Research Article

Analytical method development and validation for simultaneous estimation of amlodipine besylate and perindopril arginine in combined pharmaceutical dosage form

Bhagirath K. Patel, Subhaschandra K. Patel, Swati R. Patel*

Sat Kaival College of Pharmacy, Sarsa, Gujarat, India

*For correspondence

Swati R. Patel

Research Scholar, Sat Kaival College of Pharmacy, Sarsa, Gujarat, India.

Email: swati28492@gmail.com

 

 

 

 

 

 

 

 

 

 

 

 

 

Received: 05 April 2016

Revised: 21 April 2016

Accepted: 26 April 2016

ABSTRACT

Objective: A simple, accurate, precise and sensitive Reversed Phase High Performance Liquid Chromatography (RP-HPLC) method was developed for simultaneous estimation of amlodipine besylate and Perindopril arginine in combined pharmaceutical dosage form.

Methods: Using C18 (250 mm × 4.6 mm i.d., 5µm) column and phosphate Buffer (pH 5.0): Acetonitrile (70:30 v/v) as mobile phase at Flow Rate 1.0 ml/min with Detection Wavelength 240 nm. Retention time in RP-HPLC was found to be 10 min. The method was validated in term of accuracy, precision, linearity, limit of detection, limit of quantitation and robustness as per International Conference of harmonization (ICH) guidelines.

Results: For RP-HPLC linearity were found in range of 20 µg/ml and 10 µg/ml, respectively. Retention times in RP-HPLC were found to be 3.243 min and 7.110 min for Perindopril Arginine and Amlodipine Basylate and optimized chromatographic condition. The linearity and range data for Amlodipine Basylate and Perindopril Arginine was found to be 0.998 and 0.997 Respectively. The precision data for Perindopril Arginine and Amlodipine Basylate was found to be 0.816 and 1.063 respectively. The accuracy of the present method was evaluated at 80%, 100% and 120%. The Accuracy data for Perindopril Arginine was 99.72-100.02%, while for Amlodipine Basylate, it was found to be in range of 99.87-100.17%. LOD value was found to be 3.3 for Amlodipine Basylate-0.700 µg/ml and Perindopril Arginine - 0.564 µg/ml respectively. LOQ value was found to be 10 for The method was found to be Amlodipine Basylate 2.121 µg/ml and Perindopril Arginine 1.709 µg/ml.

Conclusions: This method can be used to utilize for routine laboratory analysis and assay of Amlodipine Basylate and Perindopril Arginine in their combined dosage form.

Keywords: RP-HPLC, Amlodipine besylate, Perindropril arginine, ICH guidelines

Introduction

Amlodipine besylate is chemically benzenesulfonic acid;3-O-ethyl 5-O-methyl 2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate (Figure 1). It is is in a group of drugs called calcium channel blockers. Amlodipine relaxes (widens) blood vessels and improves blood flow.1,2

Amlodipine is used to treat high blood pressure (hypertension) or chest pain (angina) and other conditions caused by coronary artery disease. This medication is for use in adults and children who are at least 6 years old.

Figure 1: Amlodipine besylate.

Perindopril arginine is chemically (2S,3aS,7aS)-1-[(2S)-2-[[(1S)-1 (ethoxycarbonyl)butyl]amino] propanoyl]octahydro-1H-indole-2-carboxylate (Figure 2). Perindopril is a nonsulfhydryl prodrug that belongs to the angiotensin-converting enzyme (ACE) inhibitor class of medications.3 It is rapidly metabolized in the liver to perindoprilat, its active metabolite, following oral administration. Perindoprilat is a potent, competitive inhibitor of ACE, the enzyme responsible for the conversion of angiotensin I (ATI) to angiotensin II (ATII). ATII regulates blood pressure and is a key component of the renin-angiotensin-aldosterone system (RAAS). Perindopril may be used to treat mild to moderate essential hypertension, mild to moderate congestive heart failure, and to reduce the cardiovascular risk of individuals with hypertension or post-myocardial infarction and stable coronary disease.

Figure 2: Perindopril arginine.

Materials and Methods

Chemicals and reagents

Perindopril Arginine was procured from Torrent Pharma, Amlodipine Besylate was procured from Unison Pharmaceutical, Water, Methanol, Acetonitrile, Hydrogen phosphate.

Apparatus and instruments

Model: Shimadzu HPLC System, Pump:-LC-20 AT, Column: C18 (250 mm × 4.6 mm i.d., 5µm), Injector: 20 μl fixed loop.

Detector: UV Detector

Software: Spinchrom

Analytical balance: Electronic Balance (Shimadzu AUX240)

Preparation of standard solutions

Amlodipine Besylate standard stock solution (200 μg/ml)

A 20 mg of Amlodipine Besylate was weighed and transferred to a 100 ml volumetric flask. Volume was made up to the mark with methanol.

Perindopril Arginine standard stock solution (100 μg/ml)

A 10 mg of Perindopril Arginine was weighed and transferred to a 100 ml volumetric flask. Volume was made up to the mark with methanol.

Preparation of standard solution of binary mixtures of Amlodipine Besylate (20 μg/ml) and Perindopril Arginine (10 μg/ml)

Take 1 ml from the Amlodipine Besylate stock solution and 1ml from Perindopril Arginine stock solution and transferred to 10 ml volumetric flask and volume made up to the mark by mobile phase which was used in particular trials.

HPLC method development and optimization

Selection of wavelength

Standard solution of Amlodipine Besylate (20 μg/ml) and standard solution of Perindopril Arginine (10 μg/ml) were scanned between 200-400 nm using UV-visible spectrophotometer. Both solutions were scanned between 200-400 nm. Methanol show reasonably good response at 240 nm.

Figure 3: Overlay UV Spectrum of Amlodipine Basylate and Perindopril Arginine showing selection of wavelength detection.

Optimization of RP-HPLC method4,5

The pure drug solution of were Amlodipine Besylate (20 μg/ml) and Perindopril Arginine (10 μg/ml) injected individually in to HPLC system and allow to run in different mobile phase like Methanol, Water: Methanol, Water: Acetonitrile, Phosphate Buffer: Methanol, Phosphate Buffer:Acetonitrile were tried in order to find the optimum condition for the separation of Amlodipine Besylate and Perindopril Arginine. It was found that mobile phase containing Phosphate Buffer (pH=5.0): Acetonitrile (70:30 v/v) at a flow rate of 1.0 ml/min with detecting wavelength 240 nm give satisfactory result with sharp, well defined and resolving peak with minimum tailing as compared to other mobile phases. Under these condition the retention time) 3.243 min and 7.110 min for Perindopril Arginine and Amlodipine Basylate and optimized chromatographic condition.

Figure 4: Chromatogram of Amlodipine Basylate and Perindopril Arginine in Buffer (pH 5.0): Acetonitrile 70:30 v/v) (Flow rate-1.0 ml/min).

Table 1: Optimized chromatographic conditions.

Detection

Parameter

Mode of elution

Isocratic

Mobile Phase

Buffer( pH 5.0) : Acetonitrile (70:30)

Column

C18 (250 mm × 4.6 mm i.d.,5µm)

Flow rate

1.0 ml/min

Runtime

10.0 min

Injection volume

20 µL

Detection wavelength

240 nm

Validation of RP-HPLC method6

Linearity

The linearity for Amlodipine Besylate and Perindopril Arginine were assessed by analysis of combined standard solution in range of 10-30 μg/ml and 5-15 μg/ml respectively.

Precision

A. Repeatability

Standard solution containing Amlodipine Besylate (20 µg/ml) and Perindopril Arginine(10 µg/ml) was injected six times and areas of peaks were measured and %R.S.D. was calculated.

B. Intra-day precision

Standard solution containing (10,20,30 µg/ml) of Amlodipine Besylate and (5,10,15 µg/ml) of Perindopril Arginine were analyzed three times on the same day and %R.S.D was calculated.

C. Inter-day precision

Standard solution containing (10,20,30 µg/ml) of Amlodipine Besylate and (5,10,15 µg/ml) of Perindopril Arginine were analyzed three times on the different day and % R.S.D was calculated.

Accuracy

To the pre-analyzed sample, a known amount of standard solution of pure drug Amlodipine Besylate and Perindopril Arginine were spiked at three different levels. This study was carried out at 80%, 100% and 120% level.

LOD and LOQ

The LOD and LOQ measurement of Amlodipine Besylate and Perindopril Arginine bye the use of proposed method was estimated in term of Limit of Detection (LOD) and Limit of Quantitation (LOQ). The LOD and LOQ were calculated using following equations.

LOD= 3.3*σ/S

LOQ=10* σ/S

Where,

σ= the standard deviation of the response

s= slop of the calibration curve

Robustness

Following parameters were changed one by one and their effect was observed on system suitability for standard preparation.

  1. Flow rate of mobile phase was changed (± 0.2 ml/min) 0.8 ml/min and 1.2 ml/min.
  2. pH of Mobile phase was changed (± 0.2) 5.2 and 4.8.
  3. Ratio of Mobile phase was changed (±2) Buffer: Acetonitrile (68:32) and Buffer: Acetonitrile (72:28).

Results and Discussion

The result of method development and validation study on simultaneous estimation of amlodipine besylate and perindopril arginine in current study involving phosphate buffer (pH-5.0): Acetonitrile (70:30 v/v) as mobile phase for RP-HPLC are given below.

Method development

Amlodipine besylate and perindopril arginine were completely separated on C18 column by RP-HPLC using isocratic elution of phosphate buffer and acetonitrile as mobile phase. When the increase the percentage of acetonitrileas compare to phosphate buffer at pH-5.0, a sharp pointed and well separated peak was observed.

Eventually proper resolution was achieved at a flow rate of 1.0 ml/min and using phosphate buffer (pH-5.0): Acetonitrile (70:30 v/v) as the mobile phase for RP-HPLC.

Method validation

Linearity and range

The linearity for Amlodipine Basylate and Perindopril Arginine were assessed by analysis of combined standard solution in range of 10-30μg/ml and 5-15 μg/ml respectively. Correlation co-efficient for calibration curve Amlodipine Basylate and Perindopril Arginine was found to be 0.998 and 0.997 respectively. The regression line equation for Amlodipine Basylate: y = 65.81x - 14.33 and for Perindopril Arginine: y = 156.6x - 35.33.

Table 2: Linearity data for Amlodipine Basylate.

Sr. No

Concentration (µg/ml)

Area

1

10

650.798

2

15

960.726

3

20

1313.438

4

25

1617.137

5

30

1968.044

Table 3: Linearity data for Perindopril Arginine.

Sr. No

Concentration (µg/ml)

Area

1

5

767.027

2

7.5

1105.321

3

10

1549.923

4

12.5

1909.031

5

15

2322.883

Precision

I. Repeatability

The data for repeatability of peak area measurement for Perindopril Arginine and Amlodipine Basylate, based on six measurements of same solution of Perindopril Arginine and Amlodipine Basylate are depicted in Table 4 and 5. The %RSD for Perindopril Arginine and Amlodipine Basylate was found to be 0.816 and 1.063 respectively.

Table 4: Repeatability data for Amlodipine Basylate.

Sr No.

Conc (μg/ml)

Area

Mean ± S.D (n=6)

% R.S.D

1

20

1308.159

 

1306.810 ± 13.886

 

1.063

1279.002

1313.429

1316.063

1313.422

1310.784

Table 5: Repeatability data for Perindopril Arginine.

Sr No.

Conc (μg/ml)

Area

Mean ± S.D (n=6)

% R.S.D

1

10

1543.702

 

 

 

 

1542.389 ± 12.590

 

 

 

 

0.816

1546.818

1549.959

1517.136

1549.915

1546.805

II. Intraday precision

The data for intraday precision for Perindopril Arginine and Amlodipine Basylate is shown in Table 6. The % R.S.D. for Intraday precision was found to be 0.909-1.533.forAmlodipine Basylate and 0.794-1.801 for Perindopril Arginine.

Table 6: Intraday precision data for estimation of Perindopril Arginine and Amlodipine Basylate.

 

Amlodipine Basylate

Perindopril Arginine

Sr. No.

Conc.

(µg/ml)

Area

Mean ± S.D. (n=3)

% R.S.D

Conc.

(µg/ml)

Area

Mean ± S.D. (n=3)

% R.S.D

1

10

641.867±11.563

1.801

5

757.012±11.609

1.533

2

20

1300.475±14.615

1.124

10

1534.824±14.545

0.948

3

30

1952.366±15.497

0.794

15

2300.794± 20.927

0.909

III. Interday precision

The data for intraday precision for Perindopril Arginine and Amlodipine Basylate is shown in Table 7. The % R.S.D. for interday precision was found to be 1.072-1.660 for Amlodipine Basylate and 0.900-1.936 for Perindopril Arginine.

Table 7: Interday precision data for estimation of Perindopril Arginine and Amlodipine Basylate.

 

Amlodipine Basylate

Perindopril Arginine

Sr. No.

Conc. (µg/ml)

Area Mean ± S.D. (n=3)

% R.S.D

Conc. (µg/ml)

Area Mean ± S.D. (n=3)

% R.S.D

1

10

642.713±6.874

1.069

5

757.396±6.914

0.913

2

20

1298.704±11.223

0.864

10

1532.233±10.708

0.699

3

30

1946.645±11.824

0.608

15

2295.871±21.479

0.935

Accuracy

Accuracy of the method was confirmed by recovery study from marketed formulation at three level of standard addition. Percentage recovery for Perindopril Arginine was 99.72-100.02 %, while for Amlodipine Basylate, it was found to be in range of 99.87-100.17%.

Table 8: Recovery data for Perindopril Arginine.

Sr. No.

Conc. Level (%)

Sample amount (μg/ml)

Amount Added (μg/ml)

Amount recovered (μg/ml)

% Recovery

% Mean

Recovery ± S.D

1

80 %

5

4

3.967

99.169

100.011± 0.732

2

5

4

4.020

100.497

3

5

4

4.015

100.367

4

100 %

5

5

4.966

99.318

99.759± 0.415

5

5

5

5.007

100.141

6

5

5

4.991

99.818

7

120 %

5

6

5.992

99.871

99.750 ± 0.204

8

5

6

5.971

99.515

9

5

6

5.992

99.863

Table 9: Recovery data for Amlodipine Basylate.

Sr. No.

Conc. Level (%)

Sample Amount

Amount Added

Amount recovered  (μg/ml)

% Recovery

% Mean Recovery ± S.D

1

80 %

10

8

7.916

98.949

100.006 ± 0.994

2

10

8

8.074

100.922

3

10

8

8.012

100.149

4

100 %

10

10

9.910

99.098

99.665 ± 0.603

5

10

10

10.030

100.298

6

10

10

9.960

99.599

7

120 %

10

12

12.014

100.113

99.687 ± 0.408

8

10

12

11.916

99.300

9

10

12

11.958

99.649

Table 10: Limit of detection data for Amlodipine Basylate and Perindopril Arginine.

Amlodipine Basylate

Perindopril Arginine

LOD = 3.3 x (SD / Slope)

=3.3x(13.958/65.810)

= 0.700 µg/ml

LOD = 3.3 x (SD / Slope)

=3.3x(26.761/156.6)

= 0.564µg/ml

LOD and LOQ

Calibration curve was repeated for five times and the standard deviation (SD) of the intercepts was calculated. Then LOD and LOQ were calculated as follows:

LOD= 3.3 * SD/slope of calibration curve

LOQ=10 * SD/slope of calibration curve

Where,

SD = Standard deviation of intercepts

Table 11: Limit of quantitation data for Amlodipine Basylate and Perindopril Arginine.

Amlodipine Basylate

Perindopril Arginine

LOQ = 10 x (SD / Slope)

= 10 x (13.958/65.810)

= 2.121 µg/ml

LOQ = 10 x ( SD / Slope )

= 10 x (26.761/156.6)

= 1.709 µg/ml

Table 12: Robustness data for Amlodipine Basylate.

Sr No.

Area at

Flow rate

(-0.2 ml/min)

Area at

Flow rate

(+0.2 ml/min)

Area at

pH (-0.2)

Area at

pH (+0.2)

Area at

Mobile phase(-2)

Area at

Mobile phase(+2)

1

1338.830

1275.301

1313.947

1230.315

1342.382

1249.476

2

1362.129

1264.321

1348.977

1256.842

1310.592

1280.602

3

1367.337

1291.030

1356.904

1262.155

1352.829

1289.733

% R.S.D

1.119

1.051

1.706

1.365

1.648

1.658

Table 13: Robustness data for Perindopril Arginine.

Sr No.

Area at

Flow rate

(-0.2 ml/min)

Area at

Flow rate

(+0.2 ml/min)

Area at

pH (-0.2)

Area at

pH (+0.2)

Area at

Mobile phase(-2)

Area at

Mobile phase(+2)

1

1601.086

1474.322

1582.457

1476.961

1556.343

1501.967

2

1583.736

1514.251

1567.358

1456.233

1587.000

1488.185

3

1613.390

1523.537

1601.086

1489.528

1596.276

1522.009

% R.S.D

0.931

1.739

1.067

1.140

1.323

1.131

Table 14: Analysis of marketed formulation.

Tablet

mg/Tablet powder

Assay (% of label claim*) Mean ± S. D.

Amlodipine Basylate

Perindopril Arginine

% Amlodipine Basylate

% Perindopril Arginine

Coveram

10

5

101.77±1.080

98.54± 1.42

Robustness

The effect of changes was found to be within the acceptance criteria as shown in table. The % RSD should be less than 2%.

Analysis of marketed formulation by developed method

Applicability of the proposed method was tested by analyzing the commercially available Tablet formulation Coveram. The results are shown in Table 14.

The assay results were comparable to labeled value of each drug in Tablet dosage form. These results indicate that the developed method is accurate, precise, simple and rapid. It can be used in the routine quality control of dosage form in industries.

Conclusions

Development and validation of RPHPLC method was found to be simple, accurate, precise and sensitive. These method can be applied for routine quantitative analysis of Amlodipine besylate and perindopril arginine in combined pharmaceutical dosage forms.

Acknowledgements

Authors are grateful to TORRENT PHARMA for providing gratis sample Perindopril Arginine and UNISON PHARMACEUTICAL for proving Amlodipine Besylate. Our heartily thanks Sat Kaival College of Pharmacy, Gujarat, India for the perfect logistic support and guidance they have extended to us

Funding: No funding sources

Conflict of interest: None declared

References

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