The present study described a new, simple, accurate, and precise high performance liquid chromatography method for the simultaneous determination of Ambroxol and Doxofylline in combined tablet dosage form. The chromatographic method was standardized using a BDS hypersil C18, 250 mm × 4.6 mm, 5
Ambroxol is a secretolytic agent used in the treatment of tracheobronchitis, emphysema with bronchitis pneumoconiosis, chronic inflammatory pulmonary conditions, bronchiectasis, and bronchitis with bronchospasm asthma [
Doxofylline is a new generation long acting oral methylxanthine derivative. Methylxanthines are phosphodiesterase inhibitors. It is mainly used for maintenance therapy in patients suffering from asthma and Chronic Obstructive Pulmonary Disease (COPD). Chemically, it is 7-(1,3-dioxolan-2-ylmethyl)-3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione [
From the literature survey, it was found 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 so far. Hence an attempt has been made to develop new HPLC method which is simple, rapid, reproducible, and economical method for simultaneous estimation of Ambroxol and Doxofylline in tablet dosage form.
The working standards of Ambroxol and Doxofylline were generous gift obtained from Ranbaxy Laboratories Ltd., India. The combination formulation of Ambroxol and Doxofylline (Synasma-AX) were marketed by Ranbaxy Laboratories Ltd., India; tablets were purchased from the local market. Acetonitrile, methanol, and water were used of HPLC grade make-Merck, Rankem. Potassium dihydrogen phosphate and phosphoric acid used were of analytical grade.
Chromatographic separation was performed with Shimadzu Prominence System (SPD-20AT, Shimadzu) having the following components: LC-20AT Pump, SPD-20A Detector, BDS hypersil C18, 250 mm × 4.6 mm, 5
The mobile phase consisted of potassium dihydrogen phosphate buffer pH 4.5-acetonitrile (60 : 40). To prepare the buffer solution, 6.8 gm potassium dihydrogen phosphate was weighed and dissolved in 900 mL HPLC grade water in 1000 mL volumetric flask. The pH of the buffer was adjusted to 4.5 with diluted phosphoric acid (H3PO4). The volume is made up to 1000 mL with HPLC grade water. Mobile phase was filtered through a 0.45
The mobile phase consisting of phosphate buffer-pH 4.5 (0.05 M KH2PO4): acetonitrile in the ratio (60 : 40) with an apparent pH adjusted to 4.5 using diluted phosphoric acid was selected as the optimum composition of mobile phase, because it was found that this solvent system resolved both the components ideally. The mobile phase and samples were degassed by ultrasonication for 20 min and filtered through 0.45
Twenty tablets were weighed, and the average weight was calculated. The tablets were crushed with a mortar and pestle for 10 min. A portion of powder equivalent to the weight of one tablet was accurately weighed and transferred to a 100 mL volumetric flask, diluted with mobile phase, and sonicated of 30 minutes with normal hand-shaking; then cool the flask to room temperature; afterward filter this solution through 0.45
Proper selection of the methods depends upon the nature of the sample (ionic or ionisable or neutral molecule), its molecular weight, and solubility. Ambroxol and Doxofylline were dissolved in polar solvent, so the developed method of estimation was called as reverse phase high performance liquid chromatography. To develop a rugged and suitable HPLC method for the quantitative determination of Ambroxol and Doxofylline, the analytical condition was selected after the consideration of different parameters such as diluent, buffer, buffer concentration, organic solvent for mobile phase and mobile phase composition, and other chromatographic conditions. Preliminary trials were taken with different composition of buffer and organic phase of mobile phases with pH range of 3–5. The column selection has been done by backpressure, resolution, peak shape, theoretical plates, and day-to-day reproducibility of the retention time and resolution between Ambroxol and Doxofylline peak. After evaluating all these factors, a BDS hypersil C18 column was found to be giving satisfactory results. The selection of acetonitrile and buffer were based on chemical structure of both the drugs (Figures
Chemical structure of Ambroxol.
Chemical structure of Doxofylline.
HPLC chromatogram of Ambroxol and Doxofylline standards.
HPLC chromatogram of Ambroxol and Doxofylline in tablet formulation.
Under above described experimental conditions, all the peaks were well defined and free from tailing. The concern of small deliberate changes in the mobile phase composition, flow rates, and column temperature on results was evaluated as a part of testing for methods robustness. The peak homogeneity was expressed for peak purity values and was obtained directly from the spectral analysis of the sample.
The developed analytical method was subjected to validation with respect to various parameters such as linearity, limit of quantification (LOQ), limit of detection (LOD), accuracy, precision, recovery studies, specificity and reproducibility, and robustness/ruggedness as per the ICH guidelines [
The specificity of the method was evaluated by assessing interference from excipients in the pharmaceutical dosage form prepared as a placebo solution. The specificity of the method for the drug was also established by checking for interference with drug quantification from degradation products formed during the forced degradation study. The peak purity of the Ambroxol and Doxofylline was found satisfactory under different stress conditions. There was no interference of any peak of degradation product with drug peak.
For linearity, five-point calibration curve was obtained in a concentration range from 3.75 to 11.25
Linearity study for Ambroxol.
Linearity study for Doxofylline.
Precision study was established by evaluating method precision and intermediate precision study. System precision was evaluated by analyzing the standard solution five times. Method precision of the analytical method was determined by analyzing three sets of sample preparation. Assay of all three replicate sample preparations was determined and standard deviation; % relative standard deviation was calculated.
Intermediate precision of the analytical method was determined by performing method precision on another day by another analyst under the same experimental condition. Assay of all three replicate sample preparations was determined and standard deviation; % relative standard deviation was calculated.
Data obtained from precision experiments are given in Table
Precision data for Ambroxol and Doxofylline.
Drug | Concentration ( |
Intraday ( |
C.V. | Interday ( |
C.V. |
---|---|---|---|---|---|
Ambroxol | 3.75 |
|
0.82 |
|
1.09 |
7.50 |
|
1.05 |
|
1.15 | |
11.25 |
|
0.97 |
|
0.93 | |
| |||||
Doxofylline | 50 |
|
0.99 |
|
0.86 |
100 |
|
1.20 |
|
1.04 | |
150 |
|
0.77 |
|
1.090 |
Accuracy study was assessed by determination of the % assay of Ambroxol and Doxofylline in market formulation. The mean % assay of Ambroxol was 98.42%, and the mean recovery of Doxofylline was 98.18%, that was satisfactory (Table
Results of accuracy study.
Drug | Label claim | Result | % assay | Average % assay | SD | % RSD |
---|---|---|---|---|---|---|
Ambroxol | 7.5 | 7.36 | 98.09 | 98.42 | 0.303337 | 0.308202 |
7.5 | 7.39 | 98.48 | ||||
7.5 | 7.40 | 98.69 | ||||
| ||||||
Doxofylline | 100 | 98.11 | 98.11 | 98.19 | 0.47832 | 0.487141 |
100 | 97.75 | 97.75 | ||||
100 | 98.70 | 98.70 |
Recovery of Ambroxol and Doxofylline was done at three different concentrations (corresponding to 80, 100, and 120% of test solution concentration). Known amounts of Ambroxol (3.75, 7.5, and 11.25
Results of recovery study of Ambroxol.
Concentration level | Standard: amount added in sample solution in |
Amount recovered in |
% recovery |
---|---|---|---|
80% | 6 | 5.98 | 99.71 |
80% | 6 | 6.03 | 100.57 |
80% | 6 | 6.00 | 100.04 |
100% | 7.5 | 7.49 | 99.84 |
100% | 7.5 | 7.56 | 100.84 |
100% | 7.5 | 7.53 | 100.34 |
120% | 9 | 8.95 | 99.43 |
120% | 9 | 9.11 | 101.18 |
120% | 9 | 9.01 | 100.14 |
Note: sample concentration was used 7.5
Results of recovery study of Doxofylline.
Concentration level | Standard: amount added in sample solution in |
Amount recovered in |
% recovery |
---|---|---|---|
80% | 80 | 79.72 | 99.65 |
80% | 80 | 79.69 | 99.62 |
80% | 80 | 79.98 | 99.97 |
100% | 100 | 99.78 | 99.78 |
100% | 100 | 99.60 | 99.60 |
100% | 100 | 100.28 | 100.28 |
120% | 120 | 119.25 | 99.37 |
120% | 120 | 119.94 | 99.95 |
120% | 120 | 120.11 | 100.09 |
Note: sample concentration was used 100
Statistical data for Ambroxol and Doxofylline by HPLC method.
Parameter | Ambroxol | Doxofylline |
---|---|---|
Linear range ( |
3.75–11.25 | 50–150 |
Slope | 57.06 | 34.24 |
Limit of detection ( |
0.59 | 1.80 |
Limit of quantitation ( |
4.97 | 15.06 |
The LOD was determined on the basis of signal to noise ratios and was determined using analytical response of three times the background noise. LOQ was determined as the lowest amount of analyte that was reproducibly quantified above the baseline noise following triplicate injections. Both LOQ and LOD were calculated on the peak area using the following equations:
The limit of detection and limit of quantification were evaluated by serial dilutions of Ambroxol and Doxofylline stock solution in order to obtain signal to noise ratio of 3 : 1 for LOD and 10 : 1 for LOQ. The LOD value for Ambroxol and Doxofylline was found to be 0.597
The robustness was studied by evaluating the effect of small but deliberate variations in the chromatographic conditions. The conditions studied were flow rate (altered by ±0.2/min), mobile phase composition (by using 58 : 42 and 62 : 38 v/v buffer pH 4.5: acetonitrile), buffer pH (altered by ±0.2), and use of HPLC columns from different batches. The result of robustness study of the developed assay method was established in Tables
Robustness data for Ambroxol.
Factors | % RSD | Tailing factor ( |
Efficiency (theoretical plates) | Resolution |
---|---|---|---|---|
pH of mobile phase | ||||
pH 4.3 | 1.1935 | 1.455 | 6903 | 14.724 |
pH 4.7 | 0.7549 | 1.348 | 7318 | 14.810 |
| ||||
Flow rate | ||||
0.8 mL/min | 0.7022 | 1.375 | 7090 | 14.736 |
1.2 mL/min | 0.7072 | 1.429 | 7109 | 14.745 |
| ||||
Solvent % | ||||
−2% | 1.0015 | 1.348 | 6890 | 14.698 |
+2% | 1.1029 | 1.455 | 6851 | 14.672 |
Robustness data for Doxofylline.
Factors | % RSD | Tailing factor ( |
Efficiency (theoretical plates) | Resolution |
---|---|---|---|---|
pH of mobile phase | ||||
pH 4.3 | 1.3415 | 1.362 | 7347 | 14.724 |
pH 4.7 | 0.9134 | 1.391 | 7273 | 14.810 |
| ||||
Flow rate | ||||
0.8 mL/min | 0.5180 | 1.367 | 7294 | 14.736 |
1.2 mL/min | 0.8075 | 1.386 | 7292 | 14.745 |
| ||||
Solvent % | ||||
−2% | 1.1918 | 1.348 | 7327 | 14.698 |
+2% | 0.9294 | 1.413 | 7293 | 14.672 |
The system suitability tests represent an integral part of the method and are used to ensure adequate performance of the chromatographic system. The parameters, retention time (
System suitability test parameter.
System suitability parameters | Ambroxol | Doxofylline |
---|---|---|
Retention times ( |
3.510 | 7.247 |
Theoretical plates ( |
7318 | 7293 |
Resolution ( |
14.850 | |
Tailing factor ( |
1.455 | 1.413 |
The surveillance and results obtained from each validation experiment including specificity, linearity and range, LOD and LOQ, precision, accuracy, robustness, recovery, and system suitability lie well inside the acceptance criteria of ICH guideline. Since all the results are within the limit, the developed analytical method is considered as validated and suitable for probable use.
The authors wish to confirm that there is no known conflict of interests associated with this paper. The authors confirm that they have given due consideration to the protection of intellectual property associated with this work and that there is no impediment to publication, including the trademarks mentioned in their paper.
The authors are heartily grateful to Mr. Ketan Patel, Director, Molecule Laboratory, Ahmedabad, Gujarat, for providing all the facilities to carry out the research work.