Pharmaceutica Analytica Acta

Roflumilast (Figure 1) is a chemically3-(cyclopropylmethoxy)-N- (3,5-dichloropyridin-4-yl)-4-(difluoromethoxy) benzamide and it is a selective, long acting phosphodiesterase-4 inhibitor.

Figure 1: Chemical structure of roflumilast.

Roflumilast was used for the treatment of chronic obstractive pulmonary disease [1,2] and roflumilast was freely soluble in methanol and lower alcohols. A few spectrophotometric [3-5] and HPLC [6,7] methods were reported earlier for the estimation of roflumilast in bulk and pharmaceutical dosage forms and this method was validated as per ICH guidelines [8]. Although no method was reported for the estimation of roflumilast in serum in pharmaceutical dosage forms. The present study was authors reported a simple, sensitive, accurate and precise for estimation of roflumilast in spiked human serum by using UV spectroscopy

Instrument

ElicoSL164 UV-Visible spectrophotometer with double beam detector configuration. The above instruments had automatic wavelength accuracy 0.1nm and matched quartz cells and Weighing balance (Elico, India).

Materials

Blank human serum, Roflumilast sample (99.90% purity) was purchased from Spectrum Pharma Research Solutions, Hyderabad, India. Hydrochloiric acid and water (HPLC grade) obtained from Qualigens Fine Chemicals, Mumbai, India.

Method development

Preparation of standard solution: A standard stock solution was prepared by accurately weighed 1000 mg of roflumilast in 1000ml of volumetric flask and dissolved in 0.2M of Hcl to obtain a concentration 1000 μg/ml. Further diluting 2.5ml of stock solution to 25ml with distilled water to get desired concentration of 25μg/ml. Further diluting 0.4ml of stock solution to 100ml with distilled water to get desired concentration of 40μg/ml.

Selection of wavelength for analysis of Roflumilast: Accurately measured 0.4ml of standard stock-III was transferred in to 100ml volumetric flask and diluted to 100ml to give concentration of 40μg/ml and it was used for initial spectral scan in the range of 200-400 nm to detect maximum wavelength and further dilutions for linearity was prepared from the stock solution by allegation method. The maximum wave length was found at 248nm.

Preparation of calibration plot: 5ml of serum was taken in a 250 ml separating funnel and spiked with 10ml aqueous solution contains 40-88 μg/ml of Roflumilast (40, 50, 64, 72, 88 μg/ml).

To the same solution add 20 ml of methyl acetate. The content was shaken for 15 min and the liquids were allowed to separate in to two immiscible phases.

The lower aqueous layer was discarded and the upper organic layer was collected in a beaker. Finally water free organic layer was transferred in to a dried beaker and evaporated to dryness on a hot water bath. The dry residue was reconstituted with 2ml of 0.2M HCL and transferred in to a 25ml calibrated flask. The volume was making with water.

Method validation

The developed method was validated for variouspp parameters like linearity, precision, accuracy, limit of detection, limit of quantitation, robustness and ruggedness according to ICH guidelines.

Linearity: The manifest linear relationship in the range of 40-88 μg/ml of roflumilast.

Precision: The intra day precision analysed in the same day and inter day precision analysed for three consecutive days. The results was indicated by calculated percent relative standard deviation.

Accuracy: Recovery study was carried out at three different levels 50%, 100% and 150%. The percentage recovery was calculated as mean ± standard deviation.

Limit of detection (LOD): Formula for measuring of limit of detection.

LOD = 3.3 σ/S

Where,

σ = Standard deviation

S = Slope

Limit of quantification (LOQ): Formula for measuring of limit of quantification.

LOD = 10 σ/S

Where,

σ = Standard deviation

S = Slope

Robustness: The robustness of an analytical procedure is a measure of its capacity unaffected by a small.

Ruggedness: The ruggedness to express in condition with variations like different analyst, different instruments and different days.

Selection of wavelength

The spectra of roflumilast in 0.2M hydrochloric acid showed absorption at 248 nm shown in Figure 2.

Figure 2: UV spectrum of roflumilast.

Linearity

The linearity for the proposed method was investigated at five concentration levels (40-88 μg/ml) of reference standard roflumilast. The linearity was shown in Table 1 and Figure 3.

Table 1: Linearity data of roflumilast.

Figure 3: Linearity plot of roflumilast.

Precision

The percent RSD value of intra day and inter day precision were found to be 0.031 and 0.046 respectively, as shown in Table 2.

Table 2: Precision data of roflumilast.

Accuracy

The percentage of recovery of roflumilast was found to be 99.82 %, shown in Table 3.

Table 3: Accuracy data of roflumilast.

Limit of detection

The limit of Detection (LOD) value obtained was 0.783 μg/ml, it indicates the high sensitivity of the proposed method Table 4.

Table 4: Validation parameters of roflumilast.

Limit of quantitation

The limit of quantitation (LOQ) obtained was 2.375 μg/ml, it indicates the high sensitivity of the proposed method.

Robustness

The percent RSD of roflumilast was found to be 0.966. The robustness of results were shown in Table 5.

Table 5: Robustness data of roflumilast.

Ruggedness

Percent relative standard deviation of roflumilast was found to be 0.719. The ruggedness of results were shown in Table 6.

Table 6: Ruggedness data of Roflumilat.

The proposed method was simple, linear, precise, accurate and robust developed for the estimation of roflumilast in human serum by using UV spectroscopy. The method was validated as per the ICH guidelines. Hence, The present method can be adopted for routine in quality control and reaserch industries.