STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT … · Oxidation 8644.06 9.00 Photo 7578.32 20.22 Thermal 8120.12 14.52 VALIDATION OF DEVELOPED METHOD Linearity By analysis of

www.wjpps.com Vol 9, Issue 6, 2020.

1133

Harmani et al. World Journal of Pharmacy and Pharmaceutical Sciences

STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT

AND VALIDATION FOR THE ESTIMATION OF DAROLUTAMIDE IN

PHARMACEUTICAL DOSAGE FORM

Nirav J. Harmani*, Ojas B. Patel, Neha S. Patel and Dr. Harsha U. Patel

Faculty of Pharmacy-Shree Satsangi Saketdham ―RAM ASHRAM‖ Group of Institution,

Gujarat Technological University, Mehsana-382708, Gujarat, India.

ABSTRACT

A simple, rapid, economical, precise and accurate RP-HPLC

method for Darolutamide in its Pharmaceutical Dosage Form has

been developed. A reverse phase high performance liquid

chromatographic method was developed for the Darolutamide in its

Pharmaceutical Dosage Form. The separation was achieved by

Hypersil BDS C18 (250 mm x 4..6 mm, 5µ) column and Acetonitrile:

Phosphate Buffer, pH 3.0 (20:80) as mobile phase, at a flow rate of

1 mL/min. Detection was carried out at 242 nm. Retention time of

Darolutamide was found to be 10.303 min. The method has been

validated for linearity, accuracy and precision. Linearity observed for

Darolutamide 25-75 μg/mL. Developed method was found to be

accurate, precise and rapid for estimation of Darolutamide in its

Dosage Form. The drug was subjected to stress condition of

hydrolysis, oxidation, photolysis and Thermal degradation, Considerable Degradation was

found in alkaline degradation. The proposed method was successfully applied for the

estimation of Darolutamide in Pharmaceutical dosage form.

KEYWORDS: Darolutamide, Stability Indicating RP-HPLC Method, Validation.

1. INTRODUCTION

Prostate cancer is the cancer that occurs in the prostate gland — a small walnut-shaped gland

in men from that the seminal fluid generated which promotes and carries sperm. When some

cells of the prostate become atypical, atypical cells develop and split faster than normal cell

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.632

Volume 9, Issue 6, 1133-1146 Research Article ISSN 2278 – 4357

*Corresponding Author

Nirav J. Harmani

Faculty of Pharmacy-Shree

Satsangi Saketdham "RAM

ASHRAM" Group of

Institution, Gujarat

Technological University,

Mehsana-382708, Gujarat,

India.

Article Received on

25 March 2020,

Revised on 15 April 2020,

Accepted on 06 May 2020

DOI: 10.20959/wjpps20206-16238


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because of mutations in the abnormal cells' DNA. While atypical cells continues developing,

development of the normal cells decreases and would die rapidly. Due to accumulation of

atypical cells, formation of tumor happens that can develop and attack surrounding tissue.

These atypical cells can be affect to other parts of the body after the break off and metastasize

(spread).

Darolutamide is chemically N((S)-1-(3-(3-Chloro-4-cyanophenyl)-1H-pyrazol-1-yl) propan-

2-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide (Fig. 1), is a drug used for the

treatment of Prostate Cancer from Nonsteroidal antiandrogen category. Darolutamide is

second- or third-generation nonsteroidal anti androgen (NSAA). It acts as a selective

competitive silent antagonist of the androgen receptor (AR), the biological target of

androgens like testosterone and dihydrotestosterone (DHT). Its affinity (Ki) for the AR is 11

nM and its functional inhibition (IC50) of the AR is 26 nM. It is white to yellowish white

powder. It is insoluble in water and soluble in DMSO, Methanol and Acetonitrile.

Darolutamide is not official in any pharmacopeia. Literature review reveals that the methods

reported for the estimation of Darolutamide is in mice plasma only. But no method has been

reported for the estimation of Darolutamide in Tablet dosage form by HPLC. So it is thought

of interest to develop specific, accurate, precise, rapid, simple, selective and sensitive

stability indicating HPLC method for estimation of Darolutamide in Tablet dosage form.

Fig. 1: Chemical structure of Darolutamide.

2. METHODS AND MATERIALS

Chemicals and Reagents

A sample of Darolutamide was obtained as a gift from Sotac Healthcare, India. Methanol and

Acetonitrile of HPLC grade, KH2PO4 and NaOH of AR grade, HCl and H2O2 of LR grade

and Water of Milli-Q grade were used. All the chemicals were procured from the Finar

Chemicals Limited, India.


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Apparatus and Instrumentation

Chromatographic separation achieved by the Shimadzu HPLC system (Model: LC-10 AT)

with SPD 20A UV detector and LC solution software, Hypersil BDS C18 (250 × 4.6 mm, 5µ)

column, Shimadzu analytical balance, Lab man pH meter and Ultrasonicator.

PREPARATION OF STANDARD SOLUTIONS

Darolutamide standard stock solution (500 μg/mL)

Weighed 50 mg of Darolutamide and transfer it into a 100 mL volumetric flask. Dilute it with

methanol to the mark.

Preparation of working solution Darolutamide (50 μg/mL)

Transferred 1 mL from the Darolutamide stock solution into 10 mL volumetric flask and

Dilute it up to the mark with mobile phase which was used in particular trials.

Phosphate Buffer preparation

Weighed 6.8 gm of Phosphate buffer and was transferred to 1000 mL beaker and 200 mL of

water added and shaken for few minutes than volume was made up with water, pH was

adjusted by adding 1% o-Phosphoric acid or 0.1M KOH.

Diluent: Mobile phase

Selection of wavelength

The sensitivity of HPLC method that uses UV detection depends upon proper selection of

detection wavelength. An ideal wavelength is the one that gives good response for the drugs

that are to be detected. In the present study drug solutions of Darolutamide (50 µg/mL) was

prepared in Methanol. This drug solution was then scanned in UV region of 190-400 nm and

maximum using the Methanol as blank Absorbance was recorded. Maximum absorbance was

achieved at 242 nm shown in fig. 2.


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Fig. 2: UV Spectra of Darolutamide.

HPLC Method Development

Trial contains various mobile phase which are considered of Methanol, Acetonitrile and

Water in different proportions and different volumes at different flow rate were tried. On the

basis of various trial the mixture of Acetonitrile: Phosphate Buffer, pH 3.0 (20:80 %v/v) at 1

mL/min flow rate, proved to be better than the other mixture in terms of peak shape,

theoretical plate and asymmetry shown in fig. 3. It was observed that developed

chromatographic condition provides better separation of Darolutamide at 10.303 min.

Fig. 3: Optimized Chromatogram of Darolutamide.


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Optimization of HPLC Method

The developed HPLC method was optimized with a view to develop stability indicating

method. The method was optimized on C18 (250× 4.6 mm, 5µ) Hypersil BDS with isocratic

method of elution using the mobile phase Acetonitrile: Phosphate Buffer, pH 3.0 (20:80%v/v)

at a flow rate of 1 mL/min, 20 µL injection volume and 20 min run time. The detection was

carried out at 242 nm.

Forced Degradation Studies

Stability-Indicating method are based on the characteristic structural, chemical or biological

properties of each active ingredient of a drug product, is quantitative analytical methods and

will differentiate separately active ingredient from its degradation products so that the active

ingredient content can be exactly evaluated. Degradation studies were carried out under

different stress conditions like Acidic, Alkaline, Oxidation, Photo and Thermal.

Acid degradation

Acid degradation studies were carried out by taking one mL of stock solution and transfer it in

to 10 mL of volumetric flask. Further added the Two mL of 0.1 N HCl solutions and mixed

well and kept it for 6 hrs at Room temperature. To neutralize the solution, two mL of 0.1 N

NaOH was added after time period. To get 50 μg/mL for Darolutamide, volume was make up

with diluent. Results of acid hydrolysis is shown in fig. 4

Fig. 4: Chromatogram of Acid degradation.


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Base degradation

Base degradation studies were carried out by taking one mL of stock solution and transfer it in

to 10 mL of volumetric flask. Further added the Two mL of 0.1 N NaOH solutions was added

and mixed well and kept it for 6 hrs at Room temperature. To neutralize the solution, two mL

of 0.1 N HCl was added after time period. To get 50 μg/mL for Darolutamide, volume was

make up with diluent. Results of base hydrolysis is shown in fig. 5

Fig. 5: Chromatogram of Base degradation.

Oxidation degradation

Oxidation degradation studies were carried out by taking one mL of stock solution and transfer

it in to 10 mL of volumetric flask. Further added two mL of 3% H2O2 solution and mixed well

and kept for 3 hrs at room temperature. To get 50 μg/mL for Darolutamide, volume was make

up with diluent after time period. Results of Oxidation degradation is shown in fig. 6

Fig. 6: Chromatogram of Oxidation degradation.


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Photo degradation

Photo degradation studies were carried out by taking one mL of stock solution and transfer it in

to 10 mL of volumetric flask. Volumetric flask was kept in UV Chamber for 24 hrs. To get 50

μg/mL for Darolutamide, volume was make up with diluent after time period. Results of Photo

degradation is shown in fig. 7.

Fig. 7: Chromatogram of Photo degradation.

Thermal degradation

Weighed 50 mg of Darolutamide and transfer it in to 100 mL Volumetric flask and kept it in

oven for 6 hrs at 800C temperature, then after Volumetric flask was removed and cool at

room temperature, volume was made up with Methanol, one mL of this solution was taken

and transfer it in 10 mL volumetric flask. To get 50 μg/mL for Darolutamide, volume was

make up with diluent after time period. Results of Thermal degradation is shown in fig. 8.

Fig. 8: Chromatogram of Thermal degradation.


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Table 1: % Degradation Calculation.

Darolutamide

Area of Standard 9499.42

Condition Area of Standard % Degradation

Acid 8033.05 15.44

Base 7532.88 20.70

Oxidation 8644.06 9.00

Photo 7578.32 20.22

Thermal 8120.12 14.52

VALIDATION OF DEVELOPED METHOD

Linearity

By analysis of standard solution in range of 25-75 μg/mL, the linearity for Darolutamide was

evaluated.

The graph of peak area obtained verses respective concentrations were plotted in term of slope,

intercept and correlation co-efficient value.

For Darolutamide calibration curve, Correlation co-efficient (r2) was observed 0.9993.

The regression line equation for Darolutamide is as following:

For Darolutamide y = 191.97x – 109.49

Table 2 Data for Linearity.

Sr. No. Concentration

(µg/mL) Area ± SD (n=5) % RSD

1 25 4748.76 ± 43.57 0.92

2 37.5 7072.00 ± 40.80 0.58

3 50 9469.25 ± 13.22 0.14

4 62.5 11742.99 ± 3.25 0.03

5 75 11411.17 ± 36.12 0.25


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Fig. 9: Calibration Curve of Darolutamide (25-75 μg/mL).

Fig. 10: Overlay Chromatogram of Linearity.

PRECISION

A. Repeatability

Repeatability is measured by six times injecting the standard solution containing Darolutamide

(50 μg/mL) and peak areas were determined and calculation was done to obtain % R.S.D.

The % RSD for Darolutamide (50 μg/mL) was found to be 1.34

Table 3: Repeatability data.

Sr. No. Conc. (μg/mL) Area Mean ± S.D (n=6) % R.S.D

1 50

9384.34

9515.42±127.19 1.34

9441.22

9490.07

9446.24

9602.77

9727.90


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B. Intraday Precision

Standard solution having Darolutamide concentration (25, 50, 75 μg/mL) was injected three

times in same day and peak areas were determined and calculation was done to obtain %

R.S.D.

Intraday precision for Darolutamide was found in range of 0.97-1.84 % RSD

Table 4 Intraday precision data.

Sr. No. Conc. (μg/mL) Mean ± S.D (n=6) % R.S.D

1 25 4773.05±53.43 1.12

2 50 9520.92±174.78 1.84

3 75 14234.87±137.87 0.97

C. Intraday Precision

Standard solution having Darolutamide concentration (25, 50, 75 μg/mL) was injected three

times in different days and peak areas were determined and calculation was done to obtain %

R.S.D.

Interday precision for Darolutamide was found in range of 0.70-1.12 % RSD

Table 5 Interday precision data.

Sr. No. Conc. (μg/mL) Mean ± S.D (n=6) % R.S.D

1 25 4722.21±33.25 0.70

2 50 9409.66±57.86 0.61

3 75 14238.29±158.94 1.12

Accuracy

Drug solution having concentration 25 µg/mL was taken and transfer it in to three different

flask label A, B and C. Spiking of the 80%, 100%, 120% of standard solution was carried out in

it and dilution was made up to 10 mL. At 242 nm, peak area was measured. Calculation was

carried out at each level to find out the amount of Darolutamide and Computation for the %

recoveries was carried out.

Table 6: Recovery data.

Sr.

No.

Conc.

Level

(%)

Sample

amount

(μg/mL)

Amount

Added

(μg/mL)

Amount

recovered

(μg/mL)

%

Recovery

%

RSD

1

80 %

25 20 20.21 101.07

1.15 2 25 20 19.76 98.78

3 25 20 19.94 99.72

4 100 %

25 25 24.97 99.90 0.15

5 25 25 24.93 99.70


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6 25 25 25.00 100.01

7

120 %

25 30 29.94 99.79

1.03 8 25 30 30.25 100.83

9 25 30 29.63 98.77

LOD and LOQ

Estimation of the LOD was carried out from the set of 5 calibration curves used to

determination linearity. The LOD may be calculated as,

LOD = 3.3 × {SD (i.e. Standard deviation of Y-intercepts of 5 calibration curves) / (Mean slope

of the 5 calibration curves)}

Estimation of the LOQ was carried out from the set of 5 calibration curves used to determine

Linearity. The LOQ may be calculated as,

LOQ = 10 × {SD (i.e. Standard deviation of Y-intercepts of 5 calibration curves) / (Mean slope

of the 5 calibration curves)}

Table 7: LOD and LOQ data.

LOD LOQ

LOD = 3.3 x (SD / Slope)

= 3.3 x (116.18/191.97)

= 2.00 µg/mL

LOQ = 10 x (SD / Slope)

= 10 x (116.18/191.97)

= 6.05 µg/mL

Robustness

Changing was done in following parameters one by one and their effect was observed on

system suitability for standard preparation.

1. Changes were done in flow rate of mobile phase by ± 0.1 mL/min (i.e. 0.9 mL/min and 1.1

mL/min).

2. Changes were done in ratio of Mobile phase by ±2 {i.e. Acetonitrile: Buffer (22:78) and

Acetonitrile: Buffer (18:82)}

3. Changes was done in pH of Mobile phase by ±0.2 (i.e.3.2 and 2.8)

Table 8: Robustness data.

Sr. no.

Area at

Flow rate

(- 0.2 mL/min)

Area at

Flow rate

(+ 0.2 mL/min)

Area at

Mobile

phase(-2)

Area at

Mobile

phase(+2)

Area at

pH (+

0.2)

Area at

pH

(-0.2)

1 9789.61 8979.13 9972.57 9053.06 9809.35 8884.64

2 9933.61 8904.94 9849.21 8979.90 9883.51 8949.79

3 9859.09 8872.39 9900.93 8878.25 9702.00 8970.11

%

R.S.D 0.73 0.61 0.63 0.98 0.93 0.50


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Assay

Weighed powder of tablet equivalent to 50 mg Darolutamide and transferred it in to 100 mL

volumetric flask, then 60 mL of Methanol was added and shake it for 15 minutes and

Dilution was made with methanol to make up the volume. (Stock solution 500 µg/mL),

transfer the 1 mL of this solution into 10 mL volumetric flask and Dilution was made with

mobile phase to make up the volume. (Working solution 50 µg/mL).

Table 9: Assay results.

Sr. No. Label claim

(mg) Result (mg) % Assay

average

% Assay SD %RSD

1 300 300.00 100.00

99.77 0.22 0.22 2 300 299.26 99.75

3 300 298.71 99.57

3. RESULTS AND DISCUSSION

The analytical method for the estimation of Darolutamide was developed and validation.

Also, forced degradation study was carried out. Linearity of the Darolutamide was found to

be linear and Correlation co-efficient (r2) was observed 0.9993. The % RSD values for

Repeatability, Intraday and Interday precision was found less than 2% (i.e. 1.34%, 0.97-

1.84% and 0.70-1.12% respectively). LOD and LOQ values was found 2.00 µg/mL and 6.05

µg/mL. Estimation of accuracy was carried out in terms of %Recovery, is found to be 98.77-

101.07%.

Forced degradation studies was carried out as per the ICH recommendations and the

developed stability indicating RP-HPLC method could separate drug from degradation

products formed under various stressed conditions.

Table 10: Summary of Developed method.

Sr. no. Parameter Result

1 Linearity range 25-75 µg/mL

2 Regression equation y = 191.97x – 109.49

3 Correlation coefficient r2= 0.9993

4

Precision (%RSD)

Repeatability 1.34

Intraday 0.97-1.84

Interday 0.70-1.12

5 Accuracy (% Recovery) 98.77-101.07

6 LOD 2.00 µg/mL

7 LOQ 6.05 µg/mL

8 %Assay 99.77


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4. CONCLUSION

The method validation has been performed for linearity, Specificity, Robustness, precision

and accuracy. As reported methods for estimation of Darolutamide is available in mice

plasma only, this method development is worthwhile. Developed method was found to be

specific, accurate, precise, rapid, simple, selective and sensitive for estimation of

Darolutamide.

5. ACKNOWLEDGEMENTS

The authors are thankful to the K analytical Laboratory, Gujarat India, for providing

necessary infrastructure, facilities and support.

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Documents

STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT … · Oxidation 8644.06 9.00 Photo 7578.32 20.22 Thermal 8120.12 14.52 VALIDATION OF DEVELOPED METHOD Linearity By analysis of