Method Development and Validation of Roflumilast in Tablet

J. Atoms and Molecules / 2(5); 2012 / 369–374 Ladani JJ et al

All rights reserved© 2011 www.jamonline.in 369

Research Article

Journal of Atoms and Molecules An International Online JournalAn International Online JournalAn International Online JournalAn International Online Journal ISSN ISSN ISSN ISSN –––– 2277 2277 2277 2277 –––– 1247124712471247

METHOD DEVELOPMENT AND VALIDATION OF ROFLUMILAST IN TABLET DOSAGE FORM BY UV SPECTROPHOTOMETRY

Ladani JJ* 1, Bhimani RD1, Vyas KB2, Nimavat KS2 *1Research Scholar, JJT University, Jhunjhunu, Rajasthan, India.

2Government Science College, Gandhinagar, Gujarat, India.

Received on: 10-10-2012 Revised on: 20-10-2012 Accepted on: 25–10–2012

Abstract:

Analytical method development and validation play important roles in the discovery, development

and manufacture of pharmaceuticals. A simple and reproducible UV – spectrophotometric method

for the quantitative determination of Roflumilast in tablet formulation was developed and validated

in the present work. The parameters linearity, precision, accuracy was studied. Roflumilast has the

maximum wavelength at 250 nm. Analytical calibration curves were linear within a concentration

range from 2 to 35 µg/ml. The developed method was applied directly and easily to the analysis of

the pharmaceutical tablet preparations. %RSD was found to be less than 0.3. The result of analysis

has been validated statistically. Hence the proposed method can be used for the reliable

quantification of Roflumilast in tablet formulation.

Key Words: UV-Spectrophotometry, Roflumilast, Pharmaceutical dosage form.

Introduction:

Roflumilast, which is an anti-inflammatory

medicine called phosphodiesterase 4 inhibitor.

Roflumilast reduces the activity of

phosphodiesterase 4, a protein occurring

naturally in body cells. When the activity of

this protein is reduced, there is less

inflammation in the lungs. Roflumilast is used

to treat severe COPD in adults. COPD is a

chronic disease of the lungs that results in

tightening of the airways (obstruction) and

* Corresponding author

Jatin Ladani,

Email: [email protected] Tel: +91 – 9909007832



swelling and irritation of the walls of the

small air passages (inflammation) leading to

symptoms such as coughing, wheezing, chest

tightness or difficulty in breathing.

Roflumilast is to be used in addition to

bronchodilators.

Figure: 1 Structure of Roflumilast

Material and Method:

A shimadzu UV – visible 1800 spectrometer

was used.

All the chemicals used were of analytical

grade. Methanol A.R. grade was procured

from Merck Pvt ltd.,Mumbai. An analytically

pure sample of Roflumilast was obtained from

Zydus Cadila, Ahmedabad as a gift sample.

Roflumilast standard stock solution

(100µg/ml):

A 50mg of Roflumilast standard was weighed

and transferred to a 50 ml volumetric flask. 10

ml of methanol is transferred to this

volumetric flask and sonicated for 15 min. the

flask was shaken and volume was made up to

the mark with methanol to give a solution

containing 1000µg/ml Roflumilast. From this

solution 5 ml was transferred to 50 ml

volumetric flask. The volume was adjusted to

the mark with the methanol to give the

solution containing 100µg/ml Roflumilast.

Preparation of Standard Solution:

Take 10 ml of Standard stock solution in 50

ml volumetric flask and dilute it up to 5o ml

with methanol to make concentration

20µg/ml.

Figure: 2 Spectrum of Roflumilast STD

Preparation.

Selection of analytical wavelength:

2-35 µg/ml solution of Roflumilast was

prepared in methanol and spectrum was

recorded between 200-400nm. Spectrum of

above concentration were obtained with n=10.

The overlain spectrum of Roflumilast at a

different concentration was recorded at 250

nm.

Figure: 3 Overlain Spectrum of Roflumilast

J. Atoms and Molecules / 2(5)

All rights reserved© 2011

Table.1: Result of calibration curve at 250 nm for Roflumilast in methanol

Concentration

(µg/ml)

absorbance at

250 nm

mean+sd

4 0.119 + 0.0017

6 0.179 + 0.0012

8 0.240 + 0.0008

10 0.299 + 0.0009

12 0.357 + 0.0008

Figure: 4 Calibration curve for Roflumilast

at 250 nm

Preparation of Sample solution:

Ten Tablets (Dosage: 0.5mg/tablet)

weighed and powdered. The powder

equivalent to 2.5 mg Roflumilast was

accurately weighed and transferred to the 25

ml volumetric flask. 10 ml methanol was

transferred to the volumetric flask and

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 5 10

); 2012 / 369–374

Table.1: Result of calibration curve at 250 nm for Roflumilast in methanol

absorbance at

250 nm

mean+sd

coefficient

of variation

0.119 + 0.0017 1.47

0.179 + 0.0012 0.67

0.0008 0.34

0.299 + 0.0009 0.32

0.357 + 0.0008 0.25

Figure: 4 Calibration curve for Roflumilast

Preparation of Sample solution:

(Dosage: 0.5mg/tablet) were

weighed and powdered. The powder

equivalent to 2.5 mg Roflumilast was

accurately weighed and transferred to the 25

ml volumetric flask. 10 ml methanol was

transferred to the volumetric flask and

sonicate it for 20 min. the flask was shaken

and volume was made up to the mark with

methanol.

Above solution was filtered through the

whatman filter paper (0.45

was taken and transferred to the volumetric

flask of 50 ml capacity. Volume was made up

to the mark with methanol to give a soluti

containing 20µg/ml of Roflumilast drug. This

solution was used for the estimation of the

Roflumilast drug.

System precision/system suitability:

System suitability testing is an integral part of

many analytical procedures. System

suitability test parame

of procedure being validated. System

precision is determined by measuring the

absorbance of standard solution containing

100% working concentration for six times and

calculates the % RSD. The % RSD should be

less than 2.0% The relat

of six replicate measurement of standard

solution was found to be 0.13% ( limit NMT

2.0%), which indicates that the system is

precise to analyse the sample.

Table.2: System suitability for Roflumilas

No Parameter (n=6)

1 Mean

2 Standard Deviation

3 % RSD

15

Series1

Ladani JJ et al

www.jamonline.in 371

sonicate it for 20 min. the flask was shaken

was made up to the mark with

Above solution was filtered through the

whatman filter paper (0.45µ). 10 ml of aliquot

was taken and transferred to the volumetric

flask of 50 ml capacity. Volume was made up

to the mark with methanol to give a solution

g/ml of Roflumilast drug. This

solution was used for the estimation of the

System precision/system suitability:

System suitability testing is an integral part of

many analytical procedures. System

suitability test parameters depend on the type

of procedure being validated. System

precision is determined by measuring the

absorbance of standard solution containing

100% working concentration for six times and

calculates the % RSD. The % RSD should be

less than 2.0% The relative standard deviation

of six replicate measurement of standard

solution was found to be 0.13% ( limit NMT

2.0%), which indicates that the system is

precise to analyse the sample.

Table.2: System suitability for Roflumilast

Parameter (n=6) Roflumilast

Mean 0.602

Standard Deviation 0.008

% RSD 0.13



Method Precision:

Method precision was established by

analyzing six separate samples at 100% of the

working concentration. Percent of result was

calculated against claimed label. The % RSD

of assay result of six separate from a single

batch was found to be 0.17% (limit NMT

2.0%) which indicates that the method is

precise to analyze the tablet.

Accuracy:

Accuracy was established by analyzing nine

sample solution of Roflumilast at 50%, 100%

and 150% of the working concentration (

Three Replicate for each Concentration) into a

placebo mixture and by calculating the

percent recovery of active ingradient from the

placebo solution. The percent recovery at

each level should be within 98.0% to 102.0%.

the percent recovery was calculated for nine

determination and found to be within limit.

Thus it has been concluded that the method

was accurate to analyze the tablet.

Table 3: Determination of accuracy

Level of

recovery

Amt of drug

added (mg)

Amt of drug

recovered (mg) %Recovery Mean ( % ) % RSD

0 0 0 -- -- --

50% 1 1.02 102%

1 1.02 102% 101.7% 0.0

1.02 1.02 101%

100% 2 2.00 100.5%

2 2.00 100.5% 100.0% 0.9

2.03 2.00 99.0%

150% 3 3.02 101.0%

3.10 3.09 98.1% 99.1% 1.7

3.10 3.09 98.1%



Specificity

Identification

The UV absoption spectrum of the sample

preparation for assay is concordant with the

reference spectrum of standard sample from

assay preparation.

Placebo interference

Placebo solution was prepared in the manner

as standard and sample preparation. No

interference of placebo was found.

Linearity:

Five different standard solution were prepared

covering a concentration of 2 to 35 µg/ml of

working concentration of Roflumilast and all

absorbance were recorded. A linear curve was

prepared by plotting actual concentration

(µg/ml of ppm) Vs absorbance and correlation

co-efficient was calculated. The result

obtained correlated with the concentrations

resulting co-efficient found 0.9998, which is

with in the limit (limit : NLT 0.990). Thus the

graph confirms the linearity of the method in

the range of 2 to 35 µg/ml.

Robustness:

Robustness of this method was determined by

analyzing the Roflumilast tablet in different

equipment in different day and by different

analyst. From the above mentioned data it

observed that the method is robust enough to

analyse Roflumilast tablet.

Table No. 4 Data for Robustness test

Sr no. Variable parameter Assay Result

1 Analyst – 1

Analyst - 2

99.70%

100.01%

2 Equipment – 1

Equipment – 2

100.00%

99.98%

3 Day -1

Day -2

99.85%

99.95%

Result & discussion:

The analytical method development &

validation for the drug roflumilast was done ,

which shows the best elution of the peak. The

specificity test studies shows that the analyte

chromatographic peak is not attributable to

more than one component. The linearity

calibration curve shows linear response over

the range of concentration used. The precision

data shows that the reproducibility of the

assay procedure was satisfactory. The

accuracy of the method was determined by

recovery studies. The recovery studies were

carried out of the percentage recovery was

calculated. The robustness studies show that

there were no marked changes in the

chromatogram. The ruggedness of the method

was determined for the same sample under

different laboratory, different analysis &

using operational & environment condition ;

the degree of reproducibility will shows



results within their limits. Further there was

no interference due to excipients. The system

suitability studies were also carried out to

determine peak asymmetry.

Conclusion:

From the above data it was observed that all

validation parameter (like system suitability,

method precision, accuracy, specificity,

linearity, robustness) meet the predetermined

acceptance criteria. Thus it has been

concluded that the method is validated for the

analysis of assay of Roflumilast in tablet

dosage form.

Acknowledgements:

The Author is grateful to express my sincere

thanks to Dr. Kiran S. Nimavat (Government

Sci. College, Gandhinagar) and Dr. Kartik

Vyas (Sheth L.H. College, Mansa) for their

continuous support and guideace.

References:

1) Sharma B.K., instrumental method of

chemical analysis, GOEL, publishing

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2) Chatwal R.G., Anand K.S., instrumental

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publishing house, 2002, 5th edition,

volume 1, 2.149-84.

3) Skoog D.A., holler F.J., west D.M.,

Nierman T.A., An introduction in

analytical chemistry, Singapore: Thomas

Asia pte.ltd; 1994, 5th edition, 567, 728-44

4) Available online at:

Http://en.wikipedia.org/wiki/ultraviolate-

visible-Spectroscopy on January 2010

5) ICH Topic Q 2 (R1) Validation of

Analytical Procedures: Text and

Methodology: 2005

6) Available online at :

http://www.ema.europa.eu

7) Uzunov, P. and Weiss, B.: Separation of

multiple molecular forms of cyclic

adenosine 3',5'-monophosphate

phosphodiesterase in rat cerebellum by

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Biochim. Biophys. Acta 284:220-226,

1972.

8) Weiss, B.: Differential activation and

inhibition of the multiple forms of cyclic

nucleotide phosphodiesterase. Adv. Cycl.

Nucl. Res. 5:195-211, 1975.

Documents

Method Development and Validation of Roflumilast in Tablet