Asian J. Pharm. Ana. 2011; Vol.

1: Issue 4, Pg 67-70

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ISSN- 22315667 (Print) www.asianpharmaonline.org ISSN- 22315675 (Online) 0974-3618 REVIEW ARTICLE

An Analytical Approach of Doxofylline: A Review

ABSTRACT:

Doxofylline is chemically designated as 7-(1, 3 dioxolone-2-yl methyl) theophylline. Presence of a dioxolane group in position C-7 differentiates it from theophylline. It is a new antibronchospastic drug recently introduced in therapy with pharmacological properties like theophylline; a potent adenosine receptor antagonist. doxofylline do not affect gastric acid secretion; either invivo or in-vitro; unlike theophylline. The lack of side effects with doxofylline indicates that the drug can be used safely and effectively. Some analytical methods for quantitative determination of doxofylline in pharmaceutical formulations like UV-Spectrophotometry, HPLC and LC-MS are reported. The present review deals with the various analytical methods reported as well as adopted for the estimation of doxofylline.

KEYWORDS: Doxofylline, UV, HPLC, LC-MS. INTRODUCTION:

Doxofylline is methyl xanthine derivatives 1; it is a bronchodilator and plays a direct role in bronchial relaxation of bronchial smooth muscle. Doxofylline by inhibiting the phosphodiesterase within the smooth muscle cells and cause smooth muscle relaxation, thus achieving suppression of asthma. Doxofylline is a novel bronchodilator xanthine that differs from theophylline because the presence of a dioxalane group in position C-72. Like theophylline, mechanism of action of doxofyllines is related to the inhibition of phosphodiesterase activities. However, differently from theophylline, doxofylline appears to have decreased affinities toward adenosine A1 and A2 receptors which may account for the better safety profile of the drug3. As per pharmacokinetic parameters it absorbed rapidly. In healthy adults for the first 0.4 g oral, time to peak plasma concentration (Tmax) was 1.22 hours, the peak plasma concentration (Cmax) for a 0.9 g / ml. The materials widely distributed in various organs, with the highest content in the lungs4. The phony goods to and from the form of metabolites in urine excretion, for the main metabolite of - hydroxyethyl theophylline.

The elimination half-life of the goods (t1 / 2) for 7.42 hours. Eight healthy male volunteers for each of the 0.4 g of oral, twice daily, for seven days, reaching a steady state Cmax (13.00 2.54) g / ml, Cmin (3.22 1.99) g / ml, steady Mean plasma concentration of (7.11 1.72) g / ml. Taking xanthine derivatives may cause nausea, vomiting, abdominal pain, headache, insomnia, irritability, tachycardia, period of contraction, shortness of breath, high blood sugar, and proteinuria. Excessive use will emerge as a serious arrhythmia5. ANALYTICAL APPROACH FOR THE DETERMINATION OF DOXOFYLLINE: H R Joshi and coworkers6 reported two methods for determination of Doxophylline in solid dosage form. The first method was based on UV-Spectrophotometric determination of the drug. It involves absorbance measurement at 274 nm (absorption maxima of Doxophylline) in 0.1 N hydrochloric acid. Calibration curve was linear with the correlation coefficient between 0.99 to 1.0 over a concentration range of 0.20 to 30 g/ml for the drug. The second method was based on HPLC separation of the drug in reverse phase mode using Hypersil ODS C18 column (250 X 4.6mm). The mobile phase constituted of Buffer: Acetonitrile (80:20) and pH adjusted to 3.0 with dilute orthophosphoric acid was delivered at the flow rate 1.0 ml min-1. Detection was performed at 210 nm. Separation was completed within 7 min. Calibration curve

Received on 09.09.2011 Accepted on 18.10.2011 Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 1(4): Oct. - Dec. 2011; Page 67-70

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was linear with the correlation coefficient between 0.99 to 1.0 over a concentration range of 0.165 to 30 g/ml for the drug. The relative standard deviation (R.S.D) was found 2.0% for both U.V. Spectrophotometry and HPLC methods. Both these methods have been successively applied to solid dosage pharmaceutical formulation and were fully validated according to ICH guidelines. Ashu Mittal and Shikha Parmar7 were reported a simple, selective, rapid, and economical reversed phase high performance liquid chromatography (RP-HPLC) method for the determination of doxofylline in the commercial dosage form. The separation and quantification were achieved on a HiQ Sil C 18 W column using a mobile phase of acetonitrile: buffer (50: 50), pH 3, at a flow rate of 1 mL/min with detection of analyte at 272 nm. The separation was achieved within 3.1 0.3 min for doxofylline sample. The method showed good linearity in the range of 1080 g/ml. The intra and inter day RSD ranged from 0.37 0.53%. The recovery (mean S.D.) of low, middle and high concentrations were 100.04 0.80, 100.01 0.20, 100.07 0.30 respectively. Limit of detection and limit of quantification were 0.03 and 0.1 g/mL, respectively. Anurekha Jain and coworkers8 developed HPLC method proved to be stability indicating, separating the impurity peaks from doxofylline and montelukast. Acceptable intraday - interday assay precision (<2% RSD) and accuracy (98%-102% with <1% RSD) were observed over the linear ranges for doxofylline and montelukast were 0.759 48.0 g/ml (correlation coefficient = 0.99950.0009, n=3) and 2.1 150 g/ml (correlation coefficient = 0.99910.0005, n=3), respectively. The derivative spectroscopy method also proved to be precise (Intraday - Interday RSD < 1.5 %), linear (correlation coefficient = 0.9990 0.0003, n=3) ranging between 15 g/ml 37.5 g/ml and accurate 99% - 101%, RSD< 1% for both drugs, as per the ICH and USP limits. The fvariance ratio test for HPLC and first derivative method was found to be 2.30 and 1.50, respectively. The theoretical value of students t-test and f-variance ratio test at 95% confidence limit are t=2.26 and F=3.18, respectively. R.Gannu9 reported a simple, isocratic; stability-indicating reversed-phase high-performance liquid chromatographic (RP-HPLC) method for determination of doxofylline in human serum. The method employs precipitation for isolation and sample concentration, followed by reversedphase liquid chromatographic analysis with ultraviolet (UV) detection at 275 nm. Analytes were extracted from serum with 400 l methanol. Samples containing the internal standard (metronidazole) and doxofylline were chromatographed on a C-18column with 18:82 acetonitrile phosphate buffer (12.5 mMpotassium dihydrogen orthophosphate, pH 3.0) as mobile phase at a flow rate of 1 ml min1. The retention times of doxofylline and metronidazole were 7.54 min and 4.36 min, respectively. The intra-day and in-ter-day coefficients of variation and the error of the assay were <15%. The calibration plot was

linear for concentrations in the range of 0.025 to 25 g/ml. The extraction recovery of doxofylline was >95%. The validated method was applied to a pharmacokinetic study of doxofylline in human serum after administration of a single doxofylline tablet (400 mg). The method is ideally suited to estimation of the drug for pharmacokinetic studies on human volunteers after oral administration of doxofylline. Nimmagadda Sreenivas and coworkers10 developed a highly sensitive and specific LC-MS/MS method for the estimation of doxofylline (DFL) with 300 microL human serum using imipramine as the internal standard (IS). The API-3000 LCMS/MS was operated under multiple reaction-monitoring modes using the electrospray ionization technique. The assay procedure involved direct precipitation of DFL and IS from human serum with acetonitrile. The resolution of peaks was achieved with formic acid (pH 2.5): acetonitrile (10:90, v/v) on an Amazon C-18 column. The total chromatographic run time was 3.0 min and the elution of DFL and IS occurred at approximately 1.46 and 2.15 min, respectively. The MS/MS ion transitions monitored were 267.5 --> 181.1 for DFL and 281.1 --> 86.2 for IS. The method was proved to be accurate and precise at linearity range of 1.00-5000 ng/ml with a correlation coefficient (r) of >/=0.999. The method was rugged with 1.00 ng/ml as lower limit of quantitation. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a pharmacokinetic study in human volunteers following oral administration of DFL tablet. F Tagliaro11 et al., reported a HPLC method for the simultaneous measurement of dyphylline and doxofylline in serum. Chromatography is carried out with a "Pinkerton" internal surface reversed-phase column and phosphate buffer (0.1 mol/L, pH 6.8) as mobile phase. Absorbance at 275 nm is monitored. Only 10 micro/L of serum is required to detect less than 1 mg of each drug per liter in biological samples. The standard curve for the method is linear over the range 6-100 mg/L, and precision is acceptable for both drugs, with CVs of 2% and 1.5% for respective concentrations of 6.2 and 25 mg/L. None of the 76 drugs tested for interference affected the measurement of either drug. Four samples can be injected per hour. This method provides a fast, accurate way to monitor two interesting therapeutic agents used in chronic obstructive airway diseases. Kamila and coworkers12 developed a simple, sensitive and accurate UV spectrophotometric method for the determination of an anti-asthmatic drug, doxofylline, in raw material and in tablets. The drug showed maximum absorption at 272 nm in water. Beers law was obeyed in the concentration range 5-50 g/ml of drug with an apparent molar absorptivity and Sandell sensitivity of 6.2 x 10 L.mol-cm- and 0.0363 g cm-/0.001A, respectively. The limits of detection and quantification were calculated to be 0.9623 and 2.9161 g/ml, respectively. The method was

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successfully applied to the determination of doxofylline in tablets. Results were validated statistically as per ICH guidelines. It was found that the excipients present in the commercial formulation did not interfere with the method. Lagana13 et al., developed a sensitive and selective high performance liquid chromatographic doxophylline assay with ultraviolet detection for plasma samples. The drug is isolated from biological samples with a reversed phase C18 disposable extraction column. Plasma standard curves are linear for concentrations of doxophylline from 0.03 to 10 mg/L. At the therapeutic range concentrations, the recoveries are better than 96.9%. The coefficients of variation for the procedure are 4.1% and 2.7% for the concentrations 0.03 mg/L and 10 mg/L, respectively. By this method, pharmacokinetic profiles are obtained for six adult volunteers. Xie Zi-li14 reported a method for the determination of doxofylline in doxofylline injection by capillary gas chromatography. The capillary column was DM-17(30 m0.32 mm0.25 m).The carrier gas was nitrogen,and the detector was FID.The column temperature was 2650C,and papaverine hydrochloride was selected as internal standard.The assay of doxofylline was calculated by internal standard method. Under this condition, the linearity relationship of doxofylline in the concentration range of 0.0597 to1.194 g/ml was good (r=0.9998,n=6).The average recovery (n=9) of doxofylline was 98.0%(RSD=1.4%). The proposed method is simple,convenient,and accurate in this work. It can be used for the quality control of doxofylline in bulk drug and dosage forms.

nm.The sample was separated on Waters C18(150 mm3.9 mm,4 m),eluted with acetonitrile consists of 0.1% triethylamine-0.02 molL-1 NaH2PO4 buffer(pH was adjusted to 6.80.1 with NaOH) (15: 85) as the mobile phase at flow rate 1.0 mLmin-1. The column temperature was 300C.The HPLC method was used to determinated the doxophylline in COPD patients after intravenous administration (300 mg, once-daily) from days 1 to 6. The pharmacokinetic parameters were analyzed by 3P97. It showed a good linear range of 0.05-8.0 g/ml (r=0.9999).The limited detection concentration was 0.01 g/ml.The method recovery was 103.2%-105.0%, the intraday RSD and the inter-day RSD were 0.84%-3.2% and 3.9%-7.9%(n=5).After intravenous administration of doxophylline in 10 COPD patients,the main pharmacokinetic parameters AUC0-t,Vc,T1/2 ,T1/2 and CLs were (31.066.29) g/ml, (26.5311.77) L, (0.050.01) h, (1.720.44) h and (23.645.02) Lh-1 respectively.

Wang Shujun and coworkers18 established a method for determining the concentration of doxofylline in human serum by reversed phase HPLC. A Techsphere ODS colum was used with a mobile phase of methanol water (312: 488, V/V), flow rate was 0.8 mlmin -1, the detecting wavelength was 273 nm. Serum sample was used for chromatography after protein precipitation and centrifuging. The peak area of doxofylline against doxofylline concentration was linear with a correlation coefficicent of 0.999 over the range of 0.75-20.00 mgL -1 The minimal detectable concentration of this method was 0.25 mgL -1 The average recovery was 99.21%, and the RSD within day and between day were less than 5%, respectively. This method is simple,accurate and precise. It is usable for clinical pharmacokinetic studies and Guo Junping and coworkers15 established a method to monitoring of doxofylline in human serum. determine doxofylline concentration in plasma by SEPHPLC. The doxofylline concentrations in plasma through Xu Yanggui19 established a method to determine solid-phase extraction were determined by HPLC method. doxofylline concentration in plasma by HPLC, and its RSD of intra-day and inter-day were less than 10% the pharmacokinetic properties were studied. The mobile phase accuracy accorded with the plasma concentration was composed of methol-water-triethylamine-acetic acid monitoring and pharmacokinetic study. The SPE-HPLC glacial (35:65:0.01: 0.01), flow rate was 1 ml min-1, the method to determine doxofylline concentration is a ideal detecting wavelength was 272 nm and the limit was 20 ng ml-1. All data were dealt with by "3p97" program. The method. recoveries of three doxofylline concentrations were 95. 85 Liu Yifang16 were developed a HPLC method for the 3.69% with RSD = 3. 85%, 101.30 = 3.74%, RSD = 3.69%, determination of theophylline and doxofylline in human and 100.70 5.84% with RSD = 5.80%, respectively. In the plasma.A C18 column was used,with caffeine as internal three patients, one was suitable for two-compartment standard and the mobile phase of methanol-water(23:77)at model; the others were suitable for one-compartment 273 nm.The plasma samples were injected directly after model. Conclusion; the method is found to be rapid, precipitation by methanol.The calibration curves of accurate and precise. It is suitable for clinical theophylline and doxofylline were linear in the pharmacokinetic study. concentration range of 2.5-40 g/ml.The intra-and inter-day Yao Yinxiu20 et al. reported the bioequivalence of RSDs were less than 5.2%. doxofylline produced in different companies in China was Kan Quan-cheng17 et al., established an HPLC method for studied in 8 Chinese healthy male volunteers. A randomized determination of doxophylline in serum and study its two way cross over study design was adopted in the study. pharmacokinetics in chronic obstructive pulmonary disease The 8 volunteers were randomly divided into two oral (COPD) patients after intravenous administration. The groups equally. One group took single oral dose of 300 mg doxophylline in serum was determinated by HPLC at 273 tested doxofylline, while the other took referred doxofylline

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[AJPAna.] Anurekha Jain, Amit P. Khandhar, Shweta Maheshwari, Deepika Maliwal, Analytical Method Development, Validation and Comparison of Spectrophotometric and Stability Indicating HPLC Methods for the Simultaneous Estimation of Doxofylline and Montelukast in Pharmaceutical Dosage Form, AAPS Annual Meeting and Exposition November 10, 2009 . R.Gannu, S.Bandari, S.G.Sudke,Y.M.Rao, B.P.Shankar, Development and Validation of a Stability-Indicating RP-HPLC Method for Analysis of Doxifylline in Human Serum. Application of the Method to a Pharmacokinetic Study, Acta Chromatographica, 2007, 19, 149-157. Nimmagadda Sreenivas, M Lakshmi Narasu, B Prabha Shankar, Ramesh Mullangi, Development and validation of a sensitive LCMS/MS method with electrospray ionization for quantitation of doxofylline in human serum: application to a clinical pharmacokinetic study, Biomed Chromatography, 2008, 6, 65461 . F Tagliaro, R Dorizzi, A Frigerio and M Marigo, Non-extraction HPLC method for simultaneous measurement of dyphylline and doxofylline in serum, Clinical Chemistry, 1990, 36, 113-115. Kamila,M.M., Mondal,N., Ghosh, L. K., Development and validation of spectrophotometric method for estimation of antiasthmatic drug doxofylline in bulk and pharmaceutical formulation, Indian J Chem Tech, 2007, 14, 523-525. A. Lagana, M. Bizzarri,A. Marino, M. Mancini, Solid phase extraction and high performance liquid chromatographic determination of doxophylline in plasma Biomedical Chromatography, 1990, 4, 205207. Xie Zi-li, Determination of doxofylline in doxofylline injection by GC method , J Anhui Medical and Pharmaceutical, 2006, 9. Guo Junping, Wan Hongbo, Feng Yinjie, et al., Determination of Doxofylline in Plasma by SPE-HPLC, China Pharmacist, 2005, 4. Liu Yifang, Shen Weimin, Shen Jie, Song Zhongjuan1, Xia Yun, Determination of Theophylline and Doxofylline in Human Plasma by HPLC, Chinese Journal of Pharmaceuticals, 2010, 2. Kan Quan-cheng, Li Duo-lu,SHI Xiu-qin, HPLC determination of doxophylline and pharmacokinetic study in serum of patients with chronic obstructive pulmonary disease, Chinese Journal of Pharmaceutical Analysis, 2009, 5. Wang Shujun, Wu Lifeng, Xiong Jianhua, Zhang Kangshou, Determination of doxofylline concentration in human serum by HPLC, Chinese Journal of Clinical Pharmacy, 2003, 6. Xu Yanggui, Bo Yishan, He Zhenmei, Wang Chunyang, Li Jun, Gao Zhongyang, Determination of Doxofylline Concentration in Plasma by RP-HPLC, China Pharmacist, 2002, 8. Yao Yinxiu, Peng Wenxing, Li Huande, Bioequivalence of Doxofylline in Healthy Volunteers, J China Pharmaceutical University, 1998, 5. Zeng Ming-Hui,W.U. Zheng-Zhong, Xie Yu-Qiong,et al, Rapid determination of theophylline in serum by HPLC and its clinical application, Chinese Journal of Hospital Pharmacy, 2003, 12.

at the same dosage After one week wash out period, the two 8. groups were administered orally the other preparation of doxofylline respectively. Doxofylline concentrations in serum were detected by RP HPLC. The results showed that the relative bioavailability of tested doxofylline was 102%. A simplified two one sided t test showed that the two 9. doxofylline preparations were bioequivalent. The results also indicated that an inter subject variability for absorption of the drug exists Zeng Ming21 et al., determined theophlline rapidly in serum by HPLC. HPLC was selected with acetanilide as internal standard to determine concentration of theophylline in serum. The separation was performed on -Bondapak C-18( 4.6 mm250 mm,10 m)column with methanol- 11. water(25:75) as mobile phase.The flow rate was 1.2 mLmin -1 .The wavelength for detection was 265 nm .Ethyl acetate was used to precipitate the serum protein. The 12. calibration curve had good linearity between 2.5 to 40 mgL -1 , r = 0.9994 .The average recovery was 99.5% .The clinical application was good. This method is accurate, 13. rapid and simple. Doxofylline is available and frequently used in market; hence, there is great need to determine it in bulk, dosage form as well as to estabilish pharmacokinetic parameters. The need of analytical method validation is well reflect and highlights the aspect of analysts personal habits, that from part of the developed methods, and on the possibility of a person getting very reproducible results when he carries out the analysis again and again in a similar set of conditions. Any developed method however, may be influenced by variable like different elapsed assay times, different days, reagent lots, instruments, equipments or apparatus, environmental condition like temperature etc.
10.

CONCLUSION:

14. 15. 16. 17.

18. 19. 20. 21.

REFERENCE:
1. 2. 3. 4.

5. 6.

7.

Bagnato, G.F., Tolerability of doxofylline in the maintenance therapy of Pediatric patients with bronchial asthma, Eur Rev Med Pharmacol Sci., 1999, 3, 255- 260. Franzone, J.S., Cirillo R, Barone D, Doxofylline and theophylline are xanthines with partly different mechanisms of action in animals, Clinical Chemistry,1988, 38, 128-132. Dini, F. L., Cogo R., Doxofylline: a new generation xanthine bronchodilator devoid of major cardiovascular adverse effects, J Curr Med Res Opin, 2001, 16, 258-268. Lazzaroni, M., Grossi, E., and Bianchi Porro, G., The effects of intravenous doxofylline or amino group on gastric secretion in duodent ulcer patients, J Aliment Pharmacol Ther., 1990, 4, 643649 Grossi, E., Biffignandi, P. and Franzone, J. S., Doxofylline: pharmacological profile and a review of clinical studies, J Riv Eur Sci Med Farmacol, 1988, 10, 415- 430. H.R. Joshi, A.H.Patel, A.D.Captain, Spectrophotometric and Reversed-Phase High-Performance Liquid Chromatographic Method for the Determination of Doxophylline in Pharmaceutical Formulation, In. J. Chemical and Analytical Science, 2010, 1, 141-145. Ashu Mittal and Shikha Parmar, Development and validation of rapid HPLC method for determination of doxofylline in bulk drug and pharmaceutical dosage forms, J Ana Chem, 2010, 65, , 29329.

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