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PHARM ANALYSIS
Year : 2010  |  Volume : 2  |  Issue : 3  |  Page : 289-296 Table of Contents     

Spectrophotometric and reversed-phase high-performance liquid chromatographic method for the determination of doxophylline in pharmaceutical formulations


1 Shree Swaminarayan College of Pharmacy, Kalol Highway Road, Kalol - 382 721, Gujarat, India
2 Torrent Research Centre, Bhat, Dist.: Gandhinagar - 382 428, Gujarat, India
3 A.R. College of Pharmacy and Institute of Science and Technology for Advance Research (I.S.T.A.R), Vallabh Vidyanagar - 388 120, Gujarat, India

Date of Web Publication27-Jul-2010

Correspondence Address:
A H Patel
Torrent Research Centre, Bhat, Dist.: Gandhinagar - 382 428, Gujarat
India
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DOI: 10.4103/0975-1483.66791

PMID: 21042488

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   Abstract 

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 C 18 column (250 X 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.

Keywords: Doxophylline, HPLC, reversed-phase, UV-spectrophotometry


How to cite this article:
Joshi H R, Patel A H, Captain A D. Spectrophotometric and reversed-phase high-performance liquid chromatographic method for the determination of doxophylline in pharmaceutical formulations. J Young Pharmacists 2010;2:289-96

How to cite this URL:
Joshi H R, Patel A H, Captain A D. Spectrophotometric and reversed-phase high-performance liquid chromatographic method for the determination of doxophylline in pharmaceutical formulations. J Young Pharmacists [serial online] 2010 [cited 2014 Apr 23];2:289-96. Available from: http://www.jyoungpharm.in/text.asp?2010/2/3/289/66791


   Introduction Top


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 pharmacological 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-effects with doxophylline indicates that the drug can be used safely and effectively in the treatment of chronic obstructive pulmonary disease (COPD). [3] Doxophylline inhibits phosphodiesterase (PDE IV) activities with the consequent increase of cyclic AMP, which determines relaxation of the smooth musculature. Doxophylline appears to have decreased affinities toward adenosine A1 and A2 receptors, which may account for the better safety profile of the drug. Doxophylline does not interfere with calcium influx into the cells or antagonize calcium channel blockers. [4] Unlike aminophylline, it has low secretagogue activity and is suitable for asthmatic patients with peptic ulcer disease. [5]

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.


   Experimental Procedure Top


Instrument and condition

  1. UV-visible spectrophotometer - Model UV-1700 (Shimadzu, Tokyo, Japan).
  2. HPLC system - Shimadzu LC 2010C integrated system equipped with quaternary gradient pump, 2010C UV-VIS detector, 2010C column oven and 2010C programmable auto sampler controlled by CLASS-VP software. ( Shimadzu0 USA Manufacturing Inc, 1900, SE 4 th Ave, Canby, OR, 97013-4348, North America, USA)
  3. Analytical column - Hypersil ODS C 18 (250 X 4.6 mm, 5 mm particle size), (Weber Consulting, Attila u. 38/b. H-2132 Gφd, Hungary)
  4. Detector - UV visible
  5. Chromatographic parameters- Detection at 210 nm, flow rate 1.0 ml/min.
  6. Mobile phase - Potassium dihydrogen phosphate (pH 3.0 ± 0.2 adjusted with orthophosporic acid)-acetonitrile (80:20, v/v).
  7. Diluent - 0.1 N hydrochloric acid.


Reagents

  1. Doxophylline reference standard - Assigned purity 99.24% (Cadila Healthcare Limited, Ankleshwar, Gujarat, India).
  2. Acetonitrile - AR grade (Spectrochem), Spectrochem Private Limited, Office 221, 2nd Floor, Anand Bhuvan, 17, Babu Genu Road, Princess Street, MUMBAI - 400 002.
  3. Orthophosphoric acid - AR grade (E-Merck Limited), E-Merck (India) Ltd, Shiv Sagar Estate, `A', Dr. A B Road, Worli, Mumbai, 400018, India
  4. Commercially available Doxophylline tablet - Claimed to contain 800 mg of the drug. Procured from Zydus Cadila, Ahmedabad, Gujarat, India.


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

λmax 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. 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 is then filtered through a 0.45-μm HVLP (High Vinyl Liquid Polymer) 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.

HPLC Method

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 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 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.


   Result and Discussion Top


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 [Figure 2] and [Figure 3]. It can be seen that the plot is linear over the concentration range of 0.20-30 μg-ml in UV-spectrophotometry 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 [Table 2]A and B.

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 room temperature. Please refer, Spectrum of Standard which is provided as [Figure 4] and Chromatograms of Standard and sample which are provided as [Figure 5] and [Figure 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 [Table 3]A 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 [Table 4]A and B.

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 [Table 5]A and B.

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.


   Discussion Top


For UV-spectrophotometric method

The proposed analytical method is simple, accurate and reproducible. Doxophylline showed λmax at 274 nm. The advantages lie in the simplicity of sample preparation and the cost economic reagents used. The contribution of another important factor is its limit of detection (LOD). Results from statistical analysis of the experimental results were indicative of satisfactory precision and reproducibility. Hence, this spectrophotometric method can be used for analysis of different solid dosage formulations in commercial quality control laboratories.

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.


   Acknowledgments Top


The authors are thankful to Zydus Cadila Limited, Ahmedabad, India for providing reference standards and all facilities to complete this research work. [Table 7]

 
   References Top

1.Bagnato GF. Tolerability of doxofylline in the maintenance therapy of pediatric patients with bronchial asthma. Eur Rev Med Pharmacol Sci 1999;3:255-60.  Back to cited text no. 1  [PUBMED]    
2.Chen HX, Tu B, Shu Z, Ma XJ, Jin ZM. 7-(1,3-Dioxolan-2-ylmethyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione, International Union of Crystallography. Acta Crystallographica Section E, Structure Reports Online, organic compounds. Acta Cryst 2007;63:o726-7.  Back to cited text no. 2      
3.Franzone JS, Cirillo R, Barone D. Doxofylline and Theophylline are xanthines with partly different mechanism of action in animals. Drugs Exp Clin Res 1988;14:479-89.  Back to cited text no. 3  [PUBMED]    
4.Dini FL, Cogo R. Doxofylline: A new generation xanthine bronchodilator devoid of major cardiovascular adverse effects. Curr Med Res Opin 2001;16:258-68.  Back to cited text no. 4  [PUBMED]    
5.Lazzaroni M, Grossi E, Bianchi Porro G. The effects of intravenous doxofylline or amino group on gastric secretion in duodent ulcer patients, J Aliment Pharmacol Ther 1990;4:643-9.  Back to cited text no. 5      
6.Grossi E, Biffignandi P, Franzone JS. Doxofylline: Pharmacological profile and a review of clinical studies. J Riv Eur Sci Med Farmacol 1988;10:415-30.  Back to cited text no. 6      
7.Kamila MM, Mondal N, Ghosh LK. Development and Validation of Spectrophotometric method for estimation of anti-asthmatic drug doxofylline in bulk and pharmaceutical formulation. Indian J Chem Technol 2007;14:526-8.   Back to cited text no. 7      
8.Lagana A, Bizzarri M, Marino A, Mancini M. Solid phase extraction and high performance liquid chromatographic determination of doxofylline in plasma. Biomed Chromatogr 1990;4:205-7.  Back to cited text no. 8  [PUBMED]    
9.Tagliaro F, Dorizzi R, Frigerio A, Marigo M. Non-extraction HPLC method for simultaneous determination of dyphylline and doxofylline in serum. J Clin Chem 1990;36:113-5.  Back to cited text no. 9      
10.Sreenivas N, Narasu ML, Shankar BP, Mullangi R. Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of doxofylline in human serum: Application to a clinical pharmacokinetic study. Biomed Chromatogr 2008;22:654-61.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]  


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]


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