Development and Validation of Stability indicating RP-HPLC Method for Estimation of Roflumilast in Tablet dosage form

 

Pooja Avula¹, Rajitha Galla¹*, Geetha Susmita Adepu¹, Hari Babu Vemanaboina²,

Sivaranjani Tyagarajan²

¹Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam

 (Women’s University), Tirupati.

²Analytical Development (AR&D), Orchid Health Care, IKKT, Chennai.

 

ABSTRACT:

A simple, precise, stable and economical RP-HPLC method was developed for estimation of Roflumilast in tablet dosage form and validated according to the ICH guidelines. The chromatographic separation was achieved on an Inertsil ODS-3V column of dimensions 150mm x 4.6mm, 5µm, using a mobile phase mixture of 0.01M ammonium acetate buffer and organic mixture (methanol: acetonitrile in 1:1 ratio) in the ratio of 25: 75 (v/v) at a flow rate of 1.5ml/min and UV detection at 250nm. The injection volume was maintained at 80µl with 6 min run time. Retention time (RT) was found to be 2.92±0.01min. This developed method was validated as per the ICH guidelines. Linear plot was obtained with a correlation coefficient of 0.999 in the concentration range of 5-40 µg/ml. The optimized method was accurate with 100.1% recovery and precise with %RSD - 0.1 respectively. The method was found to be specific with no interference of drug from the excipients. Stress studies or forced degradation studies were carried out at different conditions like thermal, acidic, basic, photolytic, peroxide, humidity and the results revealed that the method was stable and it can be used for routine analysis in tablet dosage formulations.

 

 

 

INTRODUCTION:

Roflumilast is chemically 3-(cyclopropylmethoxy)-N-(3, 5-dichloro pyridin-4-yl)-4-(difluoromethoxy)-benzamide is an anti-asthmatic drug which belongs to therapeutic category of phosphodiesterase-4 inhibitor (PDE-4) (Fig.1). Roflumilast is used for treatment of chronic obstructive pulmonary disorder (COPD) associated with chronic bronchitis and a history of exacerbations. It is sparingly soluble in ethanol, freely soluble in acetone and insoluble in water, hexane (water solubility 0.52-0.56mg\l at 21-22^⁰C). Roflumilast and its active metabolite (Roflumilast N-oxide) are selective inhibitors of PDE-4. Inhibition of PDE-4 activity by roflumilast and roflumilast N-oxide leads to accumulation of intracellular cyclic adenosine monophosphate (cAMP).

 

Increased cAMP activates protein kinase A (PKA) enzyme, which inactivates transcription factors involved in inflammation. It also decreases the amount of sputum neutrophils and eosinophils in COPD patients. [1,2].

 

Fig 1: Chemical structure of Roflumilast

 

The literature survey revealed that various UV [3-6], LC-MS [7-9], HPTLC [10], HPLC [11-13], RP-HPLC [14-17] methods were developed for the estimation of Roflumilast in pure and tablet formulations. But certain drawbacks were found in some of those methods like complexity in mobile phase preparation, longer run time and retention time (6 min). Therefore, an attempt was made to develop a simple RP-HPLC method for estimating Roflumilast in pure and tablet formulations that could overcome those drawbacks. The developed method was validated according to the ICH (International Conference on Harmonization guidelines) [18-21].

 

MATERIALS AND METHODS:

Instrumentation:

Shimadzu LC AHT 20AT HPLC system with Inertsil ODS-3V column of dimensions 150mm x 4.6mm, 5µm of particle size was used for better chromatographic separation. Waters alliance E 2996 HPLC equipped with PDA (Photo Diode Array) detector, auto sampler and Empower 3 software was used for development and validation of method. An electronic balance, a sonicator, centrifuge, rotary shaker and UV–Visible spectrophotometer were used in this study.

 

Chemicals and reagents:

Roflumilast standard drug was obtained as a gift sample from Orchid Healthcare and dosage form (Daliresp, Daxas manufactured by Takeda GambH) from local market. HPLC grade methanol and acetonitrile were obtained from Sdfcl and Rankem respectively. Analytical grade HCl, NaOH and H₂O₂ were obtained from Rankem. Milli-Q water was obtained from Millipore.

 

Chromatographic conditions:

Mobile phase used was a mixture of 0.01M ammonium acetate buffer and organic mixture (methanol: acetonitrile in 1:1 ratio) in the ratio of 25: 75 (v/v) with a isocratic flow rate of 1.5ml/min. The injection volume was maintained at 80µl with 6 min run time. Absorption maxima was set at 250nm with the column oven temperature at 30^⁰C.

 

Preparation of standard solution (20 ppm):

50mg of Roflumilast working standard was accurately weighed and transferred into a 100ml volumetric flask, then 40ml of methanol was added and sonicated. Then it was diluted to final volume with methanol and mixed well. About 4ml of the standard stock solution was pipetted out and taken in a 100ml of volumetric flask, diluted upto the final volume with diluent and mixed well.

 

Preparation of sample solution (20 ppm):

10 tablets were accurately weighed and transferred into a 250ml volumetric flask. 170ml of the diluent was added in volumetric flask and kept on a rotary shaker for 40 minutes at 200RPM (Rotations Per Minute). Then it was sonicated for 30 min with intermediate shaking and diluted to final volume with diluent and mixed. A portion of the above solution was centrifuged at 2500 RPM for 10 minutes. The supernatant solution was filtered through 0.45µm nylon filter by discarding the first 4ml of the filtrate.

Method development and validation:

Number of trials were carried out by using different columns, flow rates, mobile phase ratios and finally a method was developed with optimized chromatographic conditions. The developed method was validated for specificity, accuracy, precision, linearity, robustness, solution stability and system suitability according to the ICH (International Conference on Harmonization) guidelines [22,23].

 

Specificity:

Blank, standard, sample and placebo solutions of Roflumilast were prepared and injected into the HPLC system to check the interference of excipients with that of analyte peak.

 

Accuracy:

The accuracy studies were carried out by preparing three spiked levels of sample such as 50%, 100% and 150% solutions. These solutions were analyzed in triplicate injections at each level and % recovery values were noted.

 

Precision:

Repeatability (intraday) was performed by preparing six different sample solutions from single sample stock solution as per the method and injected into the HPLC system as one injection each. % assay and % RSD values were calculated. System precision was performed by preparing different standard solutions from the single standard stock solution and injected into the HPLC system. Column-column and system-system variabilities were determined in intermediate precision (interday). System suitability parameters were calculated.

 

Linearity:

The linearity was performed by preparing the solutions in different percentage levels like 25%, 50%, 75%, 100%, 150% and 200% from the standard solution and injected two times into the HPLC system. Calibration plot and square of correlation coefficient was calculated.

 

Robustness:

For robustness studies, the same standard solution was analyzed under a variety of conditions like variation in flow rate (1.5±0.2 ml/min), variation in column oven temperature (30±5^⁰C) and variation in mobile phase composition (high and low organic). The system suitability parameters were calculated.

 

Filter validation was performed by preparing single standard and three test solutions. A small portion of test solution was centrifuged and another portion of test solution was filtered through individual filters. Then the unfiltered standard solution, centrifuged test solution and filtered test solutions were injected into the HPLC system. The difference in % assay values of centrifuged, filtered test solutions and unfiltered standard solution was calculated,

 

Solution stability:

Standard, sample solutions were prepared as per the method and kept them on the bench top and under refrigerated conditions for 24 hrs. Then the standard and sample solutions were injected into the HPLC system. Similarity factor for standard solution and % assay for sample solutions was calculated against freshly prepared standard.

                                  A × WS

Similarity factor = -----------------

                                  B × WT

Where WS = weight of freshly prepared standard in mg

WT = weight of bench top or refrigerated standard in mg

B = average area of freshly prepared standard

A = area of bench top or refrigerated standard

 

System suitability:

System suitability was carried out to verify whether the resolution and reproducibility of the system were sufficient to perform the analysis. The standard solution of Roflumilast was prepared by weighing 50mg of Roflumilast working standard and taken in a 100ml volumetric flask, 40ml of methanol was added and sonicated. Diluted to final volume with methanol and mixed well. 4ml of the above solution was pipetted out and taken into a 100ml of volumetric flask, diluted to final volume with diluent and mixed well. The prepared solution was then injected 5 times into the HPLC system at a 80µl injection volume and system suitability parameters like plate count, tailing, % RSD, retention time, peak areas were evaluated.

 

Stress studies or Forced degradation studies:

These studies were performed to determine the stability of the drug in the formulations. The Roflumilast drug was subjected to different conditions like acid stress, base stress, peroxide stress, thermal stress, photolytic stress and humidity stressed conditions. % degraded values were calculated in each stress condition.

 

Acid stress studies:

Acid stress studies were carried out by taking the sample and placebo quantities as per the method in respective 250ml volumetric flasks. 5ml of methanol and 5ml of 1N HCl solution was added and kept in a water bath for 1 h at 60^⁰C. Then the solutions were cooled to room temperature for 30 min and 5ml of 1N NaOH solution was added. Then working solutions were prepared as per the method to get 20µg/ml concentration and 80µl of above solution was injected into the HPLC system. Then the chromatograms were recorded.

 

Base stress studies:

Base stress studies were carried out by taking the sample and placebo quantities as per the method in respective 250ml volumetric flasks. 5ml of methanol and 5ml of 1N NaOH solution was added and kept in a water bath for 1 h at 60^⁰C. Then the solutions were cooled to room temperature for 30 min and 5ml of 1N HCl solution was added. Then working solutions were prepared as per the method to get 20µg/ml concentration and 80µl of above solution was injected into the HPLC system. Then the chromatograms were recorded.

 

Peroxide stress studies:

Peroxide stress studies were carried out by taking the sample and placebo quantities as per the method in respective 250ml volumetric flasks. 5ml of methanol and 5ml of 3% H₂O₂ solution was added and kept in a water bath for 1 h at 40^⁰C. Then the solutions were cooled to room temperature for 30 min. Then working solutions were prepared as per the method to get 20µg/ml concentration and 80µl of above solution was injected into the HPLC system. Then the chromatograms were recorded.

 

Thermal stress studies:

Thermal stress studies were carried out by taking the sample and placebo quantities as per the method respective clean, dry petri dishes and placed in hot air oven at 60^⁰C for about 24 h. Then working solutions were prepared as per the method to get 20µg/ml concentration and 80µl of above solution was injected into the HPLC system. Then the chromatograms were recorded to assess the stability of the sample.

 

Photolytic stress studies:

Photolytic stress studies were carried out by taking the sample and placebo quantities as per the method in respective clean, dry petri dishes and placed in photo stability chamber for intensity at 200 W.hr/m2 for about 15 d. Then working solutions were prepared as per the method to get 20µg/ml concentrations and 80µl of above solution was injected into the HPLC system. Then the chromatograms were recorded.

 

Humidity stress studies:

Humidity stress studies were carried out by taking the sample and placebo quantities as per the method in desiccator containing saturated solution of potassium nitrate for about 9 d to maintain the conditions at 90% relative humidity (RH). Then working solutions were prepared as per the method to get 20µg/ml concentration and 80µl of above solution was injected into the HPLC system.

RESULTS AND DISCUSSION:

Several trials were carried out by using different columns, mobile phase ratios, flow rates and finally method was developed with optimized conditions that were shown in table no. 1. The respective optimized chromatogram for standard Roflumilast was shown in fig 2. The developed method was validated as per the ICH guidelines.

 

Fig 2: Optimized chromatogram of Roflumilast standard

 

Table 1: Optimized chromatographic conditions

S. No.	Parameters	Optimized condition
1	Mobile phase	0.01M Ammonium acetate: Organic mixture (25:75), v/v
2	Column	Inertsil ODS-3V(150mm x 4.6mm)5µm
3	Flow rate	1.5 ml/min
4	Detector wavelength	250 nm
5	Run time	6 min
6	Elution	Isocratic
7	Column oven temperature	30°C
8	Volume of injection	80µL

 

Specificity:

The chromatograms obtained from the placebo, blank shown that there was no interference from the excipients which indicated that the method was specific.

 

Accuracy:

The mean % recovery value was found to be 100.1% and values for spiked levels were shown in table no.2.

 

Precision:

Repeatability (intraday) was performed within short time interval and the results were found to be % RSD- 0.1. The system precision values were found to be % RSD - 0.04 (table 3).For intermediate precision (interday), the results of system to system variability and column to column variability were found to be %RSD-0.2 and 0.1 respectively.

 

Table 2: % recovery data in accuracy

Spiked levels with concentration	Amount added (mg)	Amount found (mg)	% Recovery
50% Injection 1	2.494	2.486	100.3
50% Injection 2	2.505	2.486	100.8
50% Injection 3	2.480	2.486	99.7
			Mean- 100.3
100% Injection 1	4.973	4.972	100.0
100% Injection 2	4.989	4.972	100.3
100% Injection 3	4.969	4.972	99.9
			Mean- 100.1
150% Injection 1	7.418	7.458	99.5
150% Injection 2	7.484	7.458	100.4
150% Injection 3	7.459	7.458	100.0
			Mean- 99.9
			Avg. of mean- 100.1

 

Table 3: Method precision and System precision data

Repeatability		System precision
Injection no.	Peak area	Injection no.	Peak area
1	2050619	1	2086179
2	2054562	2	2085564
3	2049914	3	2085423
4	2051907	4	2084088
5	2054253	5	2084401
6	2053158	Mean	2085131
Mean	2052402	Std dev	864.84
Std dev	1911.99	% RSD	0.04
% RSD	0.1

 

Linearity:

Linearity plot for Roflumilast was found to be linear in the concentration range of 5-40 µg/ml. The square of correlation coefficient was found to be 0.9997. The linearity plot was shown in fig 3.

 

 

Fig 3: Linearity plot

 

Robustness:

Robustness studies were evaluated by making small changes in the chromatographic parameters. The results for the system suitability were given in table 4 which indicated that these small changes doesn’t show much effect on the method and %RSD was found to be 0.05.In filter validation, the filter was considered as acceptable because the % assay difference values were found to be 0.15 and 0.11.

 

Solution stability:

Similarity factor values for the standard were found to be 1.008, 1.01 and % assay difference values in the sample solution were  found to be 1.84, 1.55.

 

System suitability:

System suitability parameters were calculated from the chromatograms obtained by five replicate injections of standard solution. The results for system suitability parameters were found to be plate count (3076), %RSD (0.1) and tailing factor (1.16).

 

Stress studies or Forced degradation studies:

% degraded values from stress studies indicated that the method was stable because in each stress condition the purity angle was less than the purity threshold and no purity flag was observed (Table 5) and chromatograms were shown in fig 4.

 

Table 4: Robustness data

S. No.	Name of sample	Retention time (min)	Peak area	Tailing factor	Plate count	% RSD
1	Std- Flow rate (High flow)	2.643	1825757	1.28	3064	0.02
2	Std- Flow rate (Low flow)	3.434	2384057	1.25	3646	0.04
3	Std- Column oven temperature (High temperature)	2.859	2059078	1.29	3376	0.03
4	Std- Column oven temperature (Low temperature)	2.986	2076030	1.29	3349	0.1
5	Std- Mobile phase composition (High organic)	2.116	2080780	1.18	3769	0.1
6	Std- Mobile phase composition (Low organic)	4.883	2084669	1.12	5184	0.04
						Mean-0.05

Table 5: Stress studies data of Roflumilast

S. No	Stress condition	Peak Area	Retention time (min)	% Degraded	Purity Angle	Purity Threshold	Purity Flag
1	Unstressed sample	1932393	3.029	---	0.139	0.294	No
2	Humidity stressed sample	1941993	3.029	0.5	0.134	0.295	No
3	Thermal stressed sample	1953057	3.037	1.06	0.142	0.279	No
4	Photolytic stressed sample	1935795	3.034	0.18	0.133	0.280	No
5	Peroxide stressed sample	1952773	2.971	1.96	0.133	0.270	No
6	Acid stressed sample	1941170	2.986	1.36	0.143	0.265	No
7	Base stressed sample	1927052	3.004	0.63	0.118	0.284	No

 

Fig 4: Chromatograms for stress studies

CONCLUSION:

Reverse phase HPLC method for estimation of Roflumilast has been developed, optimized and validated. The developed and validated RP-HPLC was found to be specific, accurate, precise, linear, robust and rugged. From the stress studies conducted, it was concluded that the method was stability indicating because in each condition the purity angle was less than purity threshold with absence of purity flag and could be used throughout the shelf life of the drug. The results obtained for developed and validated method were within the acceptance criteria. The method was found to have suitable application in routine laboratory analysis and also used for estimation of drug in pure and tablet dosage formulations.

 

ACKNOWLEDGEMENTS:

The authors were thankful to Orchid Healthcare, A division of Orchid Pharma Ltd, Irungattukottai, Chennai for their support.

 

CONFLICT OF INTEREST:

Declared none.

 

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Received on 06.02.2020            Modified on 24.03.2020

Accepted on 08.05.2020         © RJPT All right reserved