Development and Validation of Stability Indicating high performance liquid chromatographic Method for Olmesartan medoxomil and Indapamide in Tablet

 

Rucha A Patel1*, Meghna P. Patel1, Hasumati A. Raj1, Nehal Shah2

1Department of Quality Assurance, Shree Dhanvantry Pharmacy College, Kim, Surat, Gujarat, India

2 Dharmaj Degree of Pharmacy, Dharmaj, Anand, Gujarat, India

*Corresponding Author E-mail: ruchajigar6114@gmail.com

 

ABSTRACT:

An approach of forced degradation study was successfully applied for the development of a stability-indicating high performance liquid chromatographic method for simultaneous determination of Olmesartan medoxomil and Indapamide in a formulation in the presence of its degradation products. In the present study a simple, accurate and precise reverse phase liquid chromatographic method has been developed and validated for simultaneous estimation of Olmesartan medoxomil and Indapamide in tablet dosage form. Developed Method was achieved on symmetry C18 (150 mm × 4.6 mm, 5 μ) column using a Acetonitrile: 0.02 M Na2HPO4 (45:55 v/v) mobile phase and pH 7 adjusted with ortho phosphoric acid. Isocratic elution mode at a flow rate of 1.0 ml/min at Room temperature with a load of 20μl Injection volume. The detection was carried out at 240 nm. The linearity of the proposed method was investigated in the range of 50-250 microg/mL (r2 = 0.998) for Olmesartan medoxomil and 10-50 microg/mL (r2 = 0.998) for Indapamide. The retention time of Olmesartan medoxomil and Indapamide were found to be around 4.79 min and 7.59 min respectively. The drug substances were subjected to stress conditions of acid hydrolysis, base hydrolysis, Oxidative, photolytic and thermal. The developed RP-HPLC method was validated with respect to linearity, accuracy, precision, robustness, LOD and LOQ.

 

KEY WORDS: Olmesartan medoxomil, Indapamide, Hypertension, stability study.

 

 

INTRODUCTION:

Olmesartan medoxomil and Indapamide combination is used in cardiac disease condition like Hypertension. Olmesartan medoxomil is a new orally active Angiotensin II type 1 receptor antagonist used as an anti-hypertensive agent(1). It is a prodrug and is rapidly de-esterified during absorption to form olmesartan, the active metabolite(2). Olmesartan medoxomil [Figure 1] (a prodrug, which is hydrolyzed in body active olmesartan during absorption from the gastrointestinal tract) is chemically, 2, 3-dihydroxy‑2-butenyl 4-(1-hydroxy-1- methylethyl)-2-propyl-1-[p-(o‑1H-tetrazol-5-ylphenyl) benzyl] imidazole‑5‑carboxylate, cyclic‑2,3‑carbonate. Indapamide is an orally administered diuretic and anti‑hypertensive drug.

 

Its molecule contains both a polar sulfamoyl chlorobenzamide moiety and a lipid soluble methyl‑indoline moiety.(3) Indapamide [Figure 2]  is chemically 3-(aminosulfonyl)-4-chloro-N-(2, 3-dihydro-2-methyl-1H-indol-1-yl) benzamide [Figure 2]  It differs chemically from thiazide in a way that it does not possess the thiazide ring system and contains only one sulfonamide group. It is used for hypertension and also for oedema, including that associated with heart failure. Currently most commonly prescribed medicines for hypertension are Angiotensin receptor blockers and diuretics. Monotherapy with oral anti‑hypertensive agents is not sufficient to achieve target blood pressure levels and henceforth, a combination tablet formulation is beneficial in terms of its convenience and patient compliance. The present drug combination has promising anti‑hypertensive effect. The clinical and pharmaceutical analysis of this drug requires effective analytical procedures for quality control and pharmacodynamics and pharmacokinetic studies as well as stability study Several HPLC assay method of Indapamide bulk and in tablet dosage form are available(4-7). The objective of the present study was to develop an accurate, specific and repeatable stability indicating HPLC for simultaneous determination of Olmesartan medoxomil and Indapamide from Tablet dosage form. Olmesartan medoxomil is official in BP 2013 and Indapamide is official in IP, BP, EP, JP(8-11). The method was validated as per International Conference on Harmonization (ICH) guidelines (12)

 

Figure1. Chemical Structure of Olmesartan medoxomil

 

Figure2. Chemical Structure of Indapamide

 

MATERIALS AND METHODS:

·         Olmesartan medoxomil raw material was received as gift sample from Cadila Healthcare Limited, Ankleshwar.

·         Indapamide raw material was received as gift sample from Torrent research centre, Ahmedabad.

·         Marketed formulation OLMY-D 20 (20:1.5) and OLMY-D 40 (40:1.5) From Biocon

·         Acetonitrile (FINAR) Gradient grade

·         Hydrochloric acid (MERCK)AR grade

·         Sodium hydroxide (MERCK)AR grade

·         HPLC grade Water

·         H2O2  (MERCK) AR grade

·          Disodium hydrogen Phosphate (RANKEM) LR grade were used for development purpose.

 

INSTRUMENTS:

·         Chromatographic analysis was carried out on

Operation-semi automatic,

Pump-Single pump

Model- SPD 10 A-LC 10 AT

Company-Shimadzu, Japan

Software-Winchrome software

·         Semi micro analytical balance (Sartorius CD2250, Germany) was used for weighing purpose.

·         HPLC water was obtained using Arium®611VF (Sartorius).

·         Magnetic stirrer (1 MLH, Remi) was used for mixing purpose.

·         pH tutor (313927, Eutech Instruments) was used for pH measurement.

·         Sonications of solutions were done using Ultrasonic cleaner (D 120/1H, Trans-O-Sonic).

·         Column used was Inert Phenomenex C18  (250mm × 4.6 mm i.d.) 5μm

·         Nylon membrane filters (0.22 µm, 47 mm D)

·         All volumetric glass wares used were calibrated.

 

EXPERIMENTAL WORK AND CONDITION:

Selection of mobile phase:

From literature survey I choose mobile phase Sodium perchlorate and Triethyl amine buffer: Acetonitrile (60:40 v/v) with pH 3 but Splitting was observed and No peak resolve between two peak then I changed ratio and mobile phase [Acetonitrile: Phosphate Buffer (70:30 v/v)] but Less resolution and splitting. After many trails I choose mobile phase [0.02 M Na2HPO4 : Acetonitrile (55:45 v/v)] with pH 7 Because Olmesartan medoxomil having  pKa 4.30 and Indapamide having  pKa  8.85. Both drugs were separated with sharp peak and retention time of Olmesartan medoxomil was 4.42 min and retention time of Indapamide was 7.58 min.

 

Buffer preparation

Accurately weighed 2.83 gm Na2HPO4 was dissolved in to 1000 ml water, than pH was adjusted to 7 with ortho‑phosphoric acid.

 

A) Preparation of standard stock solution (1000 μg/mL)

10  mg  of bulk drug was weighed  accurately  and  transferred  into  a clean, dry 10 mL volumetric flask, dissolved in 1ml of  Acetonitrile and volume was adjusted to 10 mL with mobile phase and further 0.1 ml dilute up to 10 ml with mobile phase to get a concentration of  10 μg/mL

 

B)  Preparation of sample stock solution (1000 μg/mL)

Twenty tablets were powdered and sample weighed equivalent to 10 mg of olmesartan medoxomil and indapamide and transferred into a 10 mL volumetric flask separately, dissolved in 1 ml Acetonitrile and further diluted with mobile phase up to 10 ml, sonicated for 15  min, filtered through a Whatman filter paper ≠ 42 and volume was adjusted to 10 mL with Diluent to get a concentration of 10 μg/mL

 

Figure 3. Standard olmesartan medoxomil  (10 μg/mL)

 

Figure 4. Standard Indapamide  (10 μg/mL)

 

Figure 5. Standard olmesartan medoxomil  and medoxomil (10 μg/ml)

 

Figure 6 Linearity  of  Olmesartan medoxomil (50-250 μg/ml)  and  Indapamide (10-50 μg/ml)

 

Table 1:Percent degradation of olmesartan medoxomil and Indapamide retention time and area of degradation product

Sr. No.

Condition

Degradation products

Retention time(Min) and Area of impurity

% Degradation at 60 ˚c

Retention time(Min)

Area

 of impurity peak

Olmesartan

Indapamide

Untreated stock Solution (10μg/ml)

-

Olmesartan (Area)

Indapamide (Area)

4.42(182415)

7.58(245142)

1

Acid hydrolysis

IMP B

-

1.12

4729

70.41%

29.73%

IMP I

2.42

-

188517

IMP II

4.08

-

7607

OLME

4.42

-

41582

IMP A

-

5.43

5620

INDA

-

7.63

170800

2

Base

hydrolysis

IMP III

2.38

-

230436

100%

17.72%

IMP IV

2.86

-

1246

IMP C

-

3.14

75331

IMP D

-

3.45

31923

IMP V

3.87

-

78778

IMP F

-

5.14

1433

INDA

-

7.28

249826

IMP E

-

9.05

1220

3

Oxidation

IMP H

-

2.20

4673

86.18%

50.50%

IMP VI

2.37

-

200793

IMP G

-

2.85

113381

IMP I

-

3.79

1202

OLME

4.40

-

10048

IMP VII

5.59

-

19805

INDA

-

6.39

118889

4

Neutral

IMP VIII

2.61

-

180852

28.68%

20.22%

IMP J

-

3.01

998

OLME

4.70

-

165092

INDA

-

6.55

193750

5

Thermal

IMP IX

2.86

-

3654

9.30%

3.87%

OLME

4.73

-

3256759

INDA

-

7.58

1654378

6

Photolytic

OLME

4.40

 

3356759

3.82%

3.82%

INDA

 

7.25

1754378

 

Figure 7 : Acid Degradation (Mixture) – after 2 Hr AT 60˚C

 

Figure 8: Base  Degradation (Mixture) – after 2 Hr AT 60˚C

 

Figure 9: Oxidation Degradation (Mixture) – after 2 Hr AT 60˚C

 

Figure 10 : Water reflux (Mixture) – after 2 Hr AT 60˚C

 

Figure 11: Thermal Degradation (Mixture) – after 2 Hr AT 60˚C

 

Figure 12 : Photolytic Degradation (Mixture) – after 2 Hr AT 60˚C

 

 

Table 2: DEGRADATION SUMMARY

Sr. No.

CONDITION

% DEGRADATION at 60 ˚C

OLMESARTAN

INDAPAMIDE

1

Acid hydrolysis

70.41%

29.73%

2

Base hydrolysis

100%

17.72%

3

Oxidation

86.18%

50.50%

4

Water reflux

28.68%

20.22%

5

Thermal

9.30%

3.87%

6

Photolytic

3.82%

3.82%

 

Stability Indicating Property (Forced Degradation)

A. Acid Induced Degradation:

Accurately weighed 10mg bulk drug was dissolved in 1 ml Acetonitrile and add 10 ml 0.1 N HCl in it. Then this solution is kept At 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded. Samples were taken at 0 min, 10 min, 30 min, 1 Hr, 2 Hr and 3Hr. Blank solution was also injected without API.

 

B. Base Induced Degradation:

Accurately weighed 10mg bulk drug was dissolved in 1 ml Acetonitrile and add 10 ml 0.1 N NaOH in it. Then this solution is kept At 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded. Samples were taken at  0 min, 10 min, 30 min,     1 Hr, 2 Hr and 3Hr .Blank solution was also injected without API.

 

C. Hydrogen Peroxide Induced Degradation:

Accurately weighed 10mg bulk drug was dissolved in 1 ml Acetonitrile and add 10 ml 3% H2O2 in it. Then this solution is kept at 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded. Samples were taken at 0 min, 10 min, 30 min,  1 Hr, 2 Hr and 3Hr .Blank solution was also injected without API.

 

D. Water reflux:

Accurately weighed 10mg bulk drug was dissolved in 1 ml Acetonitrile and add 10 ml distill water in it. Adjust  pH 7.Then this solution is kept At 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded. Samples were taken at 0 min, 10 min, 30 min, 1 Hr, 2 Hr and 3Hr. Blank solution was also injected without API.

 

E. Thermal degradation:

Accurately weighed 10mg bulk drug was kept in a hot air oven for 3 h at a temperature of 60 °C, then made up with mobile phase. For further dilution, 1 mg sample was added to a 10 ml volumetric flask individually, and for tablet degradation, Average weight of 5 tablet powder was kept in hot air oven in same condition.10mg powder added to a 10 ml flask and made up with mobile phase and further dilute 0.1 ml in 10 ml mobile phase

F. Photochemical Degradation:

10 mg of drug was exposed to UV light for 3 hr, then made up with mobile phase up to 10ml. For further dilution, 1 ml of sample was added to a 10 ml volumetric flask individually, for tablet degradation Average weight of 5 tablet powder was exposed to UV light for 3 hr.10mg powder added to a 10 ml flask and made up with mobile phase and further dilute 0.1 ml in 10 ml mobile phase

 

METHOD VALIDATION(13)

Validation was carried out with respect to various parameters, as required under ICH guideline Q2 (R1). The developed method validated with respect to parameters such as linearity, precision, accuracy, LOD, LOQ robustness, and solution stability.

 

[A] LINEARITY

The linearity of the response of Olmesartan medoxomil was found to be between 50-250  μg/mL concentration and indapamide was found to be 10-50 μg/mL. The calibration graphs were obtained by plotting the peak area versus the concentration. Regression coefficient was found to be 0.998 for both Olmesartan medoxomil and indapamide.

 

Sr.no

Concentration (μg/ml)

Peak Area ± SD (n=6)

Olmesartan Medoxomil

Indapamide

Olmesartan Medoxomil

Indapamide

1

50

10

900265±24

482722±5586

2

100

20

1774723±738

865436±221

3

150

30

2607065±493

1297231±3580

4

200

40

3490396±220

1787321±1101

5

250

50

4492569±427

2219382±105

 

[B] PRECISION:

Method precision (intra-day precision) was evaluated by carrying out three independent measurements of standard drug solution at three times on the same day .The results obtained were within 2% Relative Standard Deviation (RSD).

 

INTRADAY:

Conc.(µg/ml)

Peak Area ± SD (n=3)

%RSD

Mean % RSD

OLME

INDA

OLME

INDA

OLME

INDA

OLME

INDA

50

10

901417±648

485271±670

0.07%

0.13%

 

0.23%

 

0.28%

100

20

1772761±2697

866080±544

0.15%

0.06%

150

30

2606748±581

1295498±3205

0.02%

0.23%

 

INTERDAY

Conc.(µg/ml)

Peak Area ± SD (n=3)

%RSD

Mean % RSD

OLME

INDA

OLME

INDA

OLME

INDA

OLME

INDA

50

10

903950±1053

508178±2913

0.11%

0.57%

0.58%

0.64%

100

20

1794179±6767

876320±114

0.37%

0.01%

150

30

2619641±7180

130313±1284

0.27%

0.75%

 

[C] ACCURACY (RECOVERY STUDY)

Accuracy  expresses  the  closeness  of  agreement  between  the  values  which  are accepted either as a conventional true value or an accepted reference value and the value found practically

 [D] LOD and LOQ

The peak area of ten solutions containing 50 µg/mL were measured at 240 nm and calculated according to equation of  LOD [3.3 x MSD/ slope] and LOQ [10 x MSD/ slope].

Sr No.

Parameter

OLME

INDA

1.

S.D of the Y-intercepts of 6 calibration curve

3018

11885

2.

Mean slope of the 6 calibration curves.

17790

43950

3.

LOD = 3.3 × (SD/Slope) (μg/ml)

0.55 μg/ml

0.89 μg/ml

4.

LOQ = 10 × (SD/Slope) (μg/ml)

1.69μg/ml

2.70   μg/ml

[E] ROBUSTNESS

 

The %RSD for Olmesartan medoxomil and indapamide for Flow rate change, wave length and ratio Change was calculated.

 

FLOW RATE CHANGE

Sr. No.

Flow rate

(ml/min)

Conc.(µg/ml)

Peak area ± SD ( n=3 )

%RSD

Mean % RSD

OLME

INDA

OLM

INDA

OLME

INDA

OLME

INDA

1

0.8

50

10

902616±1561

482882±881

0.17

0.18

0.29%

0.47%

2

1.2

50

10

905232±3887

475063±3613

0.42

0.76

 

WAVE LENGTH CHANGE

Sr. No.

wavelength

(ml/min)

Conc. (µg/ml)

Peak area ± SD (n=3 )

%RSD

Mean % RSD

OLME

INDA

OLM

INDA

OLME

INDA

OLME

INDA

1

238

50

10

903197±5233

472771±931

0.57%

0.19%

0.27%

0.53%

2

240

50

10

901264±1739

489929±3391

0.19%

0.69%

3

242

50

10

900731±10

505025±3643

0.06%

0.72%

 

RATIO CHANGE

Sr. No.

Ratio (µg/ml)

Peak area ± SD ( n=3 )

%RSD

Mean % RSD

OLME

INDA

OLM

INDA

OLME

INDA

OLME

INDA

1

140

10

2483623±12960

439522±1297

0.52%

0.29%

0.42%

0.30%

 

2

10

140

178393±581

615715±722

0.32%

0.11%

 

Determination of active ingredients in tablet formulation

Marketed formulation

Claim (mg)

Concentration taken (μg/ml)

Concentration found(μg/ml)

% Assay

 

OLME

INDA

OLME

INDA

OLME

INDA

OlmyD 20

200:15

200

15

199.87

15.03

99.93%

100.2%

Olmy D 40

400:30

400

30

400.34

29.79

100.08%

99.33%

According to USP 2013 for olmesartan medoxomil (97.5-102.0%)        

According to BP/EP for Indapamide (98-102.0%)

According to IP 2010 for Indapamide (95-105.0%)

 

OlmyD-20 tablets (10 tablets) were taken and triturated and equivalent Weight 350 mg (Tablet powder equivalent to 200 mg olmesartan medoxomil and 15 mg indapamide. i.e., 10 tablets powder was added in the flask for assay of tablets.) was transferred into 250 ml volumetric flask. Then add about 100.0 ml Mobile phase was added and sonicated for 30 min with intermittent shaking. Then volume was made up to 250ml with Mobile phase. Then 2.5 ml of standard stock solution was diluted to 10 ml with Mobile phase to make final standard concentration of olmesartan medoxomil (200 ppm) and indapamide (15 ppm) respectively and chromatogram was recorded.

 

Solution stability:

The standard and sample solutions were found stable up to 12 hr at room temperature. After 3,6,24 hr the solutions were analyzed and results related to solution stability are summarized here

 

Times

(Hr)

Area

%RSD

Olmesartan

Indapamide

Olmesartan

Indapamide

3

1854187

242536

1.34 %

0.09 %

6

1858362

242563

24

1899652

242942

 

Summary of the obtained results

Sr No.

Parameters

Olmesartan Medoxomil

Indapamide

1.

Wavelength (nm)

240 nm

2.

Linearity range (μg/ml)

50-200 µg/ml

10-50 µg/ml

3.

Standard Regression equation

Y=17790x -3018

Y=43950x + 11885

4.

Correlation coefficient (R2)

0.998

0.998

5.

Precision (%RSD)

Intraday

Interday

 

0.23

0.58

 

0.28

0.64

6.

% Recovery (Accuracy, n = 3)

99.92%

100.35%

7.

LOD (μg/ml)

0.55

0.89

8.

LOQ (μg/ml)

1.69

2.70

9.

Robustness

Flow rate change

Wavelength change

Ratio change

 

0.29

0.27

0.42

 

0.47

0.53

0.30

10.

Assay (% Label claim)

Olmy D 20

Olmy D 40

 

99.93%

100.08%

 

100.20%

99.33%

 

 

CONCLUSION:

The study shows that the developed HPLC Method is fast, precise, specific, accurate and stability indicating. The stability-indicating method resolved the drug peak and also the peaks of degradation products formed under variety of conditions. After exposure of Olmesartan medoxomil  and indapamide to stress condition like acid, base hydrolysis; oxidation, with maximum degradation of olmesartan medoxomil observed in acid hydrolysis and maximum degradation of indapamide observed in oxidation  followed by base degradation. Olmesartan IMP-I,IMP-II,IMP-III, IMP-IV, IMP-V, IMP-VI, IMP-VII, IMP-VIII and IMP- IX  were observed and in Indapamide IMP-A, IMP-B, IMP-C, IMP-D, IMP-E, IMP-F, IMP-G, IMP-H, IMP-I and IMP-J were observed Therefore this method can be employed for monitoring the stability of Olmesartan medoxomil and Indapamide drug substance commercially.

 

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4.        Sethi PD and Sethi R; “HPLC: Quantitative Analysis of Pharmaceutical Formulation, 15th  Edn; Published by CBS,2007.

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8.        United States Pharmacopoeia (USP 32- NF 29); the United States Pharmacopoeial Convention, Washington D.C., 2009, Volume II,  pp 1115-1116.

9.        The European Pharmacopoeia; 5th Edn; European Directorate for the Quality of Medicines and Health Care, 2011, Volume-II, pp 1793.

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11.     Indian Pharmacopoeia; The Indian Pharmacopoeia Commission, Ghaziabad, Govt. of India; Ministry of Health and Family Welfare,  2010, Volume II, pp 1489-1490.

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Received on 28.02.2015          Accepted on 22.03.2015        

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 5(1): Jan.-Mar. 2015; Page 15-23

DOI: 10.5958/2231-5691.2015.00003.9