A New RP-HPLC Method for Determination of Roflumilast in Bulk and Pharmaceutical Dosage Forms

Introduction

Drug profile

Roflumilast drug acts as long lasting selective inhibitor of the enzyme phosphodiesterase-4 (PDE4). It has anti-inflammatory effects and is used for the treatment of chronic obstructive pulmonary disease (COPD), inflammatory conditions of the lungs and is administered orally.¹ There are few methods for estimation of Roflumilast by UV and HPLC which are listed in references.^2-6 The RP HPLC method cited in references has retention time of around 4 to 6 min.^3-6 The new method was developed with less retention time and validated all the method parameters according to ICH.^7-11

Materials and Methods

Instrumentation

The separation was carried out by using Agilent 1120 with column (Water’s X Bridge 5 µ C18(2) 100A, 250X4.6 mm), Gradient pump (LC-10AT VP pump), Mixer (Rheodyne injector, UV detector SPD 10 A yp), Agilent syringe, Vacuum pump (SUPER FIT 110336) Millipore nylon for solvents and sample filtration, Analytical weighing balance (Shimadzu AU X220), Software (EZ-chrome elite software- double channel).

Preparation of mobile phase

Trifluroacetic acid (TFA): Acetonitrile (30:70) was prepared using HPLC grade water. The mobile phase was filtered through 0.45 µ membrane filter. The pH was adjusted to 3.8 ± 0.1 and degassed by sonicating for about 10 minutes prior to use.

Specificity

The specificity is the ability to unequivocally assess the analyte in the presence of impurities, matrix components and degradation products that may be expected to be present as per ICH. 1000 µg ml^-1 concentration was prepared for roflumilast with solvent system. By using mobile phase as solvent, working concentrations of 1035 µg ml^-1 were prepared. Mobile phase chromatogram is shown in figure 2.

Linearity

The linearity of roflumilast was determined by the analysis of analyte with concentration across 10 µg/ ml to 35 µg/ml and the area of roflumilast measured at 250 nm were plotted in the graph. The chromatograms for concentration 10 µg/ml to 35 µg/ml are shown from figure 3 to figure 8. The linearity is shown in figure 2 - 8.

Precision

Precision was studied by analyzing multiple samplings of drug. Repeatability and intermediate precision were performed to confirm reproducibility of the method. Peak area and peak symmetry parameters of each sample were measured by using HPLC. The repeatability was carried out within-day in sample duplicates and intermediate precision was done or two days. The results were observed for the drug and were in an acceptable range for both the trials. The precision was expressed as % Relative Standard Deviation (RSD). The chromatograms for precision studies is given in figure 9 to figure 12.

Accuracy

The accuracy results for roflumilast by HPLC using Trifluroacetic acid: Acetonitrile (30:70) was determined for 80%, 100% and 120% of drug and is given in figure 13 -15.

From the data obtained, the peak theoretical plates per column were calculated by the expression (theoretical plates per column) n = (5.54Vr²)/LWh²

Theoretical plates per meter were calculated by using expression

n = (5.54Vr²)/Wh²

Where, n is number of theoretical plates per meter, Wh is the width of the peak of interest at half peak height and Vr is the distance along the base line between the point of injection and a perpendicular dropped from the maximum of the peak of interest. Tailing factor or symmetry factor of peak was calculated from the expression.

 Symmetry factor = a+b/2a

Where, a = distance from leading edge to peak midpoint, b = distance from mid-point to trailing point.

Results and Discussion

Results

Limit of Detection (LOD) and Limit of Quanitation (LOQ):

LOD and LOQ were calculated as per ICH guidelines based on signal- to – noise ratio. Chromatogram signals with low concentrations of analyte were compared with the signals of blank samples. A signal to noise ratio of 3:1 and 10:1 were considered for LOD and LOQ calculations respectively.

Discussion

All the chromatographic conditions used gave good acceptable results. The validation parameters were validated and results were found within the limits as per ICH guidelines. Mobile phase, Trifluroacetic acid: Acetonitrile in a ratio of 30:70 gave retention time of 2.317 min at 250 mm UV detection. The run time of 15 min was taken to check for any interfering peaks. All the chromatographic conditions were used to validate parameters like accuracy, precision, linearity, LOD, LOQ and system suitability. The results obtained in validation were within the limits.

Conclusion

 The proposed RP- HPLC method was found to be rapid, simple, precise, accurate and economic for estimation of roflumilast in bulk and pharmaceutical formulations. This method had validated all the guidelines according to ICH and can be used in routine quality control studies of roflumilast without any interferences of excipients.