A New Analytical Method Validation and Quantification of Olmesartan Medoxomil and its related impurities in bulk drug product by HPLC

 

Tentu Nageswara Rao1*, A. Vijayalakshmi2, Karri Apparao1, N. Krishnarao1

1Department of Chemistry, Krishna University, Machilipatnam, Andhra Pradesh, India.

2Department of Marine Living Resources, Andhra University, Visakhapatnam, Andhra Pradesh, India.

*Corresponding Author E-mail: tnraochemistry@gmail.com

 

ABSTRACT:

A simple and inexpensive method was developed with high performance liquid chromatography with PDA detection for determination of olmesartan Medoxomil and its related impurities. The chromatographic separations were achieved on (250×4.6 mm), 5.0 µm make: Zorbax Eclipse XDB-C8 column employing 00.1% H3PO4 in Water: Acetonitrile in the ratio of 50:50 (v/v) as mobile phase with gradient initially A:B:70:30 and followed as Time/A/B: 25/30/70; 30/30/70; 31/75/25 and runtime is 35 mins at flow rate 1.0 mL/min was chosen. All impurities were eluted within 18 minutes. The column temperature was maintained at 30oC and a detector wavelength of 225 nm was employed.   The method was successfully validated by establishing Specificity, Linearity, Precision, Accuracy, Limit of detection and Limit of quantification.

 

KEY WORDS: HPLC, Method validation, related impurities, Olmesartan Medoxomil, LOQ, LOD.

 

 


INTRODUCTION:

Olmesartan Medoxomil is a typical antihypertensive drug used in the treatment of high blood pressure like problems. Olmesartan Medoxomil has the chemical name (5-methyl-2-oxo-2H-1,3-dioxol-4-yl)methyl 4-(2- hydroxypropan-2-yl)-2-propyl-1-({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl}methyl)-1H-imidazole-5-carboxylate. Olmesartan Medoxomil has a favorable safety and efficacy profile, with blood pressure-lowering effects comparable to those of other angiotensin receptor blockers (i.e. Losartan, Valsartan, Irbesartan)1. Its empirical formula is C29H30N6O6. Olmesartan Medoxomil is a white to light yellowish-white powder or crystalline powder with a molecular weight of 558.6. It is practically insoluble in water and sparingly soluble in methanol2.

 

Olmesartan Medoxomil is a novel selective angiotensin II receptor blocker that is approved for treatment of hypertension3,4,5. Olmesartan Medoxomil is the member of angiotensin receptor blocker approved by the Food and Drug Administration (FDA) for the treatment of hypertension6. The drug contains a medoxomil ester moiety and is cleaved rapidly by an endogenous esterase to release the active metabolite Olmesartan. Due to the fact that hydrolysis of Olmesartan in human plasma is extremely rapid. The drug works by inhibiting the effects of angiotensin II, a potent vasoconstrictor and one of the key contributors to cardiovascular and renal disease.

 

 


Sl.No

Compound Name

Structure

1.

(5-methyl-2-oxo-2H-1,3-dioxol-4-yl)methyl 4-(2- hydroxypropan-2-yl)-2-propyl-1-({4-[2-(2H-1,2,3,4- tetrazol-5-yl)phenyl] phenyl}methyl)-1H-imidazole-5- carboxylate (Olmesartan Medoxomil)

 

2.

Olmersartan acid (Impurity -1)

 

3.

Dehydro Olmesartan (Impurity-2)

 

4.

4-acetyl Impurity (Impurity-3)

 


 

MATERIAL AND METHODS:

Materials:

Standard gift samples of Olmesartan Medoxomil and impurities were provided by Dr Benarji Patrudu, Associate Professor, GITAM University, and Hyderabad. All the chemicals and reagents used were of analytical grade.

 

HPLC Chromatographic Parameters:

Chromatographic separation was performed on The HPLC-UV system used, consisted Shimadzu high performance liquid chromatography with LC- 20AT pump and SPD-20A interfaced with LC solution software, equipped with a reversed   phase C18 analytical column of 250 mm x 4.6 mm and particle size

 

5 µm (Zorbax Eclipse XDB-C8), Column oven temperature was maintained at 30°C and flow rate 1.0 mL/min. An HPLC method was developed for Olmesartan Medoxomil and related impurities by using photo diode array detector.  Olmesartan Medoxomil and all related impurities were injected into HPLC system by changing the different composition of 0.1% H3PO4 in Water: Acetonitrile in the ratio of 75:25 (v/v) initially. Time/A/B: 25/30/70; 30/30/70; 31/75/25 and runtime is 35 mins. The absorption maxima for Olmesartan Medoxomil, Impurity –1, Impurity-2 and Impurity – 3 were found to be at 225 nm and the compound was scanned form 200 – 400 nm. Injection volume was set to 20µL. Diluent was Acetonitrile: Water (50:50, v/v). By follow this analytical method conditions, Olmesartan Medoxomil and related impurities were separated. Hence, it was concluded that HPLC method was suitable for method validation.

 

Method Validation:

Specificity:

The specificity will be confirmed comparing the chromatogram of the blank run to the chromatogram of the single impurity run. Then a solution containing a mix of impurities and Olmesartan Medoxomil will be injected.

 

The Olmesartan Medoxomil peak has to be separated and has to meet the suitability parameters.

 

Preparation of Specificity Solution:

Olmesartan Medoxomil: Weigh 20.0 mg of the olmesartan Medoxomil in a100 mL volumetric flask, dissolved and diluted upto the mark with diluents. (Sol A – 200µg/mL).

 

From the above stock solution, 1 ml taken into a 100 ml volumetric flask and diluted to volume with the diluents (Sol B-2.0µg/ml) and injected into HPLC.

 

Impurity-1: Weigh 20.0 mg of the Impurity-1 in a100 mL volumetric flask, dissolved and diluted upto the mark with diluents. (Sol A – 200µg/mL).

 

From the above stock solution, 1 ml taken into a 100 ml volumetric flask and diluted to volume with the diluents (Sol B-2.0µg/ml) and injected into HPLC.

 

Impurity-2: Weigh 20.0 mg of the Impurity-2 in a100 mL volumetric flask, dissolved and diluted upto the mark with diluents. (Sol A – 200µg/mL).

 

From the above stock solution, 1 ml taken into a 100 ml volumetric flask and diluted to volume with the diluents (Sol B-2.0µg/ml) and injected into HPLC.

 

Impurity-3: Weigh 20.0 mg of the Impurity-3 in a100 mL volumetric flask, dissolved and diluted upto the mark with diluents. (Sol A – 200µg/mL).

 

From the above stock solution, 1 ml taken into a 100 ml volumetric flask and diluted to volume with the diluents (Sol B-2.0µg/ml) and injected into HPLC.

 

Selectivity Solution:

From the solutions A, prepared above 1ml from each solution was pipetted into a 100ml flask and brought to volume with dissolution phase. The latter solution was injected 6 times.

 

 

Linearity:

Preparation of Standard Stock Solution:

Weighed 20 mg of Olmesartan Medoxomil, Imp 1, Imp 2 and Imp 3 in a 100ml volumetric flask and brought to volume with dissolution phase (Sol A – 200mg/ml).

From the above solution (Solution A – 200mg/ml), dilutions were follows in Table.1

 

Table.1 Serial dilutions for linearity solutions

% of solution

Volume taken (mL)

Volume made up to (mL)

20

0.2

100

50

0.5

100

100

1.0

100

150

1.5

100

200

2.0

100

 

The above solutions were injected into HPLC in three replications. Correlation coefficient was calculated for A.I and impurities by plotting the graph between concentrations versus peak Area.

 

Precision:

The Precision will be determined in agreement with ICH guidelines, six different test solutions obtained from the homogeneous sample of impurities and Olmesartan Medoxomil were injected.

 

Precision Solution:

Weigh 20.0 mg of  Olmesartan Medoxomil, Imp 1, Imp 2 and Imp 3 in a100 mL volumetric flask, dissolved and diluted upto the mark with diluents. (Sol A –200µg/mL of Olmesartan Medoxomil, Imp 1, Imp 2 and Imp 3).

 

From the above stock solution, 1.0 ml taken into a 100 ml volumetric flask and diluted to volume with the diluents (Sol B- 2µg/mL for Olmesartan Medoxomil, Imp 1, Imp 2 and Imp 3) and injected into HPLC.

 

Accuracy:

The Accuracy will be determined as prescribed by ICH guidelines. Known quantities of impurities were added to Olmesartan Medoxomil at 50 –100 –150% of the nominal limit (0.1%) for Imp 1, Imp 2 and Imp 3. The results obtained have to meet the proposed limits.  The scheme to carry out was applied to every impurity.

 

Preparation of Test Solution:

Prepare a solution containing all the impurities at a concentration of 200 µg/ml each (10 mg/100 ml; solution from the linearity test can be used). Transfer respectively 0.5 ml, 1 ml and 1.5 ml of this solution to three different 100 ml volumetric flasks, containing 100 mg of  Olmesartan Medoxomil hydrochloride each one. The dilutions have to be carried out for each solution of the linearity test (total: 3x3 test solutions)

LOD and LOQ:

LOD and LOQ were assessed in accordance with ICH guidelines. The method chosen was based on the visual inspection of the linearity graphs for impurities at 0.1% level of  a, b, c, d, e, f  and benazepril and the signal to noise ratio, using the following formulas:

 

LOD=

      3.3 x s

 

LOQ=

    10 x s

         S

 

       S

 

RESULTS AND DISCUSSIONS:

Specificity and Selectivity:

The specificity was confirmed comparing the chromatogram of the blank run to the chromatogram of the single impurity run. Then a solution containing a mix of impurities and Olmesartan medoxomil was injected.

The Olmesartan Medoxomil peak has to be separated and has to meet the suitability parameters.

 

Linearity:

The linearity regression curve for Olmesartan Medoxomil and their impurities were drawn between concentrations and peak areas7,8,9. The correlation coefficient is above 0.99 at wavelength of 225 nm for Olmesartan medoxomil hydrochloride and their impurities. The results are mentioned in Table 2. A calibration curves were showed in Figure 1.

 

 

 

Table 2. Linearity Data of Olmesartan medoxomil and Impurities

 

Olmesartan Medoxomil

Imp 1

Imp 2

Imp 3

20%

29621

43444

20442

27532

50%

71193

93939

49136

65720

100%

142456

184887

98925

131745

150%

209505

276554

145830

194166

200%

280586

361145

194332

260555

Slope

69585.08

88953.69

48306.47

64625.63

Intercept

1935.24

6970.12

1255.53

1522.293

correlation coefficient

0.999

0.999

0.999

0.999

 

 

 

Precision:

The precision test was carried out with six homogenous solution of Olmesartan medoxomil test item and the content of  Olmesartan medoxomil  and their impurities were calculated. The results are mentioned in Table 3. 

 

 

 

 

 

Accuracy:

Preparation of Test solutions:

The accuracy test was carried out in three different levels and the percent recovery of Olmesartan medoxomil and their impurities were calculated. The representative chromatogram showed in Figure 2 and results were presented in Table 3.

 

Figure 1. Linear regression curve of Olmesartan medoxomil  and  its impurities

 

Table 3. Precision of Olmesartan medoxomil  and Impurities

Replication

Olmesartan Medoxomil

Imp 1

Imp 2

Imp 3

1

140258

177846

107734

131007

2

140989

179150

106251

135404

3

141305

180204

108111

132718

4

143447

176901

108499

134948

5

143992

185348

108638

134267

6

140297

180423

106179

130680

Average

141714.7

179978.7

107568.7

133170.7

STDEV

1613.3

2959.2

1095.3

2021.8

RSD

1.1

1.6

1.0

1.5

 

Table 4. Recovery results of benazepril and its impurities

%

Recovery in %

Imp A

Imp B

Imp C

Imp D

50

99.12

95.26

98.12

97.77

50

99.24

95.33

98.46

97.71

50

99.13

95.24

98.32

97.68

100

99.25

95.41

98.25

97.81

100

99.31

95.48

98.33

97.78

100

99.33

95.38

98.42

97.84

150

99.22

95.42

98.44

97.89

150

99.28

95.39

98.38

97.79

150

99.27

95.36

98.42

97.83

 


 

Figure. 2. Representative chromatogram of 100 % fortification level of impurities

 

 


LOD and LOQ:

The LOD and LOQ are established successfully for each impurity in  Olmesartan medoxomil  and its impurities based on Signal-to-noise ratio method7, 8, 9. The results were presented in Table 5.

 

Table 5. Limit of quantification and Limit of detection results of olmesartan medoxomil  and its impurities

Impurity

S/N Obtained

 

%

Impurity-1

10.7

LOD

0.0009

LOQ

0.0030

Impurity-2

10.0

LOD 

0.0007

LOQ

0.0022

Impurity-3

11.1

LOD

0.001

LOQ

0.0033

 

Calculations:

The Olmesartan medoxomil and impurities assay is determined by comparison of peaks areas with the following formula:

 

Percentage Benazepril impurity

=

At x C x D x PS

X 100%

Ar x W sample x Rf

 

where:

At: Peak area of impurity obtained by test solution

Ar: Peak area of Olmesartan medoxomil obtained by Standard solution

C: Olmesartan medoxomil concentration in Standard solution (mg/mL)

D: Sample dilution (mL)

W Sample: sample weight in test solution (mg)

PS: Purity of reference standard

Rf: Response factor of impurity

PS: Purity of reference standard

Fc: Response Factor of Impurity

 

% Recovery

=

Recovered Concentration

×

100

Fortified Concentration

 

 

CONCLUSIONS:

The method developed for quantitative determination of Olmesartan medoxomil and its impurities is rapid, precise, accurate and selective. The method was completely validated showing satisfactory data for all method - validated parameters tested. The mobile phase composition water showed good separation and resolution. Satisfactory validation parameters such as linearity, precision, Accuracy, LOD and LOQ were established by following ICH guidelines10. Therefore, the proposed analytical procedure could be useful for regular monitoring, pharma manufacturing labs and research scholars.

 

ACKNOWLEDGEMENT:

The authors are thankful to the Dr. Benerjee Patrudu, GITAM University, Hyderabad for providing the gift sample of Olmesartan medoxomil and providing necessary facilities to carry out the research work with keen interest and help.

 

REFERENCES:

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2.            Kuldeep Singh, Anirbandeep Bose, Gurubasavaraja Swamy PM, Divakar Goli. Method development and validation of simultaneous analysis of Olmesartan Medoxomil and hydrochlorothiazide by UV and HPLC, their cross validation. World Journal of Pharmacy and Pharmaceutical Sciences. 2015; 4(7): 905-917.

3.            Shailesh T. Prajapati, Hitesh H. Bulchandani, Dashrath M. Patel, Suresh K. Dumaniya, Chhaganbhai N. Patel. Formulation and Evaluation of Liquisolid Compacts for Olmesartan Medoxomil. Hindawi Publishing Corporation Journal of Drug Delivery. 2013; 2013: 1-9.

4.            A.T. Hemke, M.V. Bhure, K.S. Chouhan, K.R. Gupta, S.G. Wadodkar. UV Spectrophotometric Determination of Hydrochlorothiazide and Olmesartan Medoxomil in Pharmaceutical Formulation. E-Journal of Chemistry. 2010; 7(4): 1156-1161.

5.            Abdullah A Masud, Md. Mahfuzur Rahman, Moynul Hasan, Md. Kamal Hossain Ripon, Ahsanur Rahman Khan, Md. Rabiul Islam, Md. Raihan Sarkar. Validated Spectrophotometric Method for Estimation of Olmesartan Medoxomil in Pharmaceutical Formulation. International Journal of Pharmaceutical and Life Sciences. 2012; 1(3): 1-7.

6.            Chimalakonda Kameswara Rao1, , Kakumani Kishore Kumar , Maddala Vijaya Laxmi, Polisetty Srinivasulu, Gutta Madhusudhan, Khagga Mukkanti, Koduri Sai Venkata Srinivas. Development and Validation of Stability Indicating LC Method for Olmesartan Medoxomil. American Journal of Analytical Chemistry. 2012;3:153-160.

7.            G. Kumar, T.B. Patrudu, Tentu Nageswara Rao, M.V. Basaveswara Rao, A New Analytical Method Validation and Quantification of Benazepril and its Related Substance in bulk Drug Product by HPLC, Asian Journal of Pharmaceutical Analysis, 2017; 7(1): 1-5.

8.            G. Kumar, T B. Patrudu, M.V. Basaveswara Rao and Tentu. Nageswara Rao, A Novel Method Development and Validation for Related Substances of Adapalene in Bulk Drug Product by HPLC, Research J. Pharm. and Tech 2016; 9(12):2234-2240.

9.             G. Kumar, T. B. Patrudu, Tentu. Nageswara Rao, M. V. Basaveswara Rao, A new analytical HPLC method for cleaning validation of pantoprazole sodium bulk drug product,   Indo American Journal of Pharmaceutical, Research.2016:6(10), 6584-6593.

10.          International Conference on Harmonisation, Validation of Analytical Procedures. ICH Q2B.  1996.

 

 

 

 

 

 

Received on 23.05.2017       Accepted on 28.07.2017      

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech.  2017; 7 (3): 147-152.

DOI: 10.5958/2231-5713.2017.00024.1