DEVELOPMENT AND VALIDATION OF DISSOLUTION TEST …

www.wjpps.com Vol 4, Issue 10, 2015.

2034

Gupta et al. World Journal of Pharmacy and Pharmaceutical Sciences

DEVELOPMENT AND VALIDATION OF DISSOLUTION TEST

METHOD FOR ALISKIREN IN TABLET BY HPLC

Kiran N. Kale and Krishna R. Gupta*

Department of Pharmaceutical Chemistry, Smt Kishoritai Bhoyar College of Pharmacy,

New Kamptee, Nagpur (MS)

ABSTRACT

A dissolution test method was developed and validated for the Quality

control of Aliskiren in its tablet dosage form using RP-HPLC method.

The dissolution condition includes USP apparatus II at a paddle

rotation rate of 50 rpm and 900 mL of 0.1N HCL buffer at

37°C±0.5°C. The described method showed good results under

optimized conditions. The in-vitro release was evaluated on

Hyperchrom- ODS 5µ C18 column (250 X 4.6 mm) with flow rate was

1.0mL/ min and detection wavelength 280nm. The mobile phase was

prepared by mixing Acetonitrile and 0.05 M KH2PO4 Buffer

(45:55v/v) and pH was adjusted to 2.5 with 10% Ortho –phosphoric

acid. The developed method was successfully validated according to

USP Guidelines. The proposed method for dissolution test in tablet

formulation showed reliable, precise, accurate results. Hence this method could be used in

routine monitoring of the quality control of the Aliskiren in its tablet dosage form.

KEYWORDS: Dissolution test; HPLC; Aliskiren; validation.

INTRODUCTION

Dissolution is a technique in which a solid substance solubilises in a given solvent i.e. Mass

transfer from the solid surface to the liquid phase. It is the primary quality control test to

determine whether a drug product can release its active pharmaceutical ingredient(s) in a

timely manner.[1]

Dissolution test is required for various dosage forms for product release

testing. It is also commonly used as a predictor of the in vivo performance of a drug product.

The basic destination of dissolution testing is to allow the measurement of bioavailability of a

dose in addition to bioequivalence of batch to batch. The focus of dissolution testing in QC is

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 5.210

Volume 4, Issue 10, 2034-2049 Research Article ISSN 2278 – 4357

Article Received on

17 Aug 2015,

Revised on 09 Sep 2015,

Accepted on 29 Sep 2015

*Correspondence for

Author

Krishna R. Gupta

Department of

Pharmaceutical

Chemistry, Smt Kishoritai

Bhoyar College of

Pharmacy, New Kamptee,

Nagpur (MS).


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batch consistency and detection of manufacturing deviations. The test should be designed to

demonstrate that the dosage forms were manufactured according to specifications and all

critical manufacturing steps result in a consistent product. In vitro dissolution data, together

with bioavaibility and chemistry, manufacturing and control data, is a critical component of

any new drug application (NDA) submitted to the FDA.[2]

Aliskiren Hemifumarate (Alsk) chemically described as (2(2S,4S,5S,7S)-5-amino-N-(2-

carbamoyl-2,2-dimethylethyl)-4-hydroxy-7-{[4-methoxy-3-(3 methoxypropoxy)phenyl]

methyl}-8-methyl-2-(propan-2-yl) nonanamide .(Fig. 1) is an orally active rennin inhibitor

for the treatment of essential hypertension and heart failure. Aliskiren metabolized slowly in

the body resulting in stronger half lives which restrict it once a day dosing.[3,4]

Literature survey reveals that few spectrophotometric methods and HPLC methods for assay

has been reported for the estimation of Aliskiren alone or in combination with other

antihypertensive agents in pharmaceutical formulations 3-10

. Literature survey revealed no

methods are reported for dissolution test by RP-HPLC method. The present work describes

the development and validation of a RP-HPLC method for dissolution test analysis for

Aliskiren, which was then optimized on the basis of solubility and stability considerations.

METHOD AND MATERIALS

Aliskiren Hemifumarate was kindly obtained as a gift sample from Morepen Lab. Ltd.

(Delhi). Commercial tablets containing Aliskiren 150mg was procured from the local chemist

shop manufactured by Novartis. Acetonitrile, used was of HPLC grade. Double distilled

water was used for preparing both dissolution media and HPLC mobile phase. Other

chemicals were of either AR grade or GR grade.

INSTRUMENTATION


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Dissolution apparatus USPXVIII, Model no. EF 1 W, Electrolab Pvt. Ltd was used for

dissolution. Chromatographic separation was performed on Analytical Technology Limited

isocratic system consisted of hyperchrome ODS 5µ C18 column (250 X 4.6 mm), Uv-3000

detector and P-3000 Pump, Rheodyne injector with 20μL capacity. The mobile phase

comprised of 0.05M Potassium Dihydrogen phosphate Buffer: Acetonitrile, pH 2.5 (55:45) at

flow rate 1.0 mL/min. The mobile phase was filtered through a 0.45 μ membrane filter and

sonicated for 15min. Analysis was performed at ambient temperature. The detection was

monitored at 280nm.

Selection of Wavelength

The standard solutions aliskiren was prepared and subjected to U.V. spectrophotometeric

study to determine λ max of drug using mobile phase as blank solution. Aliskiren shows

maximum absorption at 280nm. The wavelength was selected as 280 nm, such that the drug

exhibit sufficient absorbance at the selected wavelength. The spectrum recorded was shown

in Fig. 2

-1

7

0

2

4

6

200 400250 300 350

Abs

Wavelength [nm]

Fig. 2: UV spectra of Aliskiren standard solution

Preparation of Mobile Phase

Preparation of 0.05M Phoshphate Buffer solution: Potassium Di hydrogen orthophosphate, 7g

was dissolved in 1000 mL of water and Mixed, pH adjusted to 2.5 using ortho-phosphoric

acid, sonicated to degas the buffer.

The mobile phase was prepared by mixing Acetonitrile and Buffer in the ratio of 45:55v/v.

The mobile phase was filtered through nylon 0.45μm membrane filter. The same mobile

phase was used as diluents.


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Aliskiren stock solution

A standard stock solution of Aliskiren was prepared in mobile having the concentration of

1mg/mL. A 2.0 mL portion of the above solution was further diluted up to 25.0 mL with

mobile phase (80μg/mL).

System Suitability Parameters

System suitability tests were carried out by making five replicate injections of a standard

solution containing 80μg/mL of aliskiren and analyzing the chromatograms for peak area,

theoretical plates, RSD and tailing factor. Triplicate of 20 μL injections were utilized for each

analysis. The results are shown in Table 1

Table 1: Result of System Suitability Studies

Solubility Studies

The in vitro studies were performed using different dissolution conditions. According to USP

32, dissolution medium may be water, an aqueous solution (typically pH 4.0 to 8.0) or a

dilute acid solution (0.001 to 0.1mol L-1 HCl). Due to the drug is a hydrophilic molecule

with high aqueous solubility, surfactants and electrolytes were not added.[10,11]

Dissolution Test Conditions

Drug release tests were carried out with paddle method (USP apparatus II) at 50 rpm and 75

rpm, dissolution volume of 900 mL. The temperature of the cell was maintained

Sr. No.

Standard

Weight

Taken(mg)

A.U.C of ASK

(mV)

1

~167.0

279.78

2 279.27

3 278.77

4 278.54

5 279.04

Mean 279.08

±S.D. 0.174

%RSD 0.1714

Theoretical plate/column 7255

Retention time 3.893

Asymmetry 1.123


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at37°C±0.5°C by using a thermostatic bath. Various dissolution media’s were tried out of

which 0.1N HCl buffer was selected. Weighed and dropped 1 tablet in each of the six

dissolution vessel containing 0.1N HCL for the respective drug under analysis. Sampling

aliquots of 10.0 mL were withdrawn at 5, 10, 15, 20, 30, 45, 60 min and infinity time interval

and replaced with an equal volume of the fresh medium to maintain a constant total volume.

After the end of each test time, sample aliquots were filtered and quantified. The percentage

content was calculated by validated HPLC method and these contents results were used to

calculate the percentage release on each time of dissolution.

Dissolution Method Parameter Optimization

Change in the USP Apparatus

0.1 N HCL was selected as dissolution media to study the effect of change in USP apparatus

due to high solubility of Aliskiren in 0.1 N HCL .A media volume of 900 mL was kept

constant as in trial and error basis the dissolution was performed on two different USP

apparatus.The results were obtained for Aliskiren. The % Drug release in different apparatus

was calculated.

Change in the Volume of Dissolution Media

The dissolution media was kept same as used in the above study where as dissolution was

performed using USP II with media volume varied from 1000 mL to 500 mL. The % release

was calculated.

Change in Dissolution Media (Buffer)

Phosphate buffer [pH 5.0] and phosphate buffer [pH 6.8] were used as dissolution media,

with a media volume of 900 mL as above. The % release was calculated.

Method Validation

The dissolution test method was validated to through the determination of linearity, precision,

accuracy, solution stability. Prior to injecting sample solutions, the column was equilibrated

for at least 30 min with the mobile phase flowing through the system.

1. Linearity


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To assess the linearity, 10%-200% level of target concentration (83.33μg/mL for Aliskiren)

solutions were prepared and chromatographed. The peak area of each solution was noted. The

plot of concentration vs area is shown in Fig. 3

Fig. 3: Graph showing linearity of Aliskiren at different concentration

2. Precision

The precision of the method was determined by measuring the precision expressed as %

RSD. Tablet samples were subjected to dissolution test conditions 900 mL of dissolution

medium (0.1 N HCL) preheated at 37°C±0.5°C, paddle with stirring rate of 50 rpm).Test

solutions obtained from dissolution were chromatographed using optimized chromatographic

conditions. Thus, the %RSD was calculated ascertaining the precision of the method.

3. Accuracy

The accuracy was evaluated for the proposed method by spiking method i.e. adding known

amount of Aliskiren and standard drug (30%-125% ) to that of target concentration in the

dissolution medium considering the label claim of respective drug at 100% drug target

concentration [167 μg of aliskiren ] and performing the dissolution test. Each solution were

analysed in triplicate. The accuracy was calculated as the percentage of the drug recovered

from the formulation matrix. Total amount of drug estimated was calculated using following

formula-

Total amt of drug estimated - Label claim

% Recovery = -------------------------------------------------- X 100

Amt of drug (mg) added


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Where, At = Peak area of test sample, As= Peak area of standard sample Ws = Weight of

standard, V1 = Volume of diluent usedfor dissolving the standard, V2 = Amount of volume

taken from ‘V1’, V3 = Volume used to dilute the ‘V2’, up to mark,V4 = Volume of diluent

used for dissolving the test, L= Label claim.

4. Range

The range study was performed to show the range of method in which method is linear,

accurate and precise. The test solutions prepared for accuracy study were used for the range

determination.

5. Ruggedness

a. Standard Solution Stability

The standard solution stability was analyzed over as specified period of time, verifying the

response of the standard solution. Five injections of standard solution were injected at 0th

hr,

24th

hr and 48th

hr and the chromatograms were recorded. The relative standard deviation

was calculated for the area of standard.

b. Test Solution Stability

The test solution stability was analyzed over a specified period of time, verifying the

response of the sample solution stored at bench top condition (25°C) and refrigeration (5°C).

The chromatograms were recorded using optimized chromatographic conditions HPLC

method from.

6. Robustness of Test Method

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by

small, but deliberate variations in method parameters and provides an indication of its

reliability during normal usage. The robustness was carried out for following parameters:

a. Change in flow rate

b. Change in pH of mobile phase

c. Change in detection wavelength

d. Change in mobile phase composition


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RESULTS AND DISCUSSION

HPLC method development and optimization

The optimized chromatographic condition mentioned below was kept constant throughout the

experimentation and mobile phase was allowed to equilibrate with stationary phase which

was indicated by a steady line.

Column - Hyperchrome ODS 5 μ C18 column (250 X 4.6mm)

Mobile Phase - Acetonitrile and Buffer pH 2.5 (45:55 v/v)

Detection Wavelength - 280nm

Flow rate - 1.0 mL/min

Temperature (Ambient) - 28-30°C

Injection volume - 20 μL

A 20 μL solution of above mix standard was injected through manual injector and

chromatogram was recorded. A standard chromatogram for Aliskiren and blank so recorded

is shown in Fig. 4a and 4b.

Optimization of Dissolution Method Parameters for Estimation of Alikiren

Various dissolutions were performed to optimize the parameters like dissolution media,

dissolution media volume, apparatus and rpm, using the optimized chromatographic

conditions and the solubility data of the drugs to select a set of parameter that will give

maximum % release of the drug. The chromatograms of dissolution analysis of formulation

under study at selected intervals recorded under optimized chromatographic parameters are

shown in Fig. (5a-5h)

Fig. 4a: Chromatogram of standard under optimized condition


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Fig. 4b: Chromatogram for Blank

Fig. 5a: Chromatogram for Test sample at 5 min

Fig. 5b: Chromatogram for Test sample at 10 min


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Fig. 5c: Chromatogram for Test sample at 15min

Fig. 5d: Chromatogram for Test sample at 20min

Fig. 5e: Chromatogram for Test sample at 30min


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Fig. 5f: Chromatogram for Test sample at 45min

Fig. 5g: Chromatogram for Test sample at 60min

Fig. 5h: Chromatogram for Test sample at infinity


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Change in USP Apparatus

The result of % release of drug is shown in Table 2. From the table, it was observed that the

release of drug in USP I was slow as compared to USP II. Also the release of drug at 50 rpm

was found to be optimum as compared to other rpm conditions; therefore USP II and 50 rpm

were selected as one of the optimized dissolution parameter and were further used in the

experimentation.

Table 2: Results Showing Effect of Change in USP Apparatus

Change in the Volume of Dissolution Media

The result of % release of drug is shown in Table 3. From the table, it can be observed that

the percent drug release in a media volume using 1000 mL and 500 mL was found to be less

as compared to a media volume of 900 mL. Hence media volume of 900 mL was selected as

one of the optimized dissolution parameter and was further used in the experimentation.

Table 3: Results Showing Effect of Change in Volume of Dissolution Media

Time

points

Mins

Dissolution Medium: 0.1N HCl Dissolution Volume:

900 mL

Apparatus: USP-I Apparatus: USP-II

% Release at RPM

50 75 50 75

Mean ±SD Mean ±SD Mean ±SD Mean ±SD

5 4.78 0.21 7.94 0.24 12.07 0.12 16.60 0.05

10 10.50 0.14 12.98 0.52 25.52 0.26 24.48 0.08

15 21.24 0.52 26.89 0.45 34.71 0.14 46.13 0.22

20 30.37 0.32 34.18 0.32 53.63 0.22 61.41 0.11

30 44.28 0.10 49.76 0.33 75.35 0.30 77.28 0.02

45 60.62 0.02 72.12 0.12 85.23 0.27 81.94 0.35

60 76.82 0.45 83.89 0.12 99.39 0.20 86.38 0.44

Infinity 79.97 0.14 86.36 0.17 101.29 0.11 95.21 0.12

ASK

Dissolution Media:0.1N HCl Apparatus: USP-II

900mL 500mL 1000mL

% Release at RPM

Time

points

50 75 50 75 50 75

Mean ±SD Mean ±SD Mean ±SD Mean ±SD Mean ±SD Mean ±SD

5 13.18 0.12 16.90 0.14 7.39 0.10 9.90 0.12 5.88 0.22 8.79 0.21

10 26.12 0.25 25.22 0.42 16.88 0.25 21.50 0.20 17.05 0.35 21.55 0.32

15 34.58 0.08 46.78 0.19 31.03 0.12 39.13 0.06 29.63 0.10 34.85 0.14

20 54.95 0.19 61.58 0.35 39.35 0.31 50.37 0.19 42.96 0.08 44.61 0.11

30 76.95 0.54 77.50 0.54 55.54 0.02 67.10 0.13 55.39 0.18 59.71 0.20


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Change in Dissolution Media (Buffer)

The result of % release of drug is shown in Table 4. From the Table, it was observed that the

release rate of the drug obtained was less in phosphate buffer (pH 5.0) and Phosphate Buffer

(pH 6.8) compared to 0.1 N HCl at Q point 60 min therefore, 0.1N HCl was selected as

optimized dissolution media and further used in the experimentation. The finalized

dissolution parameter selected for the dissolution test method of Aliskiren are shown in Table

5 and percent release of drug under final chromatographic and final dissolution parameters

are shown in Table 6 for aliskiren.

Table 4: Results Showing Effect of Change in Dissolution Medium

Table 5: Final Dissolution Method Parameters for the Drug

Table 6: Percent Release of drug Under final chromatographic condition

45 85.94 0.32 82.95 0.62 65.68 0.26 77.29 0.21 74.09 0.13 78.45 0.14

60 98.98 0.10 87.28 0.10 75.91 0.10 83.21 0.19 88.86 0.22 91.47 0.18

Infinity 101.50 0.20 95.42 0.15 82.50 0.05 89.10 0.11 90.62 0.14 93.84 0.10

ASK

Dissolution Media Volume:900 mL Apparatus: USP-II

%Release at RPM

0.1 N HCl Phosphate buffer pH 6.8 Phosphate buffer pH 5.0

Time

points

50 75 50 75 50 75

Mean ±SD Mean ±SD Mean ±SD Mean ±SD Mean ±SD Mean ±SD

5 12.90 0.20 15.40 0.32 9.21 0.40 12.21 0.34 7.18 0.12 9.90 0.23

10 25.42 0.15 26.20 0.12 21.62 0.05 32.21 0.18 20.03 0.10 23.30 0.19

15 35.19 0.12 44.56 0.34 39.95 0.12 47.42 0.19 37.16 0.19 39.40 0.11

20 54.95 0.24 62.25 0.27 52.27 0.15 59.25 0.12 61.36 0.07 63.58 0.42

30 77.30 0.39 76.85 0.10 63.68 0.19 66.05. 0.22 75.92 0.23 78.90 0.24

45 86.04 0.32 83.48 0.29 72.18 0.32 75.07 0.31 78.05 0.17 80.09 0.32

60 99.98 0.15 88.37 0.09 80.65 0.14 82.15 0.15 81.59 0.21 82.55 0.14

Infinity 100.90 0.20 95.30 0.11 85.21 0.29 88.08 0.12 84.36 0.18 86.42 0.31

Drugs Dissolution media volume USP Appratus Agitation/Rotation(RPM)

Aliskiren 0.1N HCL 900 ml II 50

Time

Point

Retention

time

A.U.C

(mV)

%Drug

Release Asymmetry

Theoretical

Plates

5 3.890 33.85 12.07 1.132 7055

10 3.865 71.96 24.84 1.130 7045

15 3.875 93.79 33.50 1.129 7015

20 3.870 151.75 54.23 1.128 6995

30 3.871 213.85 76.65 1.129 6985

45 3.880 241.05 86.31 1.130 6980

60 3.879 278.12 99.39 1.131 6965

Infinity 3.873 281.60 100.90 1.130 6950


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Validation Parameters

The peak area of linearity solutions noted was plotted against the corresponding

concentrations to obtain the calibration graphs. The coefficient of correlation for Aliskiren

was found to be 0.9999.The observation and the result of precision study for drug is

summarized in the Table 7. The proposed method was found to be precise as repeatability of

measurements was determined as percent dissolution which should not be less than 75%

release at 45 minutes and %RSD should not be more than 5.0% for drug under analysis. The

% release was found to be above the specification and %RSD of drugs was found to be

0.2145 ascertaining the precision of method. The results of accuracy data at different spike

level are shown in table 8. From the accuracy studies % recovery of drugs at each accuracy

level was found to be in the range of 98.0%-101% which is found to be in the acceptance

range of 95%-105%. Standard solutions of Aliskiren and its formulation under study are

stable for the period of up to 48 hrs. The relative standard deviation for peak areas of three

replicate injection of mix standard solution under varied condition should not be more than

5.0%.Hence, proposed method was found to be robust.

Table 7: Result of Precision Study for Drugs

Table 8: Observation and Results of Recovery Studies

Spike

Level

Amt of pure

drug added

(mg)

AUC (mV)

Total Amt. of

Drug Estimated

(mg)

Amt

Recovered

%

Recovery

30% 17.80 147.970 41.92 17.46 98.08

50% 49.40 257.955 73.08 48.56 98.29

60% 64.87 315.940 89.51 65.11 100.37

75% 86.50 391.585 110.94 86.74 100.27

100% 124.90 530.960 150.43 125.68 100.94

125% 160.95 665.320 185.84 164.29 102.07

Mean 100.03

±SD 1.196

% RSD 1.195

S.NO AREA(mv) % Dissolution

1 281.58 100.37

2 280.16 99.85

3 280.83 100.09

4 280.94 99.99

5 281.39 100.29

Mean 100.116

%RSD 0.2145


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CONCLUSION

The obtained results by the developed RP-HPLC method for Dissolution test of tablet

formulation containing Aliskiren was found to be precise, accurate and reliable. Hence the

proposed method can be adopted for routine dissolution analysis as well as Quality control

test of said drug in their formulation.

ACKNOWLEDGEMENT

I am very much thank full to my guide and Principal, S.K.B. College of Pharmacy, Kamptee

for his guidance, kind help and constant encouragement at every step during the progress of

my work.

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