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Research Article CODEN: IJPRNK IMPACT FACTOR: 4.278 ISSN: 2277-8713 N Kamble, IJPRBS, 2014; Volume 3(6): 347-361 IJPRBS

Available Online at www.ijprbs.com 347

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF NIFEDIPINE

AND ATENOLOL IN A CAPSULE FORMULATION BY RP-HPLC

N KAMBLE, S ASIRVATHAM

St. John Institute of Pharmacy and Research, Palghar (E) – 401404, Maharashtra

Accepted Date: 14/12/2014; Published Date: 27/12/2014

Abstract: A simple, rapid, precise and accurate reversed phase high performance liquid chromatographic method has been developed for simultaneous determination of Atenolol in combination with Nifedipine. This method uses a mobile phase of 0.01M phosphate buffer solution: methanol (75:25v/v). The retention times for Nifedipine and atenolol are 4.1 min and 3.08 min, respectively. The method is validated and shown to be linear. The linearity range for both Nifedipine and atenolol was found to be 10-100 μg/ml. The Percentage recovery for Nifedipine and atenolol are ranged between 99.32–100.02 and 99.10–100.4 respectively. The correlation coefficients of Nifedipine and atenolol are 0.999 and 0.999 respectively. The relative standard deviation for six replicates is always less than 2%. The Statistical analysis proves that the method is suitable for analysis of Nifedipine and atenolol as a bulk drug and in pharmaceutical formulation without any interference from the excipients. The propose method was validated as per the ICH guidelines parameters like Linearity, precision, accuracy, robustness limit of detection and limit of quantitation. The method was accurate, precise, specific and rapid found to be suitable for the quantitative analysis of the drug and dosage form.

Keywords: Atenolol, Nifedipine, validation, ICH guidelines

INTERNATIONAL JOURNAL OF

PHARMACEUTICAL RESEARCH AND BIO-SCIENCE

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Corresponding Author: MR. N. KAMBLE

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How to Cite This Article:

N Kamble, IJPRBS, 2014; Volume 3(6): 347-361



INTRODUCTION

Atenolol is a competitive, ß-1 selective adrenergic antagonist, similar to metoprolol. ß-

adrenergic antagonists counter the effect of sympathomimetic neurotransmitters (i.e.,

catecholamines) by competing for receptor sites1, 2. It antagonizes ß1 receptors at doses 50 to

100 times less than those required to block ß2 receptors3, 4, 5. This cardioselectivty is more

pronounced at low doses and is lost at high doses. It lowers the blood pressure in hypertension

and slows the heart rate. Nifedipine is the prototype of the dihydropyridine family of calcium

channel blocker6, 7. Chemically it 1, 4-dihydro-2, 6-dimethyl-4-(2-nirophenyl)-3,5-pyridine

dicarboxylic acid dimethyl ester8, 9, 10. In general, the dihydropyridine-type calcium-channel

antagonists have more prominent effects on vasodilation and coronary flow than do diltiazem

and verapamil11, 12, 13. Combined use of atenolol with nifedipine decrease the rate, conduction

and contractility of heart particularly in patients of ventricular or conduction abnormalities by

decreasing peripheral vascular resistance14, 15, 16. The chemical structures of the assayed

compounds are given below.

Fig: 1. Chemical structures of nifedipine and atenolol

The proposed method was optimized and validated in accordance with International

Conference on Hormonization (ICH) guidelines17, 18, 119. The aim of present work is to develop a

simple, rapid, precise, accurate and selective reversed phase chromatographic method and to

estimate the Atenolol and Nifedipine in bulk and its solid dosage forms.

MATERIALS AND METHODS

The reference sample of Nifedipine and Atenolol standard was kindly supplied as gift sample by

Cipla Ltd, Vikroli West, Mumbai, India and Ajanta Pharmaceutical, Mumbai, India, respectively.

All the chemicals were of analytical grade. Methanol (HPLC grade) was used of Merck

Pharmaceuticals Private Ltd., Mumbai, India. Potassium dihydrogen phosphate (monobasic)

used was of HPLC grade and purchased from Loba Chemicals. Commercial capsules of

Nifedipine and Atenolol in combination was procured from local market. Tenofed Capsule 40

Nifedipine Atenolol



mg are manufactured by Hetero drugs Pvt. Ltd. Hyderabad, A.P. The liquid chromatographic

system was of Perkin Elmer (USA), series 200, which consisted of following components: a

gradient pump, variable wavelength programmable UV/Vis detector, a manual injection facility

with 20 μl fixed loop. The chromatographic analysis was performed using Total Chrom

Navigator version 6.3 software on a HiQ Sil C8- 250×4.6 mm, particle size 5μm column.

PREPARATION OF MOBILE PHASE AND STOCK SOLUTIONS

Phosphate buffer 0.01 M solution as prepared by dissolving accurately about 1.369 gm of

potassium dihydrogen phosphate in a 1000 ml of glass double distilled water. Mobile phase was

prepared by mixing 125 ml of 0.01M potassium dihydrogen phosphate solution with 375 ml of

methanol. This mobile phase was ultrasonicated for 20 min, and then it was filtered through

0.45μm Nylon 6,47mm membrane filter paper. Stock solutions were prepared by weighing

50mg of each of reference standard of atenolol and 20mg of nifedipine and transfer to 50ml

volumetric flask. Both drugs were dissolved in 50ml of methanol with shaking and then volume

was made up to the mark with methanol to get 1000 μg/ml & 400 μg/ml of standard stock

solution of each drug. These stock solutions were filtered through 0.2 μm Nylon 6, membrane

filter paper.1 ml of above stock solution is then pipette out in 50ml volumetric flask and diluted

upto the mark with methanol to get 100 μg/ml & 40 μg/ml respectively.

Selection of analytical wavelength

By appropriate dilution of each standard stock solution with methanol, various concentrations

of atenolol and nifedipine were prepared separately. Each solution was scanned using double

beam UV visible spectrophotometer in the spectrum mode between the wavelength range of

400 nm to 200 nm and their spectra was overlaid. The wavelength selected was 237nm.

Fig: 2. Isoabsorptive point of atenolol and nifedipine



CALIBRATION CURVES FOR atenolol and nifedipine

For each drug appropriate aliquots were pipetted out from each standard stock solution into a

series of 10ml volumetric flasks. The volume was made up to the mark with methanol to get a

set of solutions for atenolol having concentration range 20, 40, 60, 80 and 100 g/ml and for

nifedipine 20, 40, 60, 80 and 100 g/ml. Triplicate dilutions of each concentration of each drug

were prepared separately. From these triplicate solutions, 20μl injections of each concentration

of each drug were injected into the HPLC system separately and chromatographed under the

conditions as described above. Carbamazepine is used as internal standard. Evaluation of both

drugs was performed with UV detector at 237 nm.

Fig: 3. Calibration curve for atenolol

Fig: 4. Calibration curve for nifedipine

y = 2216.x - 266.7 r² = 0.999

0

50000

100000

150000

200000

250000

0 20 40 60 80 100 120

Are

a U

nd

er C

urv

e

Conc. of atenolol in µg/ml

Calibration of atenolol

y = 654.5x - 49.01 r² = 0.999

0

10000

20000

30000

40000

50000

60000

70000

0 20 40 60 80 100 120

Are

a U

nd

er

Cu

rve

Conc. of nifedipine µg/ml

Calibration of nifedipine



METHODOLOGY

To optimize the RP-HPLC parameters, The pure drug of atenolol (ATN) and nifedipine (NFD)

were injected into the HPLC system and run in different solvent systems. Different mobile

phases like methanol and water, acetonitrile and water were tried in order to find the best

conditions for the separation of atenolol and nifedipine. It was found that methanol and 0.01M

potassium dihydrogen phosphate buffer gives satisfactory results as compared to other mobile

phases. Finally, the optimal composition of the mobile phase was determined to be 0.01M

phosphate buffer solution: methanol (75:25v/v). This mobile phase produced good resolution,

reasonable retention times and acceptable peak symmetry for both the drugs. Using the

optimized mobile phase, the flow rate was set to 1.3 ml/min and UV detection was carried out

at 237 nm. The mobile phase and samples were degassed by ultrasonic vibrations for 20 min

and filtered through 0.45μm Nylon, 47 mm membrane filter paper. The table 1 gives the Rt and

peak area found in the estimation. Complete resolution of the peaks with clear baseline was

obtained (fig.5). System suitability test parameters for atenolol and nifedipine for the proposed

method are reported in table 2.

Table: 1 Results of simultaneous estimation of atenolol and nifedipine

Parameter Atenolol Nifedipine Carbamazepine

Rt 4.1 3.08 2.5

Peak area 221767 8883979 3851589

Fig: 5. Graph for simultaneous estimation of nifedipine and atenolol with internal standard



Table 2: System Suitability Parameters

Analysis of capsule formulation

Twenty capsules of nifedipine and atenolol in combination were weighed. The fine powder

equivalent to 50 mg of atenolol and 20mg of nifedipine was weighed and transferred to 50 ml

volumetric flask and dissolved in methanol and the content was kept in ultrasonicator for 30

min. The volume was made up to the mark with diluent. This capsule solution was further

diluted with mobile phase to obtain mixed sample solutions in Lambert’s- Beer’s range for each

drug containing 50μg/ml of atenolol and 20μg/ml of nifedipine respectively. A 20μl volume of

each sample solution was injected into sample injector of HPLC six times under

chromatographic condition as described above. Area of each peak was measured at 237 nm.

The amount of each drug present in the sample (n=6) was determined from peak area of

nifedipine and atenolol present in the pure mixture respectively. Typical chromatogram of

nifedipine and atenolol present in capsule formulation is given in Fig: 6. and the results of

analysis of capsule formulation and its statistical evaluation are given in the Table: 3 and 4

respectively.

Fig: 6. Simultaneous estimation of nifedipine and atenolol in capsule of Tenofed

Parameter Atenolol Nifedipine

Tailing Factor 1.35 1.6

Resolution (Rs) 2.8

Separation factor 1.567

Capacity factor 2.1 3.05

Theoretical plates (N) 4475 7820

Retention Time 3.08 4.1



Table 3: Analysis of capsule formulation

Table 4: Statistical validation

Evaluation of analytical method (method validation)

The proposed method has been validated for the simultaneous determination of ATN and NFD

in capsule dosage form. Calibration curves were constructed by plotting peak areas versus

concentrations of ATN and NFD, and the regression equations were calculated. The calibration

curves were plotted over the concentration range 20-100 µg/ml for ATN and 20-100 μg/ml for

NFD. Aliquots (20μl) of each solution were injected under the operating chromatographic

conditions described as above.

I) Linearity

Suitable dilutions using methanol were made from the standard stock solutions containing 1000

μg/ml of atenolol and 400 μg/ml of nifedipine, to prepare range of standard solutions of five

different concentrations of analyte for further experimental work. In each dilution

carbamazepine of 100 μg/ml concentration was used as internal standard. Five replicates of

each concentration were injected. The linearity of the relationship between peak area and

concentration was determined by analyzing five working standards over the concentration

Sr.

No.

Label Claim mg/cap Amount found mg/cap % amount drug found

ATN NFD ATN NFD ATN NFD

1. 50 20 49.99 19.98 99.98 99.95

2. 50 20 49.96 19.96 99.92 99.92

3. 50 20 49.98 19.99 99.96 99.97

4. 50 20 49.89 19.95 99.78 99.89

5. 50 20 49.93 19.99 99.86 99.97

6. 50 20 50.06 19.99 100.12 99.98

Component %Mean Standard

Deviation

Co-efficient of

Variation

Standard

Error

Atenolol 100.61 0.6197 0.6172 0.2530

Nifedipine 99.30 0.3406 0.3413 0.1390



range 20-100 µg/ml for atenolol and 20-100 μg/ml for nifedipine. The results obtained are

shown in table 5 and 6.

Table5: Linearity of atenolol

Standard

conc. →

20 μg/ml 40 μg/ml 60 μg/ml 80 μg/ml 100 μg/ml

Replicates ↓ Peak area

1 43353.5 88707.1 1330061 176014.3 221767.9

2 43487.6 88356.9 1330487 173686.7 221143

3 43786 88846 1331124 170376 222067.4

4 43921 884898 1330695 190997 221868

5 43298.9 88297.4 1329999 171567 221556.9

Mean 43569.4 88541.1 1330473 17528.2 221680.6

SD 272.49 232.39 465.81 2312.7 352.3

%RSD 0.62 0.26 0.03 1.34 0.15

Table 6: Linearity of Nifedipine

Standard

conc.→

20 μg/ml 40 μg/ml 60 μg/ml 80 μg/ml 100 μg/ml

Replicates

↓

Peak area

1 12998.7 26017.4 39026.1 52983.8 65043.6

2 12567.3 26234 39312 52980 64990

3 12608.9 26987.1 39879 53002 65935

4 12999.9 26154.9 39107 52572 65324

5 12837 26068 39078 52434 55109

Mean 12802.5 26292.2 39280.42 52794.36 65280.32

SD 207.06 397.1 351.7 270.5 387.3

%RSD 1.61 1.51 0.89 0.51 0.59



II. Precision

One set of three different concentrations of combined working standard solution of nifedipine

and atenolol were prepared. All the solutions were analyzed thrice, in order to record any intra-

day variation in the result. The result obtained for intra-day variations are shown in the table 7

and 8. For inter-day variation study, three different concentrations of the combined standards

were analyzed for three days. The result obtained for inter-day variations are shown in the

table 9 and 10.

Table 7: Intra-day variability of Atenolol

Table 8: Intra-day variability of Nifedipine

Conc.

(μg/ml)

Peak area

Mean

SD

% RSD

Trial 1 Trial 2 Trial 3

20 12998.7 12567.3 12608.9 12724.9 237.9 1.8

40 26017.4 26234 26987.1 26292.2 397.1 1.9

60 39026.1 39312 39879 39280 434.1 1.1

Table 9: Inter-day variability of Atenolol

Conc.

(μg/ml)

Peak area

Mean

SD

% RSD

Day 1 Day 2 Day 3

20 43786 43921 43298.9 43668.6 327.2 0.74

40 88846.5 88498 88297.4 88547.3 277.8 0.31

60 1331124 1330695 1329999 1330606 567.6 0.04

Conc.

(μg/ml)

Peak area

Mean

SD

%

RSD Trial 1 Trial 2 Trial 3

20 43353.5 43487.6 43786 43569.4 272.4 0.62

40 88707.16 88356.9 88846.5 88541.1 232.3 0.28

60 1330061 1330487 1331124 1330557 535.1 0.04



Table 10: Inter-day variability of Nifedipine

Conc.

(μg/ml)

Peak area

Mean

SD

%

RSD Day 1 Day 2 Day 3

20 12608.9 12999.9 12837 12815.5 196.4 1.5

40 26987.1 26154.9 26068 26403.3 507.4 1.9

60 39879 39107 39078 39354.6 454.3 1.1

III. Accuracy

To check the accuracy of proposed method, level of recovery carried out at 80, 100 and 120 %

of the concentration as per standard addition method. To perform recovery studies of the test

concentration, a powder of preanalysed capsule sample containing 50 mg of atenolol and 20

mg of nifedipine was weighed such that it should contain 50 mg of atenolol and 20 mg of

nifedipine then transferred into 100 ml volumetric flask, add about 50 ml of methanol and

sonicated for 20 min with intermediate shaking and volume make up to the mark. 100 µg/ml

and 40 µg/ml of atenolol & nifedipine pure drugs were used as standard concentrations, finally

% recovery was calculated and results and statistical validation are shown in table 11 and 12.

Table 11: Recovery studies

Capsule sample

Level of recovery

(%)

Amount

present

( µg/ml )

Amt of std. added

( µg/ml )

Total amount recovered

( µg/ml )

% Recovery

T

E

N

O

F

E

D

ATN NFD ATN NFD ATN NFD ATN NFD

80 100 40 80 32 178.66 71.17 99.25 99.84

80 100 40 80 32 179.51 71.83 99.72 99.76

80 100 40 80 32 178.41 71.37 99.11 99.12

100 100 40 100 40 198.24 79.81 99.20 99.76

100 100 40 100 40 199.71 79.86 99.62 99.82

100 100 40 100 40 198.88 79.90 99.44 99.87

120 100 40 120 48 219.97 87.91 99.98 99.96

120 100 40 120 48 218.98 87.79 99.53 99.76

120 100 40 120 48 219.87 87.84 99.94 99.81



Table 12: Statistical validation

IV. Specificity

A blend of commonly used excipients was treated as per developed procedure and the

chromatogram showed no inferring peaks at retention time of the both drugs and internal

standard.

Fig 7: Blend of excipients with atenolol, nifedipine and carbamazepine

V) Robustness

Robustness of the method was determined by carrying out the analysis under conditions during

which mobile phase ratio and ambient temperature were altered. Variations of mobile phase

pH and ratio are seemed to have greater impact on resolution and hence it should be

meticulously controlled.

Capsule

Sample

Type of recovery %

(%) Mean

SD

Std. error of mean

Tenofed

ATN NFD ATN NFD ATN NFD

80 99.36 99.57 0.319 0.394 0.321 0.396

100 99.42 99.81 0.210 0.055 0.2119 0.005

120 99.94 99.84 0.249 0.104 0.332 0.104



Fig 8: Chromatogram of nifedipine and atenolol in mobile phase of methanol: potassium

dihydrogen phosphate buffer pH 4 (60:40 v/v)

Regression parameters are mentioned in Table 5.

Table 5: Summary for Validation Parameters

Parameters Atenolol Nifedipine

Linearity range 10-100 μg/ml 10-100 μg/ml

Correlation

Coefficient

0.999 0.999

Slope (m) 14128.76 7966

Intercept 2000 -628.072

Specificity No Interference at Rt of the

analyte peak

No Interference at Rt of the

analyte peak

Method Precision (%Rsd) 0.1 0.0

Accuracy (%Rsd) 1.424 2.466

Robustness (%Rsd) 16.5 15.8

LOD 0.6 μg/ml 0.35 μg/ml

LOQ 2.02 μg/ml 1.07 μg/ml



RESULTS AND DISCUSSION

A RP-HPLC method was developed and validated for the determination of NFD and ATN in

capsule dosage forms on a HiQ Sil C8- 250×4.6 mm, particle size 5μm column with variable

wavelength detection at 237 nm. The retention times for Nifedipine and Atenolol are 7.7 min

and 1.8 min, respectively. The LOD and the LOQ for Nifedipine and Atenolol were found to be

0.15 and 0.05 μg/ml and 0.22 and 0.075 μg/ml, respectively. These data show that method is

sensitive for the determination of Nifedipine and Atenolol The recovery experiment was

performed by the standard addition method The Percentage recovery for Nifedipine and

Atenolol are ranged between 99.10–100.4 and 99.32–100.02 respectively. The results of

recovery studies indicate that the proposed method is highly accurate. The proposed validated

method was successfully applied to determine Nifedipine and Atenolol in their capsule dosage

form. The results obtained for Nifedipine and Atenolol were comparable with the

corresponding labelled amounts. No interference of the excipients with the absorbance of

interest appeared; hence, the proposed method is applicable for the routine simultaneous

estimation of Nifedipine and Atenolol in pharmaceutical dosage forms. A simple, linear,

accurate, specific and selective RP-HPLC method was developed and validated for estimation of

Nifedipine and Atenolol in their combined dosage form. In this proposed method the linearity

range for both Nifedipine and Atenolol was found to be 10-100 μg/ml with coefficient of

correlation, (r2)=0.999 and (r2)=0.999 for Nifedipine and Atenolol, respectively at 237 nm. The

result of the analysis of pharmaceutical formulation by the proposed method is highly

reproducible and reliable and it is in good agreement with the label claim of the drug. The

method can be used for the routine analysis of the Nifedipine and Atenolol in combined dosage

form without any interference of excipients.

CONCLUSION

In the present investigation, we have developed a simple, sensitive, precise and accurate RP-

HPLC method for the quantitative estimation of Nifedipine and Atenolol in bulk drug and

pharmaceutical formulations and a simple, sensitive, precise and accurate RP-HPLC method for

the simultaneous estimation of Nifedipine and Atenolol in bulk drug and pharmaceutical

formulations. These methods can be used for the routine determination of Nifedipine and

Atenolol bulk drug and in pharmaceutical formulations.

REFERENCES

1. Christian, G.D. , Analytical Chemistry, 5 th Edn., John Wiley and Sons, Inc, Year,1-3.1-

19,555-571, 604-632

2. Skoog, D.A., West, D.M., Holler, F.J. and Crouch, S.R., In; Fundamentals of Analytical

Chemistry, 8th Edn., Thomson Asia Pvt. Ltd., Year 160-167, 971-995.



3. Connors, K.A., In; A Textbook of Pharmaceutical Analysis, 3rd Edn, John Wiley and Sons,

1982, (173,187, 221-222),385 -414 4.

4. Ashutosh Kar, Phar.maceutical Drug Analysis,New age International (P) Ltd,Publishers,,452-

473.

5. Galen. W. Ewing, Instumental Methods of Chemical Analysis, 5thEdition,HILL International

Editions-Chemisrty series,340-347,375-393.

6. Willard, H.H., Merritt, y L.L. (Jr)., Dean, J.A., Settle, F.A.(Jr)., In; Instrumental Methods of

Analysis, 6th Edn., CBS Publishers, Year 82,83, 169-172

7. Sharma, Y.R., In; Introduction of Organic Spectroscopy, 4th Edn, CBS Publishers and

Distributors, New Delhi, 1991, 22.

8. Beckett, A.H. and Stenlake, J.B., In; Practical Pharmaceutical Chemistry, 4th Edn., Part 2, CBS

Publishers and Distributors, 2002, 290- 300.

9. Skoog, D.A., Holler, F.J., Timothy, A. and Nieman, N.W., In; Principle of Instrumental

Analysis, 3rd Edn., Stanford University, Saunders College Publications, London, 1985, 1-4.

10. ICH, Q2A, Text on Validation of Analytical Procedures, International Conference on

Harmonization, Geneva, 1-5.

11. ICH, Q2B, Validation of Analytical Procedures: Methodology, International Conference on

Harmonization, Geneva, 1-8.

12. Sethi, P.D., In; Quantitative Analysis of Drugs in Pharmaceutical Formulations, 3rd Edn., CBS

Publishers and Distributors, New Delhi, 1997, 64

13. Sethi, P.D., In; HPLC‘High Performance Liquid Chromatography’, Quantitative Analysis of

Pharmaceutical Formulations, 1st Edn., CBS Publishers and. Distributors, New Delhi, 2001, 3-72,

116-120.

14. Jeffery, G.H., Bassette, J., Mendham, J. and Denney, R.C., In; Vogel’s Textbook of

Quantitative Chemical Analysis, 5 th Edn., Longman Publication,1998,668.

15. Chatwal. G., Anand. K.S., Hi Himalaya Publishing House, 1.1-1.24, 2.566-2.586, 2.624-2.639.

16. www.\An Introduction To Analytical Method Development For Pharmaceutical

Formulations Pharmainfo_net.htm



17. www.\Analytical Method Development and Validation.htm

18. www.\Reverse Phase HPLC Basics for LC-MS-IMP.htm

19. Indian Pharmacopoeia, Vol I, The Controller Of Publication, New Delhi. 1996, 72.

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