method development and validation for related …

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838

METHOD DEVELOPMENT AND VALIDATION FOR RELATED

SUBSTANCES OF BOSUTINIB MONOHYDRATE BY RP-HPLC

METHOD

Yogendra B. Parmar*, Dharmesh Shah, Yashraj A. Majmudar, Ketul C. Kaka, Arpan

S. Patel, Pankaj D. Kankad and Uday G. Sartanpara

BDR Lifesciences Pvt. Ltd. R.S No.578, Near Effluent Channel, Luna Village, Taluka-Padra,

Vadodara District, Gujrat-391440, India.

ABSTRACT

An accurate, sensitive and rapid gradient reverse phase high

performance liquid chromatography (RP-HPLC) method has been

developed and validated for related substances of Bosutinib

monohydrate. HPLC analysis was performed on Inertsil ODS 3V (250

x 4.6) mm, 5 m. Column temperature maintained at 40°C conditions.

Chromatographic separation was achieved with mobile phase gradient

program at flow rate of 0.7mL/min. the injection volume was 15μl.

The UV detection wavelength was 250nm. The method suitability was

checked and validated according to the ICH guidelines for specificity,

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

Limit of detection each impurity is less than 0.05% w/w indicating that

the developed method is highly sensitive. The calibration curve was

found to be linear within the concentration range of 0.5µg/mL to 10µg/mL. The regression

data for calibration curve shows good linear relationship. Correl coefficient (r2) of each

impurity is greater than 0.9991. The experiment results are given in detail in this research

article.

KEYWARD: RP-HPLC, Method validation, Method Development, BST, Related

substances.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.632

Volume 10, Issue 1, 838-854 Research Article ISSN 2278 – 4357

*Corresponding Author

Yogendra B. Parmar

BDR Lifesciences Pvt. Ltd.

R.S No.578, Near Effluent

Channel, Luna Village,

Taluka-Padra Vadodara

District, Gujrat-391440,

India.

Article Received on

24 October 2020,

Revised on 14 Nov. 2020,

Accepted on 04 Dec. 2020

DOI: 10.20959/wjpps20211-17727


839

Parmar et al. World Journal of Pharmacy and Pharmaceutical Sciences

INTRODUCTION

Bosutinib Monohydrate is chemically known as 4-(2, 4-dichloro-5-methoxyanilino)-6-

methoxy-7-[3-(4-methylpiperazin-1-yl) propoxy] quinoline-3-carbonitrile; hydrate, molecular

formula is C26H31Cl2N5O4 and molecular weight is 548.45g/mole. Bosutinib[3]

marketed

under the trade name Bosulif[1]

is a small molecule and tyrosine kinase inhibitor used for the

treatment of chronic myelogenous leukemia. Originally synthesized by Wyeth, it is being

developed by Pfizer.[2,7]

Bosutinib received US FDA and EU European Medicines Agency

approval on September 4, 2012 and 27 March 2013 respectively for the treatment of adult

patients with Philadelphia chromosome-positive[6]

(Ph+) chronic myelogenous leukaemia

(CML) with resistance, or intolerance to prior therapy is approved to treat: Chronic

myelogenous leukaemia (CML) that is Philadelphia chromosome positive.[4]

It is used in

patients with newly diagnosed chronic phase CML. In patients with chronic phase,

accelerated phase, or blastic phase CML that cannot be treated with or that did not respond to

other treatment.

Bosutinib is also being studied in the treatment of other types of cancer.[5]

MATERIALS AND METHODS

The chemical structure of Bosutinib Monohydrate is shown in Figure 1.

N

N

O N

O CN

NH

ClCl

O

H2O

Figure 1: Structure of bosutinib monohydrate.

Molecular formula: C26H31Cl2N5O4

Molecular weight: 548.45g/mol

IUPAC Name: 4-(2, 4-dichloro-5-methoxyanilino)-6-methoxy-7-[3-(4-methylpiperazin-1-yl)

propoxy] quinoline-3-carbonitrile; hydrate.

https://en.wikipedia.org/wiki/Tyrosine_kinase_inhibitor

https://en.wikipedia.org/wiki/Chronic_myelogenous_leukemia

https://en.wikipedia.org/wiki/Wyeth

https://en.wikipedia.org/wiki/Pfizer

https://en.wikipedia.org/wiki/Food_and_Drug_Administration

https://en.wikipedia.org/wiki/European_Medicines_Agency

https://en.wikipedia.org/wiki/Philadelphia_chromosome

https://en.wikipedia.org/wiki/Chronic_myelogenous_leukemia

https://pubchem.ncbi.nlm.nih.gov/compound/Bosutinib


840


There is no single pharmacopeial monograph available for this drug substance or drug

product and no HPLC method is available in literature for quantification of Bosutinib

Monohydrate related substances. In this research paper, development of HPLC method for

the simultaneous detection and quantitative determination of the four impurities in Bosutinib

Monohydrate drug substance has been reported. The chemical structure of Bosutinib

Monohydrate Related substances Impurity-1 to Impurity-5 are shown in Figure 2.

Name Structure

Impurity-1

O–

N+

O

O

Cl

OCH3

Impurity-2 O

NH OCH3

ClCl

N

Impurity-3 2

3

4

5

1

6

7NO2

O

O

CH3

N

NCH3

8

9

10

1112

Impurity-4 2

3

4

5

1

6

7NH2

O

O

CH3

N

NCH3

8

9

10

11

13

12

Impurity-5

2

3

4

5

1

6

7NH

O

O

CH3

N

NCH3

NCO

NH

ClCl

OCH3

8

9

10

11

13

12

14

15

18

16

17

Figure 2: structure of Bosutinib Monohydrate Related substances Impurity-1 to

Impurity-5.


841


Limit of detection (LOD), limit of quantification (LOQ) and linearity were established as per

ICH guidelines. The limit of unknown impurity have been considered as 0.3% in accordance

with ICH guideline based on maximum daily dose.[8]

The developed chromatographic method

can resolve related substances with acceptable resolution to achieve good chromatography

and the optimized methodology have been validated to accomplish ICH guidelines on

validation.[9]

Bosutinib monohydrate related substances impurity-1 to impurity-5 properties

Impurity-1

Molecular Formula : C10H8Cl2N2O2

Molecular Weight : 259.089g/mol

Chemical Name: 2-Cyano-N-(2, 4-Dichloro-5-methoxyphenyl) Acetamide

Impurity-2

Molecular Formula : C10H12ClNO4


Chemical Name: 2-(3-Chloropropoxy)-1-methoxy-4-nitrobenzene.

Impurity-3

Molecular Formula : C15H23N3O4


Chemical Name: 1-[3-(2-methoxy-5-nitrophenoxy) propyl]-4-methylpiperazine

Impurity-4

Molecular Formula : C15H25N3O4


Chemical Name: 1-[3-(2-methoxy-5-aminophenoxy) propyl]-4-methylpiperazine

Impurity-5

Molecular Formula : C26H31N5O4Cl2


Chemical Name: {E}-2-cyano-{N}-(2, 4-dichloro-5-methoxyphenyl)-3-[4-methoxy-3-[3-(4-

methylpiperazin-1-yl) propoxy] anilino] prop-2-enamide

Instrumentation and Chromatographic conditions

A Thermo scientific Ultimate 3000 HPLC with PDA detector was used for separation and

detection of analyte. Data was collected using CHROMELION software. HPLC analysis was

performed on column Inertsil ODS 3V (250 x 4.6) mm, 5 m. Column temperature


842


maintained at 40°C conditions. Chromatographic separation was achieved with mobile phase

gradient program at flow rate of 0.7mL/min. the injection volume was 15μl. The UV

detection wavelength was 250nm.

Buffer preparation: 20mM Potassium Dihydrogen Orthophosphate. Adjust pH=7.0 ± 0.05

with triethylamine and mix well.

Mobile Phase-A: Buffer: Methanol (80:20) (V/V)

Mobile Phase-B: Methanol: Acetonitrile (65:35) (V/V)

Gradient programme

Time (min) Mobile Phase A (%) Mobile Phase B (%)

0.0 50 50

12.0 50 50

40.0 0 100

48.0 0 100

49.0 50 50

60.0 50 50

Preparation of solutions

Diluent: Water: Acetonitrile: Methanol (25:50:25) (V/V/V)

Standard stock solution: Accurately weigh and transfer about 50mg of BST working or

reference standard into 50mL volumetric flask. Add 25mL of diluent and sonicate to dissolve

and dilute up to mark with diluent. Further dilute 3.0mL of this solution into 100mL

volumetric flask and dilute up to mark with diluent and mix well.

Standard solution: Transfer 1.0mL of standard stock solution into 10mL volumetric flask

and dilute up to mark with diluent and mixed well.

Test solution: Accurately weigh and transfer about 50mg of test sample into 50mL

volumetric flask. Add 25mL of diluent and sonicate to dissolve and dilute up to mark with

diluent and mix well.

System suitability criteria

1. %RSD for Bosutinib peak area from six replicate standard and with bracketing standard

solution should not be more than 5.0%.

2. Theoretical plates for Bosutinib in standard solution should not be less than 2000.

3. Tailing factor for Bosutinib in standard solution should not be more than 2.0.


843


RT, RRT & RRF are mentioned in below table.

Name of content ~ RT (min) RRT RRF

Impurity-1 20.40 0.74 0.43

Impurity-2 11.13 0.41 0.64

Impurity-3 13.90 0.51 0.39

Impurity-4 6.45 0.24 0.26

Impurity-5 35.75 1.30 0.38

Bosutinib (BST) 27.84 1.00 --

Calculation

AT WS 3 1 50 1 P

%Known impurity= --------X--------- X -------- X-------X --------X---------X-------X 100

AS 50 100 10 WT RRF 100

AU WS 3 1 50 P

% Unknown impurity= --------X--------- X -------- X-------X --------X---------X 100

AS 50 100 10 WT 100

Total impurities = Sum of all known impurities + unknown impurities.

Where,

AT = Area of known impurity from test solution chromatogram.

AU = Area of unknown impurity from test solution chromatogram.

AS = Average area of six replicate of standard solution.

WS = Weight of working/ reference standard in mg.

WT = Weight of test sample in mg.

P = Potency of standard

RRF= Relative response factor.

RESULT AND DISCUSSION

Selection of detection wavelength

The sensitivity of RP-HPLC method with UV detection depends upon proper selection of

detection wavelength. An ideal wavelength is one that gives good response for the drugs that

are to be detected. BST showed maximum absorption at 250nm and it was selected as

detection wavelength for RP-HPLC analysis.

Optimization of mobile phase

Proper selection of HPLC method depends upon the nature of the sample (ionic, ionizable or

neutral molecule), its molecular weight and solubility. The drug selected for the present study

is polar in nature and hence either reversed phase or ion pair or ion-exchange


844


chromatography can be used. Reversed phase HPLC was selected for the initial separation

because of its simplicity and suitability. The optimization of mobile phase was to resolve

chromatographic peaks for active drug ingredients with less asymmetric factor. There were

many mobile phases that we tried to resolve peaks, in that first mobile phase-A with 20mM

KH2PO4, pH 2.5 and mobile phase-B with acetonitrile but impurity-3 peak was found gradient

pattern for BST. To improve these, mobile phase-A with buffer (20mM KH2PO4, pH 7.0):

Methanol (80:20 v/v) and mobile phase-B with methanol but all peaks were found very poor.

Further improve these combination of mobile phase were tried in that mobile phase-A with

buffer (20mM KH2PO4, pH 7.0): Methanol (80:20 v/v) and mobile phase-B with methanol:

acetonitrile (50:50 v/v), were tried but we found impurity-4 peak near dad volume and

baseline not proper. Further improve these combination of mobile phase were tried in that

mobile phase-A with buffer (20mM KH2PO4, pH 7.0): Methanol (80:20 v/v) and mobile

phase-B with methanol: acetonitrile (65:35 v/v), with gradient program was found to give a

good resolution and symmetric peaks for BST and all related substances as shown in Figure4.

Figure 4: Chromatogram of Blank and BST mix standard with mobile phase-A with

buffer (20mM KH2PO4, pH 7.0): Methanol (80:20 v/v) and mobile phase-B with

methanol: acetonitrile (65:35 v/v) gradient program.


845


Method validation

The proposed method was subjected to validation for various parameters like Specificity,

precision, LOD, LOQ, linearity and accuracy in accordance with international conference on

harmonization guidelines.

Specificity

Specificity is the ability to assessing unequivocally of analytic in the presence of components

which may be expected to be present. For determination of specificity, blank, all individual

related substances solutions were prepared and injected to confirm the individual retention

times. The solutions of BST drug substance (Control Sample) and BST spiked with known

related substances at specification level (Spiked Sample) were prepared and injected into

HPLC. Peak purity was established by using Chromeleon Software. A typical representative

HPLC chromatogram of BST drug substance spiked with all related substances is shown in

Figure 5. The specificity results are tabulated in Table 1.

Preparation

Standard stock solution: Weigh accurately about 10.0mg of Bosutinib Monohydrate

working or reference standard into a 100mL volumetric flask. Add diluent and sonicate to

dissolve and dilute up to mark with diluent.

Standard solution

Further dilute 1mL Standard Stock Solution into 100mL volumetric flask and dilute up to

mark with diluent mix well. (Concentration of Bosutinib Monohydrate i.e. to 1ppm).

Test and Test spike preparation describe in accuracy parameter. And all individual related

substances preparation from stock solution of precision parameter.


846


Figure 5: A typical representative HPLC chromatogram of BST drug substance spiked

with related substances.

Table 1: Specificity experiment results.

Name of content ~ RT (min) RRT Peak purity match

Impurity-1 20.40 0.74 968

Impurity-2 11.13 0.41 1000

Impurity-3 13.90 0.51 990

Impurity-4 6.45 0.24 999

Impurity-5 35.75 1.30 995

Bosutinib (BST) 27.84 1.00 994

BST Sample Spike - - 995

System suitability

System suitability test was an integral part of the method development to verify that the

system is adequate for analysis of BST to be performed. Six replicate injections of standard

preparation were injected, then asymmetry, number of theoretical plates and relative standard

deviation of peak area were determined. The system suitability results are shown in Table 2.

Preparation

As per specificity parameter.

Table 2: system suitability experiment results.

Standard BST Area

Injection -1 99.01

Injection -2 99.25

Injection -3 98.75

Mean 99.00

%RSD 0.25

Tailing factor 1.00

Theoretical plates 57284


847


Precision

The Repeatability of sample application was assessed by Transferring 1 mL from standard

stock solutions of BST into a 100 mL volumetric flask and diluted up to the mark with

diluent to get the concentration of 1μg/mL of BST, impurity-1, impurity-2, impurity-3,

impurity-4 and impurity-5. This standard solution was chromatographed for 60 minutes using

mobile phase gradient at a flow rate of 0.7mL/min for three times. All the Results were

reported in terms of % RSD. The precision preparation and experiments results are given in

Table 3.

Table 3: Precision preparation and experiment results.

Name Area

Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 BST

Injection -1 41.51 64.39 38.14 23.85 37.94 99.01

Injection -2 42.65 63.76 37.83 23.47 38.93 99.25

Injection -3 41.48 64.24 38.51 24.05 36.84 98.75

Mean 41.88 64.13 38.16 23.79 37.90 99.00

%RSD 1.59 0.51 0.89 1.24 2.76 0.25

LOD, LOQ

The limit of detection and limit of quantitation shall be determined based on Standard

deviation of response and slop value obtained from the linearity study of related substance for

impurity-1, impurity-2, impurity-3, impurity-4, impurity-5 and BST. LOD and LOQ values

are presented in Table 4.1 to Table 4.6.

Linearity

The linearity of analytical method is its ability to elicit test results that are directly

proportional to concentration of analyte in sample within given range. The linearity is

expressed in terms of correlation co-efficient of linear regression analysis. Prepare the

solution to gate concentration 0.5-10µg/mL for impurity-1, impurity-2, impurity-3, impurity-

Impurity

Name

Wt.

(mg)

Dilution

(mL)

Taken

mL

Dilution (mL)

(stock-2)

Taken

mL

Dilution

(mL)

Conc.

in ppm

BST 20.514 20 1

100 1 10

1.0257

Impurity-1 20.312 20 1 1.0156

Impurity-2 20.265 20 1 1.0133

Impurity-3 20.125 20 1 1.0063

Impurity-4 20.327 20 1 1.0164

Impurity-5 20.221 20 1 1.0111


848


4, impurity-5 and BST. The graph was plotted for peak area vs. conc. for the drug. The

statistical values are presented in Table 4.1 to Table 4.6 and Figure 6.

Table 4.1: Statistical evaluation of linearity and LOD/LOQ for Bosutinib.

ppm mL of stock-2 solution Dilution Actual. ppm Bosutinib

Area

0.500 0.5 10 0.5129 50.36

1.000 1.0 10 1.0257 99.25

2.000 2.0 10 2.0514 234.76

5.000 5.0 10 5.1285 521.47

10.00 Used as such stock-2 solution 10.2570 1047.20

Slope 101.7076

Intercept 4.6175

Correlation Coefficient (r2) 0.9991

STEYX 14.3802

LOD (ppm) 0.4666

LOQ (ppm) 1.4139

LOD (%) 0.0467

LOQ (%) 0.1414

Table 4.2: Statistical evaluation of linearity and LOD/LOQ for Impurity-1.

ppm mL of stock-2 solution Dilution Actual. ppm Impurity-1

Area

0.500 0.5 10 0.5078 23.31

1.000 1.0 10 1.0156 41.63

2.000 2.0 10 2.0312 89.98

5.000 5.0 10 5.0780 223.39


Slope 43.4592

Intercept 0.5205


STEYX 2.3796

LOD (ppm) 0.1807

LOQ (ppm) 0.5476

LOD (%) 0.0181

LOQ (%) 0.0548

RRF 0.43



Area

0.500 0.5 10 0.5066 33.06

1.000 1.0 10 1.0133 64.97

2.000 2.0 10 2.0265 133.75

5.000 5.0 10 5.0663 326.63



849


Slope 64.8324

Intercept 0.1687


STEYX 1.8302

LOD (ppm) 0.0932

LOQ (ppm) 0.2823

LOD (%) 0.0093

LOQ (%) 0.0282

RRF 0.64



Area

0.500 0.5 10 0.5031 20.31

1.000 1.0 10 1.0063 40.00

2.000 2.0 10 2.0125 80.63

5.000 5.0 10 5.0313 199.83


Slope 40.0127

Intercept -0.2307


STEYX 0.8470

LOD (ppm) 0.0699

LOQ (ppm) 0.2117

LOD (%) 0.0070

LOQ (%) 0.0212

RRF 0.39



Area

0.500 0.5 10 0.5082 11.35

1.000 1.0 10 1.0164 23.26

2.000 2.0 10 2.0327 50.44

5.000 5.0 10 5.0818 128.30


Slope 26.1598

Intercept -2.9684


STEYX 1.2411

LOD (ppm) 0.1566

LOQ (ppm) 0.4744

LOD (%) 0.0157

LOQ (%) 0.0474

RRF 0.26


850




Area

0.500 0.5 10 0.5055 20.51

1.000 1.0 10 1.0111 38.13

2.000 2.0 10 2.0221 76.26

5.000 5.0 10 5.0553 195.34


Slope 38.8701

Intercept -0.85333


STEYX 1.3541

LOD (ppm) 0.1150

LOQ (ppm) 0.3484

LOD (%) 0.0115

LOQ (%) 0.0348

RRF 0.38


851


Figure 6: Linearity plot for BST and Related substances Impurity-1 to Impurity-5.

Accuracy

The accuracy of the method was determined by analyzing BST samples spiked with related

substances at different levels (10%, 20%, 40%, 100% and 200% of specification levels). The

percentage recovery values for all the impurities are calculated and tabulated in Table 5.1 to

table 5.6.

Standard solution

As per specificity parameter.


852


Table 5.1: Accuracy preparation.

Impurity Name Wt. (mg) Dilution (mL) Taken

mL

Dilution (mL)

(Recovery stock)

Conc. in

ppm

Impurity-1 20.312 20 5

50

101.560

Impurity-2 20.265 20 5 101.325

Impurity-3 20.125 20 5 100.625

Impurity-4 20.327 20 5 101.635

Impurity-5 20.221 20 5 101.105

Table 5.2: Accuracy system suitability standard solution.

Standard BST Area

Injection -1 99.01

Injection -2 99.25

Injection -3 98.75

Mean 99.00

%RSD 0.25

Table 5.3: Accuracy for impurity-1.

Rec

overy

L

evel

Des

ign

ati

on

Wei

gh

t of

Sam

ple

(mg)

mL

of

Rec

ov

ery

stock

solu

tion

Dil

uti

on

(m

L)

Act

ual

Con

. (p

pm

)

Act

ual

Ad

ded

Am

ou

nt

(%)

Are

a

% R

esu

lt

Rec

overe

d

%R

esu

lt

Rec

overy

(%

)

As such Test 20.0

20

9.32 0.0225

10% 20.4 1.0 20 0.5078 26.39 26.39 0.0623 0.0398 78.38

20% 20.4 2.0 20 1.0156 44.98 44.98 0.1062 0.0837 82.41

40% 20.4 4.0 20 2.0312 89.7 89.7 0.2119 0.1894 93.25

100% 20.2 10.0 20 5.0780 216.35 216.35 0.5161 0.4936 97.20

200% 20.5 Used 10ppm soln. 10.1560 1.0156 436.98 1.0272 1.0047 98.93

Table 5.4: Accuracy for impurity-2.

Rec

overy

L

evel

Des

ign

ati

on

Wei

gh

t of

Sam

ple

(mg)

mL

of

Rec

ov

ery

stock

solu

tion

Dil

uti

on

(m

L)

Act

ual

Con

. (p

pm

)

Act

ual

Ad

ded

Am

ou

nt

(%)

Are

a

% R

esu

lt

Rec

overe

d

%R

esu

lt

Rec

overy

(%

)

As such Test 20.0

20

ND NA

10% 20.4 1.0 20 0.5066 0.0507 30.81 0.0489 0.0489 96.53

20% 20.4 2.0 20 1.0133 0.1013 54.66 0.0867 0.0867 85.56

40% 20.4 4.0 20 2.0265 0.2027 114.69 0.1820 0.1820 89.81

100% 20.2 10.0 20 5.0663 0.5066 328.63 0.5267 0.5267 103.96

200% 20.5 Used 10ppm soln. 10.1325 1.0133 652.51 1.0305 1.0305 101.70


853


Table 5.5: Accuracy for impurity-3. R

ecovery

L

evel

Des

ign

ati

on

Wei

gh

t of

Sam

ple

(mg)

mL

of

Rec

ov

ery

stock

solu

tion

Dil

uti

on

(m

L)

Act

ual

Con

. (p

pm

)

Act

ual

Ad

ded

Am

ou

nt

(%)

Are

a

% R

esu

lt

Rec

overe

d

%R

esu

lt

Rec

overy

(%

)

As such Test 20.0

20

ND NA

10% 20.4 1.0 20 0.5031 0.0503 17.21 0.0448 0.0448 89.05

20% 20.4 2.0 20 1.0063 0.1006 35.37 0.0921 0.0921 91.52

40% 20.4 4.0 20 2.0125 0.2013 75.64 0.1970 0.1970 97.89

100% 20.2 10.0 20 5.0313 0.5031 188.4 0.4955 0.4955 98.48

200% 20.5 Used 10ppm soln. 10.0625 1.0063 389.5 1.0095 1.0095 100.32

Table 5.6: Accuracy for impurity-4

Rec

overy

L

evel

Des

ign

ati

on

Wei

gh

t of

Sam

ple

(mg)

mL

of

Rec

ov

ery

stock

solu

tion

Dil

uti

on

(m

L)

Act

ual

Con

. (p

pm

)

Act

ual

Ad

ded

Am

ou

nt

(%)

Are

a

% R

esu

lt

Rec

overe

d

%R

esu

lt

Rec

overy

(%

)

As such Test 20.0

20

ND NA

10% 20.4 1.0 20 0.5082 0.0508 13.05 0.0510 0.0510 100.35

20% 20.4 2.0 20 1.0164 0.1016 25.58 0.0999 0.0999 98.29

40% 20.4 4.0 20 2.0327 0.2033 51.4 0.2008 0.2008 98.78

100% 20.2 10.0 20 5.0818 0.5082 128.52 0.5070 0.5070 99.77

200% 20.5 Used 10ppm soln. 10.1635 1.0164 259.9 1.0104 1.0104 99.41

Stability of solutions

Standard solution and sample solution spiked with impurities were prepared and analyzed

initially and at different time intervals by keeping the solutions at room temperature (~ 25°C).

Experimental results show that Standard solution is stable up to 24 hours at 25°C+2°C and

sample solution is also stable at 25°C+2°C.

CONCLUSION

A reverse phase stability indicating HPLC method was developed and validated for the

quantitative determination of related substances of Bosutinib Monohydrate. The present

research work will help the manufacturers and suppliers of Bosutinib Monohydrate to

quantify and qualify the quality in terms of purity based on experimental results. Thus, it can

be used for routine analysis, quality control and for determining quality during the stability

studies of pharmaceutical analysis.


854


ACKNOWLEDGEMENT

I am thankful to analytical development department of BDR Lifesciences Pvt. Ltd. for

providing me research facility for this project.

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