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Original Article

Determination and characterization of process impuritiesfor Eprosartan mesylate

Sitaram Cheekatla b,c, Ravichandrababu Rupakula a,*, Bommineni Narasimha Reddy c,Musty Sharada b

aAssociate Professor, Department of Chemistry, Gitam Institute of Science, Gitam University, Visakhapatnam,

Andhra Pradesh 530 045, IndiabDepartment of Chemistry, Gitam Institute of Science, Gitam University, Visakhapatnam, Andhra Pradesh 530 045, IndiacAnalytical Research and Development Department, Ogene Systems (I) Pvt Ltd, GSR Estates, Balanagar, Hyderabad 018,

Andhra Pradesh, India

a r t i c l e i n f o

Article history:

Received 26 March 2013

Accepted 25 April 2013

Available online 20 May 2013

Keywords:

Characterization

Determination

Development

Eprosartan mesylate

Validation

* Corresponding author.E-mail address: rrcbabu7@yahoo.in (R. Ru

0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.04.033

a b s t r a c t

Aim: The present work aim to develop a novel, sensitive and selective HPLC method for the

determination of process impurities of Eprosartan mesylate Drug substance (EPM) and

characterization of impurities using Mass Spectrometry and NMR.

Methods: EPM and its impurities were determined by Agilent 1200 series HPLC with PDA

detector. A phenomenex GeminieC18 (250 mm � 4.6 mm � 5.0 mm) column was employed

for the separation of impurities from EPM. The mobile phase consists of 10 Mm Ammo-

nium acetate buffer (pH to 3.0) with acetic acid as solventeA and Acetonitrile as solventeB

in gradient programme.

Results: All the impurities were well resolved from one another and EPM peak indicating the

specificity of the proposed method to quantify EPM and its four impurities. Precision,

method and intermediate precision for EPM was checked at specification level and the %

RSD was found to be 0.36, 0.29 and 0.52.

Conclusion: The developed HPLC method was found to be simple, sensitive, and selective.

Detection limit for impurities was found to be as low as 0.01% and was found to have

excellent resolution for four impurities indicating high sensitivity and selectivity of the

validated method.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction elicit a higher reduction in systolic blood pressure than other

Eprosartan mesylate (EPM) is chemically monomethane sulfo-

nate of (E)-2-butyl-1-(p-carboxybenzyl)-a-2-thienylmethylimida

zole-5-acrylicacid (Fig. 1) is a new antihypertensive agent as an

angiotension II receptor antagonist that is highly selective to

pakula).2013, JPR Solutions; Publi

antihypertensive drugs.1,2 The drug acts on the renin-

angiotension system in two ways to decrease total peripheral

resistance. First, it blocks the binding of angiotension II to AT1

receptors in vascular smooth muscle, causing vascular dilata-

tion.Second, it inhibitssympatheticnorepinephrineproduction,

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

1

2

3

4 5

N6

N7

8

910

11

(E)

12

13

14

15

16

S17

COOH

1819

2021

22

23 24

HOOC25

H

26

1

2

3

4 5

N6

N7

8

910

11

(E)

12

13

14

15

16

S17

1819

2021

22

23 24

H

1

2

3

4 5

N6

N7

8

910

11

(E)

12

13

14

15

16

S17

COOCH3

1819

2021

22

23 24

H3COOC25

H

26

1

2

3

4 5

N6

N7

8

910

11

(E)

12

13

14

15

16

S17

COOCH3

1819

2021

22

23 24

H

25

1

2

3

4

5

N6

N7

8

910

11

(E)

S

COOH

H

12

18

16

13

14

15

17

Eprosartan mesylate

.HSO3CH3

DEE DME

EME

1920

25

24

23

22

21

HOOC26

EPI

O

O

25

26

O

O

29

30

27

27 28

O

26

O

27

28

29

28

Fig. 1 e Process related impurities of (E) Eprosartan mesylate.

j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 5 0 4e5 0 9 505

further reducing blood pressure.3,4 A very few spectrophoto-

metric methods5e7 and HPLC, LCeMS methods in different

matriceshavebeen reported for thedeterminationof Eprosartan

in literature.8�12

Literature survey reveals that no reference exists for the

quantitative determination and characterization of process

impurities of EPM drug substance. Presence of impurities in

drug substance can have significant impact on the quality,

safety and efficacy. Hence it was felt necessary to develop an

accurate, rapid, selective and sensitive method for the deter-

mination of EPM and its process impurities. The newly

developed method was validated according to ICH guide-

lines13,14 considering four impurities to demonstrate speci-

ficity, precision, linearity and accuracy of the method.

j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 5 0 4e5 0 9506

2. Materials and methods

2.1. Chemicals and reagents

The investigated samples EPM and its Process impurities were

supplied by Ogene Systems (I) Pvt. Ltd., Hyderabad, India. The

HPLC grade acetonitrile, methanol, ortho phosphoric acid and

Ammonium acetate were purchased from Merck Specialty

Chemicals, India. Water used was obtained by Milli Q water

purification system.

2.2. HPLC method

EPM and its impurities were determined by Agilent 1200 series

HPLC with PDA detector (Agilent Technologies, Deutschland,

Waldron, Germany) instrument with EZ-Chrome elite soft-

ware.AphenomenexGeminieC18 (250mm� 4.6mm� 5.0 mm,

Phenomenex, Torrance, CA, USA) column was employed for

the separation of impurities from EPM. Separation was ach-

ievedusingagradientmobilephase10mMammoniumacetate

inwater. pH is adjusted to 3.0withacetic acid as solventeAand

Acetonitrile as solventeB in gradient mode (Time/Sol-A: B)

0e5/80: 20, 9/60: 40, 17e28/15: 85, 32e35/80: 20 (v/v). The flow

rate of the mobile phase was set to 1.0 mL/min with detected

wavelength fixed at 250 nm. The injection volume was 10 ml.

Methanol was used as diluent.

2.3. Mass and NMR spectroscopy

The LCeMS/MS analysis has performed onQuattroMicro�API

mass spectrophotometer (Waters, Seoul, Korea). The analysis

was performed in the scan mode with electrospray ionization

source (ESþ) and triple Quadrapole mass analyzer. The anal-

ysis parameters for capillary, cone voltage were 3.50 kV and

Minute

0 2 4 6 8 10 12 14 16 18

mA

U

-50

-25

0

25

50

75

100

125

150

175

200

EP

M

EP

I

DM

E

Fig. 2 e HPLC chromatogram 0.15% impurities ble

25 V, respectively. Source, dessolvation gas temperatures

were 95 �C and 350 �C, dessolvation gas flow fixed at 450 L/h.

The mass spectrum data was processed by using Mass Lynx

software. The 1H and 13C NMR experimentswere performed in

DMSO at 25 �C temperature using mercury plug 300 MHz FT

NMR spectrometer, Bruker, Bio Spin Corporation, Billerica,

MA, USA. The 1H and 13C chemical shifts were reported on the

d scale in ppm relative to tetra methyl silane and DMSO,

respectively.

2.4. Preparation of stock solutions

1.0mg/mL EPMwas prepared by dissolving 10.0mg in 10mL of

diluent for determination of purity. 0.15% impurities blend

solution was spiked w.r.t. 1 mg/mL of EPM was prepared in

diluent (Fig. 2) (Methanol was used as the diluent).

2.5. Method development

The main target of the method is to identify the possible

process impurities and get well resolutions between EPM and

its process impurities. The blend solution of 0.15% EPM pro-

cess impurities was prepared by spiking to 1.0mg/mL EPM test

solution and it was run through C18 column with phosphate

buffer in the pH range of 3.0e6.0 along with acetonitrile. Best

results were achieved using phenomenex GeminieC18

(250 mm � 4.6 mm � 5.0 mm) column.

2.6. Solution stability

The solution stability of EPM and its impurities in diluents

were determined by leaving 0.15% spiked sample solution in a

tightly capped volumetric flask at room temperature for 48 h

and measuring the amounts of the compounds for every 12 h

and comparing the results with those obtained from freshly

s

20 22 24 26 28 30 32 34

mAU

-50

-25

0

25

50

75

100

125

150

175

200

EM

E

DE

E

nd solution spiking to Eprosartan mesylate.

Table 1 e Regression weights for EPM process impurities.

Concentration(%)

DEE DME EME EPI

Peakarea

Intercept r2 Peakarea

Intercept r2 Peakarea

Intercept r2 Peakarea

Intercept r2

0.03 (QL) 123696 11657 0.994 117030 5502.2 0.994 121708 23574 0.995 117308 13686 0.998

0.04 164870 148186 184870 173876

0.075 287741 265632 267541 286841

0.110 401611 450125 401611 401611

0.150 618482 585154 608542 586542

0.187 762352 723428 712406 708306

0.220 873225 845115 843123 843123

0.260 958903 926851 956403 976211

0.300 1158964 1161602 1088964 1114524

j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 5 0 4e5 0 9 507

prepared solution. The % RSD values for were found to be 0.98

and 0.93 respectively. All the samples were found to be stable

up to 48 h.

Table 2 e % Recovery study for EPM process impurities.

Parameter DEE DME EME EPI

LOD 0.011 0.013 0.012 0.014

LOQ 0.036 0.034 0.038 0.035

Precision (% RSD) at LOQ 0.85 0.91 0.86 0.91

Intermediate precision at

LOQ (% RSD)

1.25 1.19 1.27 1.18

Accuracy (% recovery) at

LOQ 99.34 99.65 99.43 99.78

100% 99.52 99.32 99.69 99.53

150% 99.81 99.57 99.48 99. 27

3. Results and discussion

3.1. Method validation

The present method is validated as per ICH guidelines. The

impurities mixture solution 0.15% was injected and the limit

of detection (LOD) and the limit of quantification (LOQ) values

were determined at the lowest concentrations at which

signal-to-noise ratio is 3 and 10, respectively. LOD and LOQ

values for all the impurities were found to be 0.01% and 0.03%

respectively. Linearity test solutions for impurities were pre-

pared individually at six concentration levels in the range of

LOQ to 200% of the specification level viz. 0.15%. The peak area

versus concentration data was subjected to least-squares

linear regression analysis (Table 1). System precision and

precision of themethod for EPM at specification level i.e. 0.15%

impurities spiked EPM was determined by analyzing six

replicate injections and the relative standard deviation was

calculated for each impurity. Precision at LOQ is also deter-

mined by injecting individual preparations of EPM spiked at

LOQ level of its impurities. The intermediate precision of the

method was also verified on six different days in the same

laboratory using the specification and LOQ levels. The % RSD

values for intermediate precision were found to be 0.52 and

1.2, respectively. The percentage recovery of all impurities in

drug substance has been calculated and the percentage it is

found to be within the range as per ICH. The low% RSD values

via peak areas confirm the good precision of the developed

method. The recovery experiments were conducted to deter-

mine the accuracy of EPM impurities for their quantification.

The study was carried out in triplicate at LOQ, 100% and 150%

with respect to specification level viz. 0.15%. The recovery data

presented in (Table 2) indicates the accuracy of the method

The robustness was illustrated by getting the resolution be-

tween any two compounds to be greater than 2.0, when mo-

bile phase flow rate (�0.2 mL/min), wavelength (�2 nm) and

column temperature (�2 �C) were deliberately varied. The

specificity of the developed method was checked in the

presence of its process impurities. All the impurities werewell

resolved from one another and EPM peak indicating the

specificity of the proposed method to quantify EPM and its

four impurities.

4. Characterization of impurities

4.1. LCeMS/MS method

LCeMS/MS analysis of EPM process related impurities iden-

tified as (ethyl 4-[(5-((E)-2-(ethoxy carbonyl)-3-(thiophen-

2-yl)prop-1-enyl)-2-butyl-1H-imidazol-1-yl)methyl]benzoate)

DEE, (methyl 4-[(5-((E)-2-(methoxy carbonyl)-3-(thiophen-2-

yl)prop-1-enyl)-2-butyl-1H-imidazol-1-yl)methyl]benzoate)

DME, (ethyl 4-((5-((E)-2-(methoxy carbonyl)-3-(thiophen-2-yl)

prop-1-enyl)-2-butyl-1H-imidazol-1-yl)methyl)benzoate) EME

and (4-((2-butyl-4-((E)-2-carboxy-3-(thiophen-2-yl)prop-1-enyl)-

1H-imidazol-1-yl)methyl)benzoic acid) EPI (Fig. 1). DEE shows

nominal molecular ion peak as [M þ H]þ in electron spray

positive ionization at m/z 481 and DME atm/z 453. EME and EPI

shows m/z nominal molecular ion peak as [M þ H]þ and as

sodium adduct [M þ Na]þin electron spray positive ionization

mode at m/z 467, 489 and 425 respectively. Based on this mass

spectral data these impurities are identified as process related

impurities of EPM.

4.2. Structural elucidation (NMR spectroscopy)

The chemical shift assignments, the results of 1HNMR and the13C NMR spectrum of the four impurities were briefly showed

in Table 3.

Table 3 e 1H NMR and 13C NMR chemical shift assignments for EPM process impurities.

DEE DME EME EPI

Carbon. No 1H (ppm)/J ¼ Hz 13C Carbon. No 1H (ppm)/J ¼ Hz 13C Carbon. No 1H (ppm)/J ¼ Hz 13C Carbon. No 1H (ppm)/J ¼ Hz 13C

1 (3H) t/0.79 (J ¼ 18, 2.4) 14.13 1 (3H) t/0.79 (J ¼ 14.4, 7.2) 13.51 1 (3H) t/0.78 (J ¼ 18, 2.4) 14.15 1 (3H) t/0.78 (J ¼ 14.4, 7.2) 13.42

2 (2H) m/1.59 21.68 2 (2H) m/1.32e1.24 25.11 2 (2H) m/1.42e1.40 23.39 2 (2H) m/1.37e1.21 24.07

3 (2H) m/1.90 29.24 3 (2H) m/1.58e1.51 28.84 3 (2H) m/1.57e1.56 29.04 3 (2H) m/1.60e1.49 27.76

4 (2H) m/2.66 28.27 4 (2H) m/2.89e2.84 28.04 4 (2H) m/2.87e2.84 28.15 4 (2H) m/2.830 21.477

5 e 151.51 5 e 150.60 5 e 151.05 5 e 148.60

8 (1H) s/7.4 124.02 8 (1H) s/7.66 124.33 8 (1H) s/7.58 124.17 8 (1H) s/7.45 124.12

9 e 131.64 9 e 141.20 9 e 137.92 9 e 129.88

10 (1H) s/6.8 129.11 10 (1H) s/7.4 128.31 10 (1H) s/7.63 128.71 10 (1H) s/7.96 130.75

11 e 129.68 11 e 136.24 11 132.96 11 133.11

12 (2H) s/3.99 28.2 12 (2H) s/4.0 28.82 12 (2H) s/4.12 28.51 12 (2H) s/4.169 28.07

13 e 140.99 13 e 138.12 13 e 139.55 13 e 139.57

14, 16 (2H) m/7.14e7.7 126.30

125.57

14 (1H) m/7.31e7.30 126.90 14, 16 (2H) m/7.28e7.34 126.66

126.23

14, 16 (2H) m/6.88e6.85 124.29

122.91

15 (1H) t/6.9 (J ¼ 9) 126.09 15 (1H) m/6.73e6.72 126.62 15 (1H) t/6.7 (J ¼ 9) 126.35 15 (1H) m/7.27e7.26 125.29

e e e 16 (1H) m/6.91e6.89 124.97 e e e e e e

17 e e e e e e e e e e e

18 (2H) s/5.44 45.92 18 (2H) s/5.44 46.61 18 (2H) s/5.43 48.86 18, 26 (2H) s/13.081 167.69

165.86

19 e 142.46 19 e 140.19 19 e 141.32 19 (2H) s/5.46 49.79

20, 24 (2H) m/(7.33e7.29) 129.67

129.11

20, 24 (2H) d/7.93 (J ¼ 8.1) 129.56

129.23

20, 24 (2H) m/(7.38e7.35) 128.73

128.50

20 e 140.02

21, 23 (2H) d/7.96 (J ¼ 9) 126.63

126.84

21, 23 (2H) d/7.19 (J ¼ 8.1) 127.94

127.83

21, 23 (2H) d/7.98 (J ¼ 9) 128.19

127.99

21, 25 (2H) d/7.34 (J ¼ 7.8) 127.67

127.74

25 e 165.33 22 e 127.12 25 e 166.21 22, 24 (2H) d/7.94 (J ¼ 7.8) 129.92

129.91

26 (2H) q/4.10 (J ¼ 15.6) 60.54 25 e 166.72 26 (3H) t/1.15 (J ¼ 15.6) 51.84 23 e 127.72

27 (3H) t/1.12 (J ¼ 15.6) 13.99 26 (3H) s/3.663 52.34 27 e 167.64 e e e

28 e 167.67 27 e 166.74 28 (2H) q/4.15 (J ¼ 15.6) 60.64 e e e

29 (2H) q/4.31 (J ¼ 15.6) 60.75 28 (3H) s/3.84 52.26 29(3H) t/1.15 (J ¼ 15.6) 13.81 e e e

30 (3H) t/1.25 (J ¼ 15.2.4) 13.63 e e e e e e e e e

Note: For carbon numbers refer Fig. 1.

journalofpharmacy

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j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 5 0 4e5 0 9 509

5. Conclusion

A convenient, rapid, accurate and precise HPLC method has

been developed for estimation of EPM drug substance along

with four unknown impurities. Detection limit for impurities

was found to be as low as 0.01% and was found to have

excellent resolution indicating high sensitivity and selectivity

of the validated method.

Conflicts of interest

All authors have none to declare.

Acknowledgment

The authors wish to thank Dr. B M Choudary, Managing di-

rector, Ogene Sys (I) Pvt Ltd, Hyderabad for providing facilities.

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