SIMULTANEOUS ESTIMATION OF DOXOFYLLINE AND AMBROXOL IN TABLET DOSAGE FORM BY REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

SIMULTANEOUS ESTIMATION OF DOXOFYLLINE AND AMBROXOL IN TABLET DOSAGE FORM BY REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

1. Sonia ^*1, M. Nappinnai ² and K. Manikandan¹

Department of Pharmaceutical Analysis ¹, SRM College of Pharmacy, Kattankulathur, Tamil Nadu, India.

Department of Pharmaceutics ², Surya College of Pharmacy, Vikravandi, Villupuram, Tamil Nadu, India.

ABSTRACT: A new, simple, accurate, linear, precise, efficient and reproducible Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method for the simultaneous determination of Ambroxol and Doxofylline in combined tablet dosage form. The chromatographic method was standardized using a Kromosil C18, 150mm × 4.6 mm, 3.5 ? (particle size), Thermo scientific from Germany with isocratic conditions, and mobile phase containing potassium dihydrogen orthophosphate buffer-pH 6.8 (0.01M KH₂PO₄): acetonitrile (25: 75) at flow rate of 1ml/min using UV detection at 257 nm. The retention times of Ambroxol and Doxofylline were 5min and 2min, respectively. The method was linear over the concentration range for Ambroxol 0.6–36 ?g/mL and for Doxofylline 16-96 ?g/mL. The recovery of Ambroxol and Doxofylline was found to be in the range of 98.13–99.85% and 98.09–99.66%, respectively. The validation of method was carried out using ICH guidelines. The described HPLC method was successfully employed for the analysis of pharmaceutical formulations containing combined dosage form. From the comprehensive validation conducted, it was concluded that the method is stable and could be used throughout shelf life of the drug.

Ambroxol, Doxofylline, RP-HPLC, method development and validation

INTRODUCTION: A combination drug or fixed-dose combination is a formulation of two or more active ingredients combined in a single dosage form, available in certain fixed doses. Fixed-dose combination therapies are hypothesized to enhance compliance by decreasing the number of required pills. Fixed dose combinations of drugs used to   treat   different   cardiovascular   diseases   are   available   in   different   dosage   forms   like   tablet,   capsules, modified release formulations, controlled release formulations and prolonged release combinations.

As the physio-chemical properties of the combined drugs lead to practical difficulties during execution of assay specified for the individual drugs in official compendia, necessity is present to separate, identify and determine the relative amount of the component in sample mixture. Monitoring of pharmaceutical product for the identity, strength, quality and purity, is necessary to ensure the safety, efficacy throughout the shelf life including storage, distribution and use.

A combination drug or fixed-dose combination is a formulation of two or more active ingredients combined in a single dosage form, available in certain fixed doses. Fixed-dose combination therapies are hypothesized to enhance compliance by decreasing the number of required pills. Fixed dose combinations of drugs used to treat   different   cardiovascular   diseases   are   available   in   different   dosage   forms   like   tablet,   capsules, modified release formulations, controlled release formulations and prolonged release combinations. As the physio-chemical properties of the combined drugs lead to practical difficulties during execution of assay specified for the individual drugs in official compendia, necessity is present to separate, identify and determine the relative amount of the component in sample mixture. Monitoring of pharmaceutical product for the identity, strength, quality and purity, is necessary to ensure the safety, efficacy throughout the shelf life including storage, distribution and use¹.

Ambroxol (Fig. 1.A) is a secretolytic agent used in the treatment of respiratory diseases associated with viscid or excessive mucus. It is chemically Trans-4 - (2-Amino-3, 5-dibrombenzylamino)-cyclohexanol ². Ambroxol is a very potent inhibitor of the neuronal Na+ channels. It is clinically proven systemically active mucolytic agent. When administered orally onset of action occurs after about 30 minutes. The breakdown of acid muco-polysaccharide fibers makes the sputum thinner and less viscous and therefore more easily removed by coughing. Although sputum volume eventually decreases, its viscosity remains low ^{3, 4}. It is white, hygroscopic and crystalline powder. Freely soluble in water, methanol and ethanol. Doxofylline (Fig. 2) (also known as doxofylline) is a xanthine derivative drug used in the treatment of asthma. It is phosphodiesterase inhibitor ^{5, 6}.

It is white to beige, odourless, crystalline solid. Its mechanism of action is related to the inhibition of phosphodiesterase activities, resulting in bronchodilating effects ^{7, 8}. Chemically it is 7-(1, 3-dioxolan-2-ylmethyl)-1, 3-dimethylpurine-2, 6-dione ⁹. The literature survey reveals that many methods have been reported for estimation of Ambroxol and Doxofylline individually and in combination with other drugs, and no HPLC method for simultaneous estimation of Ambroxol and Doxofylline has been reported. Hence an attempt has been made to develop new HPLC method which is simple, rapid, accurate, reproducible and linear method for simultaneous estimation of Ambroxol and Doxofylline in combined tablet dosage form has been validated ¹⁰.

MATERIALS AND METHODS:

Chemicals and reagent:

Methanol, Acetonitrile of HPLC grade was purchased from Merck (India) Ltd., Mumbai. Potassium di hydrogen phosphate, Sodium di hydrogen phosphate, Sodium hydroxide, Hydrochloric acid, ortho phosphoric acid of Analytical grade was obtained from Merck (India) Ltd., Mumbai. Triethylamine of Analytical grade was obtained from Spectrochem. Doxofylline and Ambroxol sample obtained from Chandra labs Pvt., Ltd., Hyderabad. Prandimet tablets containing 400mg Doxofyllinee and 15 mg Ambroxol procured from the local market.

Apparatus and chromatographic condition:

The chromatographic separation was carried out on HPLC system (Shimadzu LC-2010 Japan) with UV- Visible dual absorbance detector (PDA), Kromosil C₁₈(150x4.6mm,3.5µ SS column). The mobile phase consisting of 295 mg of potassium dihydrogenorthophosphoric acid in 100ml (pH 4.2 adjusted with ortho phosphoric acid) and acetonitrile were filtered through 0.45μ membrane filter before use, degassed and were pumped from the solvent reservoir in the ratio of 25:75 v/v was pumped into the column at a flow rate of 1 ml/min. The detection was monitored at 257nm. The volume of injection loop was 20 μl prior to the injection of the drug solution; the column was equilibrated for at least 20 min. with the mobile phase ¹¹.

 Preparation of mobile phase:

0.01mole of sodium dihydrogen phosphate was dissolved in 1000ml of water and adjust the pH-6.8 using acetonitrile; mix filtered and degassed mixture of acetonitrile and buffer in the ratio of 25:75.

 Preparation of Doxofylline working standard solution:

40mg of Doxofylline working standard was weighed and transferred into 100 ml volumetric flask. 15 ml of mobile phase was added sonicated about 10 min until all the content has been dissolved, then the volume was made up to the mark with mobile phase. 2 ml of above solution was diluted to 50 ml with mobile phase to get concentration of 16µg/ml ^{12, 13}.

 Preparation of Ambroxol working standard solution:

1.5mg of Ambroxol working standard was weighed and transferred into 100 ml volumetric flask.15 ml of mobile phase was added sonicate about 10 min until all the content has been dissolved, then the volume was made up to the mark with mobile phase. 2 ml of above solution was diluted to 50 ml with mobile phase to get concentration of 0.6 µg/ml.

Preparation of sample solution:

Weigh about 10 tablets and powdered. From that an equivalent amount of 16 mg of Doxofylline and 0.6 mg of Ambroxol was taken into 100 ml volumetric flask. Add about 10 ml of mobile phase and sonicate until the content was dissolved. Filter the content by using 0.45µ membrane filter by applying vacuum. Made the volume up to the mark with the mobile phase.

 Method Validation:

The objective of validation of an analytical procedure is to demonstrate its intended purpose were proposed according to ICH guidelines, the validation parameters were:

 System Suitability:

It is essential for the assurance of the quality performance of chromatographic system. From the standard solution of Doxofylline and Ambroxol five replicated injections were made. Various system suitability parameters like plate number (N), asymmetry factor, retention time, resolution, tailing factor, were evaluated from the standard chromatogram. Standard deviation & % RSD were also being calculated for the standard drug solutions. It was observed that all the values are within the limits ^{14, 15}.  The results were tabulated in Table 1.

TABLE 1: SYSTEM SUITABILITY FOR DOXOFYLLINE AND AMBROXOL

Specificity:

Specificity is the ability to assess unequivocally of an analyte in the presence of components that may be expected to be present. For the simultaneous determination of Doxofylline and Ambroxol the specificity requires that the method should not be affected by the presence of other components. Specificity is performed under stressed conditions. In addition, there was no any interference at the retention time of in the chromatogram of placebo solution. In peak purity analysis with PDA, purity angle was always less than purity threshold for the analyte. This shows that the peaks of analyte were pure and excipients in the formulation does not interfere the analyte ^{16, 17}.

1. i) Treating with Acids:

To 1 ml of stock solution, add 1 ml of 0.1M hydrochloric acid into a 10 ml flask, made up to the volume with the mobile phase. Observed for any change took place in the retention of the peak.

1. ii) Treating with Base:

To 1 ml of stock solution, add 1 ml of 0.1 M sodium hydroxide into a 10 ml flask, made up to the volume with the mobile phase.  Observed for any change took place in the retention of the peak.

iii) Heating:

To 1 ml of stock solution taken in a 10 ml flask, made up to the volume with the mobile phase. The solution should be heated at 40 ^°C for a period of 30 min.

The results were tabulated in Table 2.

 TABLE 2: SPECIFICITY RESULTS FOR DOXOFYLLINE AND AMBROXOL UNDER STRESS CONDITIONS

Linearity and Range:

Various concentrations of the Doxofylline was made by diluting stock solution to get the concentration of 16 to 96 μg and Ambroxol was made by diluting stock solution to get the concentration of 0.6 to 3.6 μg  it was shown in Table 3. The plots of peak area of each sample against respective concentration of Doxofylline and Ambroxol were found to be linear (Fig.2.A  and 2.B).  The dilution volumes used and peak area obtained are presented below. The relationship between the concentration and the peak response of Doxofylline was linear in specific range and the regression coefficient was found to be 0.999 and for Ambroxol is 0.998. It observed that correlation coefficient and regression analysis are within the limits.

TABLE 3: LINEARITY DATA FOR AMBROXOL

Preparation of sample stock solution:

About 40mg of doxofylline and1.5mg of ambroxol were weighed and transferred into 50ml volumetric flask. 15 ml of mobile phase was added sonicate about 10min until all the content has been dissolved, then the volume was made up to the mark with mobile phase. Diluted to 50 ml with mobile phase to get concentration of 0.8mg/ml of doxofylline and 0.03 mg/ml of ambroxol.

 Preparation of linearity solution-I:

Transfer 0.2 ml from stock solution to 10 ml with mobile phase (the solution contains 16 mcg of Doxofylline and 0.6 mcg of Ambroxol).

 Preparation of linearity solution-II:

Transfer 0.4ml from stock solution to 10 ml with mobile phase (the solution contains 32mcg of Doxofylline and 1.2mcg of Ambroxol).

 Preparation of linearity solution-III:

Transfer 0.6ml from stock solution to 10 ml with mobile phase (the solution contains 48mcg of Doxofylline and 1.8 mcg of Ambroxol).

 Preparation of linearity solution-IV:

Transfer 0.8 ml from stock solution to 10 ml with mobile phase (the solution contains 64mcg of Doxofylline and 2.4mcg of Ambroxol).

 Preparation of linearity solution-V:

Transfer 1ml from stock solution to 10 ml with mobile phase (the solution contains 96mcg of Doxofylline and 3.6 mcg of Ambroxol).

 Accuracy:

Accuracy for the Doxofylline and Ambroxol was carried out at three different levels. The recovery data for Doxofylline and Ambroxol is shown in table below. The accuracy of an analytical method is determined by applying the method to analyzed samples, to which known amounts of analyte have been added. The accuracy is calculated from the test results as the percentage of analyte recovered by the assay.

The accuracy of the method is determined by recovery with spiked concentration of pure drug at three levels for Doxofylline and Ambroxol.

 Preparation of Stock Solution:

Weigh 400mg of Doxofylline and 15 mg of Ambroxol in 50 ml volumetric flask dilute to volume with mobile phase.

 Preparation of Spiking standard:

Transfer 1 ml from stock solution to 100 ml with mobile phase.

 Preparation of Accuracy solution 1:

Transfer 0.8ml from stock solution to 100 ml with mobile phase (the solution contains 64mcg of Doxofylline and 2.4 mcg of Ambroxol) and add 1 ml of spiking standard.

Preparation of Accuracy solution 2:

Transfer 1ml from stock solution to 100 ml with mobile phase (The solution contains 80mcg of Doxofylline and 3mcg of Ambroxol) and add 1 ml of spiking standard.

Preparation of Accuracy solution 3:

Transfer 1.2ml from stock solution to 100 ml with mobile phase (The solution contains 96mcg of Doxofylline and 3.6mcg of Ambroxol) and add 1 ml of spiking standard. The recovery of drug is well within the acceptance limits of 97-103%.It was observed that the mean percentage recoveries were found to be for Doxofylline and Ambroxol respectively which demonstrated that the method was highly accurate. The results were tabulated in Table 4.

 Precision:

The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the homogenous sample under the prescribed conditions. The repeatability of the analytical method is done by estimating the assay for 5 sample proportion of the same batch under normal operating conditions. The % RSD for Doxofylline and Ambroxol were found to be 0.2148 and 0.4921 respectively. Hence, the precision of the system as found to be well within the acceptance criteria (not less than 2%).The results were tabulated in Table 5.

TABLE 4: ACCURACY RESULTS FOR DOXOFYLLINE AND AMBROXOL

 TABLE 5: PRECISION DATA FOR DOXOFYLLINE AND AMBROXOL

Reproducibility:

Reproducibility data for Doxofylline and Ambroxol were shown in Table 6. This indicated that method was highly precise. From the precision data of the method, the % RSD for Doxofylline and Ambroxol were found to be 0.46 and 0.29 respectively. The results were tabulated in Table 6.

 Intermediate Precision:

Intermediate precision study was carried out by repeating the completed experiment with different analyst, on different days to verify that in the same laboratory the method provides the same results once the development phase is over. Intermediate precision for Doxofylline and Ambroxol were shown in Table 7 by using different analyst on different days. This indicated that method was highly precise.

TABLE 6: METHOD PRECISION FOR DOXOFYLLINE AND AMBROXOL

 TABLE 7: INTERMEDIATE PRECISION FOR DOXOFYLLINE AND AMBROXOL

TABLE 8: RESULTS FOR DOXOFYLLINE AND AMBROXOL

Robustness:

Preparation of Sample Solution:

400 mg of Doxofylline and 15 mg of Ambroxol were accurately weighed into 50 ml volumetric flask and dilute to volume with mobile phase. Transfer 1ml from stock solution to 100 ml with mobile phase (the solution becomes 80mcg of Doxofylline and 3mcg of Ambroxol).

 Effect of variation of flow rate:

A study was conducted to determine the effect of variation in flow rate. The flow rate was varied at 0.9 ml/min to 1.1ml/min. On evaluation of the above results, it can be concluded that the variation in flow rate and wavelength not affected the method significantly. Hence it indicates that the method   is suitable even by change in the flow rate and wavelength ±10%. The robustness of the method established by making minor variations in the method parameters like, change   in flow rate by ± 10 % of actual flow rate.

DISCUSSION: The Specificity studies under stressed condition no degradation occurs for both the drug product and drug substance. Various concentrations of the Doxofylline and Ambroxol were made by diluting stock solution to get the concentration. The relationship between the concentration and the peak response of Doxofylline was linear in specific range and the regression coefficient was found to be 0.999 and for Ambroxol is 0.998. Accuracy for the Doxofylline and Ambroxol was carried out at three different levels. The accuracy of the method is determined by recovery with spiked concentration of pure drug at three levels for Doxofylline and Ambroxol. The mean recovery for Ambroxol is 99.59% and Doxofylline is 99.44%. The % RSD for Doxofylline and Ambroxol is 0.2148 and 0.4921 respectively. In method precision the % RSD for Doxofylline and Ambroxol is 0.46 and 0.291 respectively. The flow rate was varied at 0.9 ml/min to 1.1ml/min. On evaluation of the results, it is concluded that the variation in flow   rate and wavelength not affected the method significantly. Hence it indicates that the method   is suitable even by change in the flow rate and wavelength ±10%.

CONCLUSION: The Proposed study describes new and simple RP-HPLC method capable of analyzing a large number of samples in a short time period with good accuracy and precision. The main purpose of the study was to develop accurate, precise and economic methods for the determination of Doxofylline and Ambroxol. Spectrophotometric technique, and post column derivatization method were applied without using any prior chemical pretreatment in the presence of the strongly overlapping spectra can generate large amounts of data within a short period of analysis.

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    How to cite this article:

    Sonia K, Nappinnai M and Manikandan K: Simultaneous Estimation of Doxofylline and Ambroxol in Tablet Dosage Form by Reverse Phase High Performance Liquid Chromatography. Int J Pharm Sci Res 2016; 7(9): 3721-27.doi: 10.13040/IJPSR.0975-8232.7(9).3721-27.

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