INTRODUCTION:

Doxofylline is methyl xanthine derivatives¹; 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 μ 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 β - 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) μ g / ml, Cmin (3.22 ± 1.99) μ g / ml, steady Mean plasma concentration of (7.11 ± 1.72) μ 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 µg/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 was linear with the correlation coefficient between 0.99 to 1.0 over a concentration range of 0.165 to 30 µg/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 10–80 μg/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 μg/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 µg/ml (correlation coefficient = 0.9995±0.0009, n=3) and 2.1 – 150 µg/ml (correlation coefficient = 0.9991±0.0005, 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 µg/ml – 37.5 µg/ml and accurate 99% - 101%, RSD< 1% for both drugs, as per the ICH and USP limits. The f-variance ratio test for HPLC and first derivative method was found to be 2.30 and 1.50, respectively. The theoretical value of student’s 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 reversed-phase liquid chromatographic analysis with ultraviolet (UV) detection at 275 nm. Analytes were extracted from serum with 400 µl 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 min⁻¹. 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 µg/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 LC-MS/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. Beer’s law was obeyed in the concentration range 5-50 μg/ml of drug with an apparent molar absorptivity and Sandell sensitivity of 6.2 x 10³ L.mol^-¹cm^-¹ and 0.0363 μg cm^-²/0.001A, respectively. The limits of detection and quantification were calculated to be 0.9623 and 2.9161 μg/ml, respectively. The method was 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 C-18 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 m×0.32 mm×0.25 μm).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 µg/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.

Guo Junping and coworkers¹⁵ established a method to determine doxofylline concentration in plasma by SEP-HPLC. The doxofylline concentrations in plasma through solid-phase extraction were determined by HPLC method. RSD of intra-day and inter-day were less than 10% the accuracy accorded with the plasma concentration monitoring and pharmacokinetic study. The SPE-HPLC method to determine doxofylline concentration is a ideal method.

Liu Yifang¹⁶ were developed a HPLC method for the determination of theophylline and doxofylline in human plasma.A C18 column was used,with caffeine as internal standard and the mobile phase of methanol-water(23:77)at 273 nm.The plasma samples were injected directly after precipitation by methanol.The calibration curves of theophylline and doxofylline were linear in the concentration range of 2.5-40 μg/ml.The intra-and inter-day RSDs were less than 5.2%.

Kan Quan-cheng¹⁷ et al., established an HPLC method for determination of doxophylline in serum and study its pharmacokinetics in chronic obstructive pulmonary disease (COPD) patients after intravenous administration. The doxophylline in serum was determinated by HPLC at 273 nm.The sample was separated on Waters C18(150 mm×3.9 mm,4 μm),eluted with acetonitrile consists of 0.1% triethylamine-0.02 mol·L-1 NaH2PO4 buffer(pH was adjusted to 6.8±0.1 with NaOH) (15: 85) as the mobile phase at flow rate 1.0 mL·min-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 µg/ml (r=0.9999).The limited detection concentration was 0.01 µg/ml.The method recovery was 103.2%-105.0%, the intra-day 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,T1/2α,T1/2β and CLs were (31.06±6.29) µg/ml, (26.53±11.77) L, (0.05±0.01) h, (1.72±0.44) h and (23.64±5.02) L·h-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 ml·min -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 mg·L -1 The minimal detectable concentration of this method was 0.25 mg·L -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 monitoring of doxofylline in human serum.

Xu Yanggui¹⁹ established a method to determine doxofylline concentration in plasma by HPLC, and its pharmacokinetic properties were studied. The mobile phase was composed of methol-water-triethylamine-acetic acid glacial (35:65:0.01: 0.01), flow rate was 1 ml min-1, the detecting wavelength was 272 nm and the limit was 20 ng ml-1. All data were dealt with by "3p97" program. The recoveries of three doxofylline concentrations were 95. 85 ± 3.69% with RSD = 3. 85%, 101.30 = 3.74%, RSD = 3.69%, and 100.70 ± 5.84% with RSD = 5.80%, respectively. In the three patients, one was suitable for two-compartment model; the others were suitable for one-compartment model. Conclusion; the method is found to be rapid, accurate and precise. It is suitable for clinical pharmacokinetic study.

Yao Yinxiu²⁰ et al. reported the bioequivalence of doxofylline produced in different companies in China was studied in 8 Chinese healthy male volunteers. A randomized two way cross over study design was adopted in the study. The 8 volunteers were randomly divided into two oral groups equally. One group took single oral dose of 300 mg tested doxofylline, while the other took referred doxofylline at the same dosage After one week wash out period, the two 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 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 μ-Bondapak C-18( 4.6 mm×250 mm,10 μm)column with methanol-water(25:75) as mobile phase.The flow rate was 1.2 mL·min -1 .The wavelength for detection was 265 nm .Ethyl acetate was used to precipitate the serum protein. The calibration curve had good linearity between 2.5 to 40 mg·L -1 , r = 0.9994 .The average recovery was 99.5% .The clinical application was good. This method is accurate, rapid and simple.

CONCLUSION:

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 analyst’s 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.

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Received on 09.09.2011 Accepted on 18.10.2011

Asian J. Pharm. Ana. 1(4): Oct. - Dec. 2011; Page 67-70