70 30

10 70 30

15 65 35

20 50 50

30 60 40

40 10 90

50 0 100

55 0 100

58 70 30

65 70 30

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Observation:



Way forward:


Experiment 8:







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Wavelength 225 nm



Column YMC pack ODS, 250X4.6mm, 5µm

Gradient Program

Time %A %B

0.01 70 30

10 70 30

15 65 35

20 50 50

30 60 40

40 10 90

50 0 100

55 0 100

58 70 30

65 70 30

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Observation:



Way forward:


Experiment 9:







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Wavelength 225 nm



Column Waters symmetry shield RP18, 250X4.6mm, 5µm

Gradient Program

Time %A %B

0.01 70 30

10 70 30

15 65 35

20 50 50

30 60 40

40 10 90

50 0 100

55 0 100

58 70 30

65 70 30

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Observation:

Significant improvement was observed in the resolution in waters symmetry shield

RP18, 250X4.6mm, 5µm

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Optimized final method:

Buffer: 0.1% of 85% Orthophosphoric Acid in water (1ml of 85% OPA in 1000ml

Milli-Q water)


Mobile Phase B: Acetonitrile::Methanol :: 90:15

Diluent: Methanol: Water :: 70:30

Sample preparation: Cetirizine 200 ppm, Montelukast: 1000 ppm.

Sample preparation was carried out by transferring 20mg of Cetirizine and 100mg of

Montelukast in amber colored 100ml volumetric flask, sonicated for 5minutes to

dissolve and diluted to volume with diluent.

Standard stock Preparations:

Standard stock preparation was carried out by adding weighed quantities of 50 mg of

Cetirizine and 100 mg Montelukast standards into separate volumetric flasks

respectively, 50mL of diluent added in each of the volumetric flasks, subjected to

ultra sonication for about 5 mins, cooled and made upto the volume with diluent.

Standard Preparation: Cetirizine: 1ppm, Montelukast: 2ppm

2 mL from each of the standard stock solutions were pipetted out into a 100mL

volumetric flask, 50mL of diluent added and shaken well and madeup to the volume

with the diluent.




Inj Volume 30µL

Wavelength 225 nm

Sample temperature 10°C

Columns Waters symmetry shield RP18, 250X4.6mm, 5µm

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Gradient Program

Table 5.5-Individual limit of impurities considered for method validation

Cetirizine

Imp Name/No Limit

Imp A 0.10%

Imp B 0.10%

Imp C 0.10%

Imp D 0.10%

Imp E 0.10%

Imp F 0.10%

Montelukast

Imp 1 0.10%

Imp 2 0.15%

Imp 3 0.15%

Imp 4 0.10%

Imp 5 0.10%

Time %A %B

0.01 75 25

10 70 30

15 65 35

20 50 50

30 35 65

40 20 80

50 0 100

55 0 100

58 75 25

65 75 25

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Figure 5.13 Chromatogram showing all peaks in optimized method

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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).

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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 Montelukast, Cetirizine and their impurities. Hence

the method is specific for the determination of Montelukast, Cetirizine 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 Montelukast peak

was 2.1% and for Cetirizine peak it was 1.16%. This proves the system precision of

the test method. The precision of the method for the determination of impurities

related to Montelukast and Cetirizine 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 Montelukast, Cetirizine and its impurities were found to be optimum

Thus proves that this method is precise. The results are given in Table 5.6.

Table 5.6- Precision results for Montelukast impurities

Impurity

name

(Montelukast)

RRF of

impurities

%Imp of

SPL-1

%Imp of

SPL-2

%Imp of

SPL-3

%Imp of

SPL-4

%Imp of

SPL-5

%Imp of

SPL-6

%RSD

Impurity 1 0.77 0.116 0.104 0.102 0.106 0.095 0.097 7.13

Impurity 2 1.10 0.166 0.159 0.158 0.150 0.135 0.132 9.14

Impurity 3 0.75 0.169 0.152 0.147 0.144 0.136 0.130 9.16

Impurity 5 1.17 0.099 0.097 0.097 0.095 0.092 0.090 3.45

Impurity 6 0.80 0.110 0.103 0.103 0.101 0.097 0.096 6.95

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Table 5.7- Precision results for Cetirizine impurities

Impurity

name

(Cetirizine)

RRF of

impurities

%Imp of

SPL-1

%Imp of

SPL-2

%Imp of

SPL-3

%Imp of

SPL-4

%Imp of

SPL-5

%Imp of

SPL-6

%RSD

Impurity A 1.70 0.109 0.113 0.114 0.114 0.113 0.112 1.72

Impurity B 1.26 0.118 0.119 0.116 0.115 0.115 0.117 1.22

Impurity C 0.55 0.088 0.083 0.088 0.087 0.092 0.093 4.01

Impurity D 2.50 0.110 0.115 0.115 0.114 0.111 0.113 1.70

Impurity E 1.20 0.100 0.092 0.090 0.093 0.092 0.092 3.65

Impurity F 0.45 0.094 0.093 0.093 0.094 0.095 0.093 0.91

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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 Montelukast, Cetirizine and their

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.

Linearity of Montelukast and its Impurities

Table 5.8 Linearity table for Montelukast

%Level

API

Conc

(ppm) area

50 1 44494

75 1.5 62215

100 2 81877

150 3 123580

200 4 161445

300 6 241958

intercept 3539

Bias at 100% 4.3223

Correlation coefficient 0.9999

Figure 5.14- Linearity graph for Montelukast

y = 39676x + 3539.R² = 0.999

0

50000

100000

150000

200000

250000

300000

0 1 2 3 4 5 6 7

Montelukast

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Table 5.9-Linearity table for Montelukast Impurity 1

IMP 1

% Level conc(ppm) area

50 0.5 15693

75 0.75 24388

100 1 33432

150 1.5 50316

200 2 66870

300 3 104907

intercept 397

Bias at 100% 1.1875


Figure 5.14- Linearity graph for Montelukast impurity 1

y = 35428x - 397.R² = 0.999

0

20000

40000

60000

80000

100000

120000

0 0.5 1 1.5 2 2.5 3 3.5

Impurity 1

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IMP 2


50 0.75 30909

75 1.125 46554

100 1.5 62363

150 2.25 94701

200 3 127600

300 4.5 191949

intercept 1824

Bias at 100% 2.925



y = 43050x - 1824.R² = 1

0

50000

100000

150000

200000

250000

0 1 2 3 4 5

Impurity 2

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IMP 3


50 0.75 36571

75 1.125 55638

100 1.5 74477

150 2.25 112545

200 3 149943

300 4.5 224523

intercept 696.3

Bias at 100% 0.9349



y = 50124x - 696.3R² = 1

0

50000

100000

150000

200000

250000

0 1 2 3 4 5

Impurity 3

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IMP 5


50 0.5 23344

75 0.75 34977

100 1 46897

150 1.5 72874

200 2 99892

300 3 151532

intercept 651

Bias at 100% 3707



y = 51630x - 3707.R² = 0.999

0

20000

40000

60000

80000

100000

120000

140000

160000

0 0.5 1 1.5 2 2.5 3 3.5

Impurity 5

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IMP 6


50 0.5 19336

75 0.75 28854

100 1 39267

150 1.5 58960

200 2 78437

300 3 116161

intercept 205.6

Bias at 100% 0.5236



y = 38832x + 205.6R² = 0.999

0

20000

40000

60000

80000

100000

120000

140000

0 0.5 1 1.5 2 2.5 3 3.5

Impurity 6

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Linearity of Cetirizine and its Impurities

Table 5.14-Linearity table for Cetirizine

API


50 0.5 17649

75 0.75 27055

100 1 35915

150 1.5 54116

200 2 71444

300 3 107398

intercept 63.86

Bias at 100% 0.1778


Figure 5.19- Linearity graph for Cetirizine

y = 35794x + 63.86R² = 0.999

0

20000

40000

60000

80000

100000

120000

0 0.5 1 1.5 2 2.5 3 3.5

Cetrizine

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Table 5.15-Linearity table for Cetirizine Impurity A

IMP A


50 0.12 5808

75 0.18 8167

100 0.24 11410

150 0.36 15496

200 0.48 22647

300 0.72 33973

intercept 260.7

Bias at 100% 2.2848


Figure 5.20- Linearity graph for Cetirizine Impurity A

y = 47174x - 260.7R² = 0.996

0

5000

10000

15000

20000

25000

30000

35000

40000

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Are

a

Impurity A

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Table 5.16-Linearity table for Cetirizine Impurity B

IMP B


50 0.12 4214

75 0.18 6533

100 0.24 8569

150 0.36 13219

200 0.48 17332

300 0.72 26251

intercept 132.5

Bias at 100% 1.546


Figure 5.21- Linearity graph for Cetirizine Impurity B

y = 36626x - 132.5R² = 0.999

0

5000

10000

15000

20000

25000

30000

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Impurity B

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Table 5.17-Linearity table for Cetirizine Impurity C

IMP C


50 0.12 1295

75 0.18 2038

100 0.24 2720

150 0.36 4294

200 0.48 5668

300 0.72 8700

intercept 196.1

Bias at 100% 7.2096


Table 5.22-Linearity table for Cetirizine Impurity C

y = 12330x - 196.1R² = 0.999

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Are

a

Impurity C

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Table 5.18-Linearity table for Cetirizine Impurity D

IMP D


50 0.12 9910

75 0.18 13982

100 0.24 17843

150 0.36 26181

200 0.48 34026

300 0.72 49754

intercept 512

Bias at 100% 2.8695


Table 5.23-Linearity table for Cetirizine Impurity D

y = 66486x + 2012.R² = 0.999

0

10000

20000

30000

40000

50000

60000

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Impurity D

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Table 5.19-Linearity table for Cetirizine Impurity E

IMP E


50 0.12 3202

75 0.18 4902

100 0.24 6546

150 0.36 9436

200 0.48 12232

300 0.72 18387

intercept 363.4

Bias at 100% 5.5515


Table 5.24-Linearity table for Cetirizine Impurity E

y = 25012x + 363.4R² = 0.999

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Impurity E

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Table 5.20-Linearity table for Cetirizine Impurity F

IMP F


50 0.12 1199

75 0.18 1510

100 0.24 2083

150 0.36 3470

200 0.48 4220

300 0.72 6370

intercept 64.88

Bias at 100% 3.1147


Table 5.25-Linearity table for Cetirizine Impurity F

y = 8791.x + 64.88R² = 0.995

0

1000

2000

3000

4000

5000

6000

7000

0 0.2 0.4 0.6 0.8

Are

a

Impurity F

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Accuracy of test method

Accuracy of the test method was determined by analyzing Motelukast, Cetrizine drug

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 below tables

individually for Motelukast, Cetrizine and all the impurities

Table 5.21- Accuracy results for Montelukast

Level Amount Added Amount Found %Recovery

50% 0.9093 1.0090 110.97

75% 1.4397 1.4109 98.00

100% 1.8943 1.8568 98.02

150% 2.8793 2.8026 97.33

200% 3.7886 3.6613 96.64

300% 5.6829 5.4872 96.56

Table 5.22- Accuracy results for Montelukast Impurity 1


50% 0.4733 0.4636 97.95

75% 0.7573 0.7204 95.13

100% 0.9466 0.9876 104.33

150% 1.4200 1.4864 104.68

200% 1.8933 1.9754 104.34

300% 2.8399 3.0990 109.12

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50% 0.7601 0.7010 92.22

75% 1.2162 1.0558 86.81

100% 1.5202 1.4143 93.03

150% 2.2804 2.1476 94.18

200% 3.0405 2.8937 95.17

300% 4.5607 4.3531 95.45



50% 0.8116 0.8294 102.20

75% 1.2985 1.2618 97.17

100% 1.6232 1.6890 104.06

150% 2.4348 2.5523 104.83

200% 3.2463 3.4004 104.75

300% 4.8695 5.0918 104.57



50% 0.5163 0.5294 102.54

75% 0.8260 0.7932 96.03

100% 1.0325 1.0635 103.00

150% 1.5488 1.6526 106.70

200% 2.0651 2.2654 109.70

300% 3.0976 3.4365 110.94

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50% 0.4101 0.4385 106.93

75% 0.6562 0.6544 99.73

100% 0.8202 0.8905 108.57

150% 1.2303 1.3371 108.68

200% 1.6404 1.7788 108.44

300% 2.4606 2.6343 107.06

Table 5.27- Accuracy results for Cetirizine


50% 0.4786 0.4912 102.64

75% 0.7577 0.7529 99.36

100% 0.9970 0.9995 100.25

150% 1.5154 1.5060 99.38

200% 1.9940 1.9883 99.71

300% 2.9910 2.9889 99.93

Table 5.28- Accuracy results for Cetirizine Impurity A


50% 0.1014 0.0951 93.82

75% 0.1521 0.1664 109.44

100% 0.2027 0.1868 92.14

150% 0.3041 0.3355 110.32

200% 0.4055 0.3707 91.42

300% 0.6082 0.5562 91.45

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Table 5.29- Accuracy results for Cetirizine Impurity B


50% 0.1003 0.0931 92.86

75% 0.1504 0.1443 95.95

100% 0.2005 0.1893 94.40

150% 0.3008 0.2920 97.08

200% 0.4011 0.3828 95.45

300% 0.6016 0.5798 96.38

Table 5.30- Accuracy results for Cetirizine Impurity C


50% 0.0721 0.0655 90.87

75% 0.1081 0.1031 95.35

100% 0.1442 0.1376 95.44

150% 0.2163 0.2173 100.49

200% 0.2883 0.2868 99.47

300% 0.4325 0.4402 101.78

Table 5.31- Accuracy results for Cetirizine Impurity D


50% 0.0966 0.1103 114.18

75% 0.1449 0.1556 107.38

100% 0.1932 0.1986 102.79

150% 0.2898 0.2914 100.55

200% 0.3864 0.3788 98.03

300% 0.5796 0.5539 95.56

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Table 5.32- Accuracy results for Cetirizine Impurity E


50% 0.0824 0.0743 90.15

75% 0.1236 0.1137 91.97

100% 0.1648 0.1518 92.09

150% 0.2473 0.2188 88.49

200% 0.3297 0.2837 86.05

300% 0.4945 0.4264 86.23

Table 5.33- Accuracy results for Cetirizine Impurity F


50% 0.0711 0.0742 104.35

75% 0.1067 0.0934 87.56

100% 0.1422 0.1288 90.56

150% 0.2133 0.2146 100.60

200% 0.2844 0.2597 91.30

300% 0.4267 0.3939 92.32

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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

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

the LOD and LOQ values.

Table 5.34- LOD and LOQ of Montelukast impurities

Impurity Name

LOQ

LOD

Impurity 1 0.006% 0.002%

Impurity 2 0.008% 0.003%

Impurity 3 0.008% 0.003%

Impurity 5 0.006% 0.002%

Impurity 6 0.005% 0.002%

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Table 5.35- LOD and LOQ of Cetirizine impurities

Impurity Name

LOQ

LOD

Impurity A 0.019% 0.005%

Impurity B 0.010% 0.003%

Impurity C 0.017% 0.006%

Impurity D 0.003% 0.001%

Impurity E 0.023%

0.008%

Impurity F

0.041% 0.015%

Figure 5.26- Chromatogram showing LOQ level peaks

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FORCED DEGRADATION STUDY

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

development or method validation in order to understand which are 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 same 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 excipients in drug

product.

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

following conditions

Acid degradation

A tablet powder sample containing approx 20 mg of Cetirizine and 100mg of

Montelukast 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


Montelukast was weighed and 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.

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Oxidative degradation


Montelukast was weighed and transferred into a 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.

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

Array detector. The results are summarized in below table

Table 5.36-Forced degradation Cetirizine

Degradation

Type

Degradation Condition Net

degradation

Purity

angle

Purity

threshold

Acid Exposed for 1hrs with 1N

HCl at 60°C 1.8% 0.073 0.269

Base Exposed for 1hr with 1N

NaoH at 60°C 0.05% 0.068 0.268

Peroxide Exposed for 1hr with 1%

H2O2 at 60°C

2.3%

0.072 0.269

of 305

Table 5.37-Forced degradation Montelukast

Degradation

Type

Degradation

Condition

Net

degradation

Purity

angle

Purity

threshold

Acid Exposed for 1 hrs with

1N HCl at 60°C 1.9% 3.481 4.298

Base Exposed for 1hr with

1N NaoH at 60°C 0.51% 2.935 4.164

Peroxide Exposed for 1hr with

1% H2O2 at 60°C

18.0%

2.439 4.050

Chromatograms for forced degradation study.

Figure 5.27- Chromatogram for Sample in as such condition

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Figure 5.28- Chromatogram for Cetrizine degradation in IN HCl

Figure 5.29-Chromatogram for Montelukast degradation in IN HCl

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Figure 5.30-Chromatogram for Montelukast and Cetirizine In 1n HCl

Figure 5.31-Chromatogram for Cetirizine In 1N NaOH

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Figure 5.32-Chromatogram for Montelukast in 1N NaOH

Figure 5.33-Chromatogram for Montelukast and Cetirizine in 1N NaOH

of 305

Figure 5.34-Chromatogram for Cetirizine in 1% H2O2

Figure 5.35-Chromatogram for Montelukast in 1% H2O2

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Figure 5.36-Chromatogram for Montelukast and Cetirizine in 1% H2O2

Conclusion:

A method for determination of Cetirizine, Montelukast, and their related substances

has been successfully developed by HPLC. 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.

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