Atoms and Molecules / 2(5); 2012 / 369–374 Research Article

Ladani JJ et al

Journal of Atoms and Molecules
An International Online Journal
ISSN – 2277 – 1247


Research Scholar, JJT University, Jhunjhunu, Rajasthan, India. 2 Government Science College, Gandhinagar, Gujarat, India. Revised on: 20-10-2012 Accepted on: 25–10–2012

Received on: 10-10-2012 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 * Corresponding author Jatin Ladani, Email: jatin_ladani@yahoo.co.in Tel: +91 – 9909007832 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

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J. Atoms and Molecules / 2(5); 2012 / 369–374 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.

Ladani JJ et al 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

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 (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 Figure: 3 Roflumilast Overlain Spectrum of standard stock solution 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.

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J. Atoms and Molecules / 2(5) 2012 / 369–374 ); Table.1: Result of calibration curve at 250 nm for Roflumilast in methanol Concentration (µg/ml) absorbance at 250 nm mean+sd 0.119 + 0.0017 0.179 + 0.0012 0.240 + 0.0008 0.299 + 0.0009 0.357 + 0.0008 1.47 0.67 0.34 0.32 0.25 coefficient of variation

Ladani JJ et al 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 10 ml of aliquot (0.45µ).

4 6 8 10 12

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 solution containing 20µg/ml of Roflumilast drug. This g/ml solution was used for the estimation of the Roflumilast drug. System precision/system suitability: System suitability testing is an integral part of

0.4 0.35 0.3 0.25 0.2 Series1 0.15





suitability test parame 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 relat relative standard deviation

of six replicate measurement of standard
0 0 5 10 15

solution was found to be 0.13% ( limit NMT 2.0%), which indicates that the system is

Figure: 4 Calibration curve for Roflumilast at 250 nm Preparation of Sample solution:

precise to analyse the sample. Table.2: System suitability for Roflumilas Roflumilast No Parameter (n=6) Mean Standard Deviation % RSD Roflumilast 0.602 0.008 0.13

Ten Tablets (Dosage: 0.5mg/tablet) were weighed and powdered. The powder 1 2 3

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 All rights reserved© 2011

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J. Atoms and Molecules / 2(5); 2012 / 369–374 Method Precision: Method precision was established by

Ladani JJ et al 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.

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%

Table 3: Determination of accuracy Level of recovery 0 50% Amt of drug added (mg) 0 1 1 1.02 100% 2 2 2.03 150% 3 3.10 3.10 Amt of drug %Recovery recovered (mg) 0 1.02 1.02 1.02 2.00 2.00 2.00 3.02 3.09 3.09 -102% 102% 101% 100.5% 100.5% 99.0% 101.0% 98.1% 98.1% 99.1% 1.7 100.0% 0.9 101.7% 0.0 --Mean ( % ) % RSD

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J. Atoms and Molecules / 2(5); 2012 / 369–374 Specificity Identification The UV absoption spectrum of the sample preparation for assay is concordant with the reference spectrum of standard sample from assay preparation. Equipment – 1 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 Result & discussion: The analytical method 3 Day -2 Day -1 2 Equipment – 2 Analyst – 1 1 Analyst - 2

Ladani JJ et al Table No. 4 Data for Robustness test Sr no. Variable parameter Assay Result 99.70% 100.01% 100.00% 99.98% 99.85% 99.95%



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

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.

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

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J. Atoms and Molecules / 2(5); 2012 / 369–374 results within their limits. Further there was no interference due to excipients. The system suitability studies were also carried out to determine peak asymmetry. 5) ICH Topic Conclusion: From the above data it was observed that all validation parameter (like system suitability, method precision, accuracy, specificity, http://www.ema.europa.eu Analytical 4) Available online at:

Ladani JJ et al

Http://en.wikipedia.org/wiki/ultraviolatevisible-Spectroscopy on January 2010 Q 2 (R1) Validation of Procedures: Text and

Methodology: 2005 6) Available online at :

linearity, robustness) meet the predetermined acceptance criteria. Thus it has been

7) Uzunov, P. and Weiss, B.: Separation of multiple adenosine molecular forms of cyclic

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.


phosphodiesterase in rat cerebellum by polyacrylamide gel electrophoresis.

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.

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volume 1, 2.149-84. 3) Skoog D.A., holler F.J., west Nierman T.A., An D.M., in


analytical chemistry, Singapore: Thomas Asia pte.ltd; 1994, 5th edition, 567, 728-44 All rights reserved© 2011 www.jamonline.in 374

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