Spectrophotometric and Reversed-Phase High-Performance Liquid Chromatographic Method for the Determination of Doxophylline in Pharmaceutical Formulations

Two methods are described for determination of Doxophylline in a solid dosage form. The first method was based on ultraviolet (UV)-spectrophotometric determination of the drug. It involves absorbance measurement at 274 nm (λmax of Doxophylline) in 0.1 N hydrochloric acid. The calibration curve was linear, with the correlation coefficient between 0.99 and 1.0 over a concentration range of 0.20–30 mg/ml for the drug. The second method was based on high-performance liquid chromatography (HPLC) separation of the drug in reverse-phase mode using the Hypersil ODS C18 column (250 × 4.6 mm, 5 mm). The mobile phase constituted of buffer acetonitrile (80:20) and pH adjusted to 3.0, with dilute orthophosphoric acid delivered at a flow rate 1.0 ml/min. Detection was performed at 210 nm. Separation was completed within 7 min. The calibration curve was linear, with the correlation coefficient between 0.99 and 1.0 over a concentration range of 0.165–30 mg/ml for the drug. The relative standard deviation was found to be <2.0% for the UV-spectrophotometry and HPLC methods. Both these methods have been successively applied to the solid dosage pharmaceutical formulation, and were fully validated according to ICH guidelines.


INTRODUCTION
Doxophylline is chemically designated as 7(1, 3 dioxolone-2-yl methyl) theophylline. Presence of a dioxolane group in position 7 differentiates it from theophylline. [1] The chemical structure of Doxophylline is provided herewith [ Figure 1]. [2] It is a new antibronchospastic drug recently introduced in therapy, with phar macological properties like theophylline, a potent adenosine receptor antagonist. Doxophylline does not affect gastric acid secretion, either in vivo or in vitro, unlike theophylline. The lack of side-Joshi, et al. J Young Pharm. 2010;2(3): [289][290][291][292][293][294][295][296] Doxophylline is indicated for the treatment of bronchial asthma and COPD. [6] Some analytical methods for quantitative determination of Doxophylline in pharmaceutical formulations are described in the literature, like ultraviolet (UV)spectrophotometry [7] and LC-MS (Liquid Chromatography-Mass Spectroscopy). [8][9][10] At present, no high-performance liquid chromatography (HPLC) and UV-spectrophotometric methods are reported for the estimation of Doxophylline in a tablet dosage form. The purpose of this work is to develop and validate the proposed methods for routine analysis in a quality control laboratory.

Standard preparation
For UV-spectrophotometric and HPLC methods Standard stock solution of 400 µg/ml was prepared by dissolving 40 mg working standard of Doxophylline in 100 ml of diluent. The working standard solution of Doxophylline had a final concentration of 20 µg/ml and was prepared by appropriate dilution from the stock solution.

Sample preparation
For UV-spectrophotometric and HPLC methods Twenty tablets were weighed and crushed into fine powder. An accurately weighed quantity of powder equivalent to about 125 mg of Doxophylline was transferred into a 250 ml volumetric flask. Add 100 ml of diluent and sonicate it for 30 min with continuous shaking. Make the volume up to the mark with 0.1 N HCl. This solution was filtered through a 0.45 µm HVLP nylon filter. Make an appropriate dilution to get the final concentration of Doxophylline 20 μg/ml . Appropriated aliquots were subjected to the above methods and the amount of Doxophylline was determined.

UV-spectrophotometric method
Construction of the calibration curve λ max of Doxophylline (20 µg/ml) was determined by scanning the drug solution in diluent and was found to be at 274 nm. To construct Beer's plot for Doxophylline, dilutions were made in diluent using stock solution at different concentration (4, 12, 16, 20, 24, and 30 µg/ml) levels. The drug followed linearity within the concentration range of 4-30 µg/ml.

Assay of the tablet formulation
Twenty tablets were weighed and crushed into fine powder.

Construction of the calibration curve
To construct Beer's plot for Doxophylline, dilutions were made in the diluent using stock solutions at different concentration (4, 12, 16, 20, 24 and 30 µg/ml) levels. The drug followed linearity within the concentration range of 4-30 µg/ml for Doxophylline at 210 nm.

Assay of the tablet formulation
Twenty tablets were weighed and crushed into fine powder. An accurately weighed quantity of powder equivalent to about 125 mg of Doxophylline was transferred into a 250ml volumetric flask. Add 100 ml of diluent and sonicate it for 30 min with continuous shaking. Make the volume up to the mark with 0.1 N HCl. This solution was filtered through a 0.45-µm HVLP nylon filter. Make appropriate dilution to get the final concentration of Doxophylline 20 µg-ml. Appropriated aliquots were subjected to the above methods and the amount of Doxophylline was determined.

System suitability and system precison (For HPLC)
This parameter has been performed before starting any validation parameter each time. The purpose of this parameter is to ensure that system is working properly and it can be used further for analysis and validation. For more details, Table 1.

Linearity
The plot of absorbances against concentration is shown in Figures 2 and 3. It can be seen that the plot is linear over the concentration range of 0.20-30 µg-ml in UVspectrophotometry and 0.165-30 µg/ml in HPLC for Doxophylline, with correlation coefficients (r 2 ) of 0.99798 and 0.99629, respectively. The obtained results are presented in Tables 2A and 2B.

Standard and sample solution stability
Standard and sample solution stabilities were evaluated at room temperature for 48 h. The relative standard deviation (RSD) was found to be below 2.0%. It shows that the standard and sample solutions were stable up to 48 h at       Figure 4 and Chromatograms of Standard and sample which are provided as Figures 5 and  6 respectively.

Method precision
The RSD for six replicates of the sample solution was <2.0%, which met the acceptance criteria established for the spectrophotometric and HPLC methods. The obtained results are presented in Tables 3A and 3B.

Accuracy
Accuracy was performed at three levels: 50, 100 and 150%. Percentage recovery and low RSD value show the accuracy of the spectrophotometric and HPLC methods. The data are presented in Tables 4A and 4B.

Method ruggedness
Ruggedness test was determined between two different analysts, instruments and columns. The value of RSD below 2.0% showed ruggedness of the developed spectrophotometric and HPLC methods. The results of ruggedness are presented in Tables 5A and 5B.

Method robustness
The method was found to be robust as small but deliberate changes in the method parameters had no detrimental effect on the method performance, as shown in Table 6. The content of the drug was not adversely affected by these changes, as evident from the low value of RSD, indicating that the method is robust.

Specificity
There was no interference from sample placebo, and peak purity of Doxophylline was 0.99629. This indicates that the developed analytical method was specific for its intended purpose.

For HPLC
Considering the efficiency of HPLC, an attempt has been made to develop simple, accurate, precise, rapid and economic methods for estimation of Doxophylline in a solid dosage form. Thus, the method described enables quantification of Doxophylline. The advantages lie in the simplicity of sample preparation and the cost-economic reagents used. The contribution of another important factor is its LOD. Results from statistical analysis of the experimental results were indicative of satisfactory precision and reproducibility. Hence, this HPLC method can be used for the analysis of different solid dosage formulations in commercial quality control laboratories.
The comparative advantages and disadvantages of the UV-spectrophotometric method and reverse-phase HPLC method has been provided herewith.