# Development and Validation of High Performance Liquid Chromatography Method for Simultaneous Estimation of Ambroxol and Doxofylline in Their Combined Tablet Dosage Form

Authors: Neha Singhal; Kalpesh Gaur; Anoop Singh; Karni Singh Sekhawat
Journal: ISRN Analytical Chemistry
 (2013)

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## Abstract

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 μ (particle size), Thermo scientific from Germany with isocratic conditions, and mobile phase containing potassium dihydrogen orthophosphate buffer-pH 4.5 (0.05 M KH2PO4): acetonitrile (60 : 40) at flow rate of 1 ml/min using UV detection at 254 nm. The retention times of Ambroxol and Doxofylline were 3.510 min and 7.247 min, respectively. The method was linear over the concentration range for Ambroxol 3.75–11.25 μg/mL and for Doxofylline 50-150 μg/mL. The recovery of Ambroxol and Doxofylline was found to be in the range of 99.42–101.18% and 99.37–100.28%, 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.


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## Body

## 1. Introduction

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 [1]. Chemically, it is trans-4-[(2-Amino-3,5-dibromobenzyl)amino]cyclohexanol hydrochloride [2]. Literature survey revealed that few spectroscopic methods RP-HPLC, HPTLC, and UPLC [3–10] methods have been reported for the estimation of Ambroxol with other drugs.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 [11]. Literature survey revealed that few spectrophotometric methods and RP-HPLC method have been reported for the estimation of Doxofylline with other drugs [12–15].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.


## 2. Materials and Methods

### 2.1. Chemicals and Reagents

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.


### 2.2. HPLC Instrumentation

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 μ (particle size), Thermo scientific, Rheodyne manual Injector (20 μL Capacity), Hamilton Syringe (25 μL), and Chromatograms, and data were recorded by means of Spinchrom CFR Software.


### 2.3. Preparation of Mobile Phase and Standard Solution

#### 2.3.1. Mobile Phase Preparation

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 μm nylon membrane (Millipore Pvt. Ltd., Bangalore, India) and degassed in an ultrasonic bath (Toshcon by Toshniwal).


#### 2.3.2. Standard Preparation

Stock Solution of Ambroxol. Ambroxol standard stock solution containing 75 μg/mL was prepared in a 100 mL volumetric flask by dissolving 7.5 mg of Ambroxol in small volume of mobile phase; then make up the volume up to 100 mL with mobile phase. This solution is then sonicated for 10 minutes.Stock Solution of Doxofylline. Doxofylline standard stock solution containing 1000 μg/mL was prepared in a 100 mL volumetric flask by dissolving 100 mg of Doxofylline in small volume of mobile phase; then make up the volume up to 100 mL with mobile phase. This solution is then sonicated for 10 minutes.Working Standard Preparation. Take 1 mL of Ambroxol stock solution and 1 mL of Doxofylline stock solution and dilute with mobile phase up to 10 mL. Then solution was filtered through 0.45 μm nylon syringe filter. The concentration obtained was 7.5 μg/mL of Ambroxol and 100 μg/mL of Doxofylline.


### 2.4. Chromatographic Conditions

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 μm Nylon 66 (N66) 47 mm membrane filter paper. The measurements were carried out with an injection volume of 20 μL, flow rate was set to 1.0 mL/min, and UV detection was carried out at 254 nm. All determinations were done at ambient column temperature (27°C). The chromatograms of the prepared standard stock solutions of Ambroxol and Doxofylline were recorded under the above optimized chromatographic conditions (Figure 3).


### 2.5. Test Preparation

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 0.45μm nylon syringe filter. The concentration obtained was 7.5 μg/mL of Ambroxol and 100 μg/mL of Doxofylline. The chromatogram of the prepared solution of Ambroxol and Doxofylline was recorded under the above optimized chromatographic conditions (Figure 4).


## 2.1. Chemicals and Reagents

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.


## 2.2. HPLC Instrumentation

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 μ (particle size), Thermo scientific, Rheodyne manual Injector (20 μL Capacity), Hamilton Syringe (25 μL), and Chromatograms, and data were recorded by means of Spinchrom CFR Software.


## 2.3. Preparation of Mobile Phase and Standard Solution

### 2.3.1. Mobile Phase Preparation

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 μm nylon membrane (Millipore Pvt. Ltd., Bangalore, India) and degassed in an ultrasonic bath (Toshcon by Toshniwal).


### 2.3.2. Standard Preparation

Stock Solution of Ambroxol. Ambroxol standard stock solution containing 75 μg/mL was prepared in a 100 mL volumetric flask by dissolving 7.5 mg of Ambroxol in small volume of mobile phase; then make up the volume up to 100 mL with mobile phase. This solution is then sonicated for 10 minutes.Stock Solution of Doxofylline. Doxofylline standard stock solution containing 1000 μg/mL was prepared in a 100 mL volumetric flask by dissolving 100 mg of Doxofylline in small volume of mobile phase; then make up the volume up to 100 mL with mobile phase. This solution is then sonicated for 10 minutes.Working Standard Preparation. Take 1 mL of Ambroxol stock solution and 1 mL of Doxofylline stock solution and dilute with mobile phase up to 10 mL. Then solution was filtered through 0.45 μm nylon syringe filter. The concentration obtained was 7.5 μg/mL of Ambroxol and 100 μg/mL of Doxofylline.


## 2.3.1. Mobile Phase Preparation

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 μm nylon membrane (Millipore Pvt. Ltd., Bangalore, India) and degassed in an ultrasonic bath (Toshcon by Toshniwal).


## 2.3.2. Standard Preparation

Stock Solution of Ambroxol. Ambroxol standard stock solution containing 75 μg/mL was prepared in a 100 mL volumetric flask by dissolving 7.5 mg of Ambroxol in small volume of mobile phase; then make up the volume up to 100 mL with mobile phase. This solution is then sonicated for 10 minutes.Stock Solution of Doxofylline. Doxofylline standard stock solution containing 1000 μg/mL was prepared in a 100 mL volumetric flask by dissolving 100 mg of Doxofylline in small volume of mobile phase; then make up the volume up to 100 mL with mobile phase. This solution is then sonicated for 10 minutes.Working Standard Preparation. Take 1 mL of Ambroxol stock solution and 1 mL of Doxofylline stock solution and dilute with mobile phase up to 10 mL. Then solution was filtered through 0.45 μm nylon syringe filter. The concentration obtained was 7.5 μg/mL of Ambroxol and 100 μg/mL of Doxofylline.


## 2.4. Chromatographic Conditions

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 μm Nylon 66 (N66) 47 mm membrane filter paper. The measurements were carried out with an injection volume of 20 μL, flow rate was set to 1.0 mL/min, and UV detection was carried out at 254 nm. All determinations were done at ambient column temperature (27°C). The chromatograms of the prepared standard stock solutions of Ambroxol and Doxofylline were recorded under the above optimized chromatographic conditions (Figure 3).


## 2.5. Test Preparation

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 0.45μm nylon syringe filter. The concentration obtained was 7.5 μg/mL of Ambroxol and 100 μg/mL of Doxofylline. The chromatogram of the prepared solution of Ambroxol and Doxofylline was recorded under the above optimized chromatographic conditions (Figure 4).


## 3. Result and Discussion

### 3.1. Method Development and Optimization

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 1 and 2). The acidic pH range was found suitable for solubility, resolution, stability, theoretical plates, and peak shape of both components. Best results were obtained with 0.05 M potassium dihydrogen orthophosphate pH 4.5 with phosphoric acid solution that improved the peak shapes of Ambroxol and Doxofylline. For the selection of organic constituent of mobile phase, acetonitrile was chosen to reduce the longer retention time and to attain good peak shape. Therefore, final mobile phase composition consisting of a mixture of buffer-pH 4.5 (0.05 M KH2PO4): acetonitrile (60 : 40), set at a flow rate of 1.0 mL/min was selected for the chromatographic analysis. Optimized mobile phase proportion was providing good resolution between Ambroxol and Doxofylline.Figure 1
Chemical structure of Ambroxol.Figure 2
Chemical structure of Doxofylline.Figure 3
HPLC chromatogram of Ambroxol and Doxofylline standards.Figure 4
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.


### 3.2. Method Validation

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 [16–21] (Table 5).


#### 3.2.1. Specificity

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.


#### 3.2.2. Linearity and Range

For linearity, five-point calibration curve was obtained in a concentration range from 3.75 to 11.25μg/mL for Ambroxol and 50–150 μg/mL for Doxofylline. The response of the drug was found to be linear in the investigation concentration range, and the linear regression equation for Ambroxol was y=57.06x-12.67 with correlation coefficient 0.997 (Figure 5) and for Doxofylline was y=34.24x-60.39 with correlation coefficient 0.998 (Figure 6) where x is the concentration in μg/mL and y is the peak area in absorbance unit.Figure 5
Linearity study for Ambroxol.Figure 6
Linearity study for Doxofylline.


#### 3.2.3. Precision (Repeatability and Reproducibility)

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 Table1 for intraday and interday precision study for both Ambroxol and Doxofylline. The RSD values for intraday precision study and interday precision study were <2.0% for Ambroxol and Doxofylline that confirm the method was precise.Table 1
Precision data for Ambroxol and Doxofylline.
Drug
Concentration (μg/mL)
Intraday (n=3) average ± SD
C.V.
Interday (n=3) average ± SD
C.V.
Ambroxol
3.75
209.47
±
1.71
0.82
209.88
±
2.29
1.09
7.50
419.91
±
4.43
1.05
421.32
±
4.84
1.15
11.25
629.12
±
6.11
0.97
627.44
±
5.84
0.93
Doxofylline
50
1689.00
±
16.78
0.99
1691.66
±
14.54
0.86
100
3384.19
±
40.70
1.20
3396.37
±
35.26
1.04
150
5067.86
±
38.96
0.77
5055.92
±
55.21
1.090


#### 3.2.4. Accuracy (% Assay)

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 (Table2).Table 2
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


#### 3.2.5. Recovery Studies

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 11.25μg/mL) and Doxofylline (50, 100, and 150 μg/mL) were added to a diluent preparation, and the amount of Ambroxol and Doxofylline recovered was calculated. For each concentration, three sets were prepared and injected in duplicate. % recovery was calculated at each level and recorded as shown in Tables 3 and 4. The mean recovery of Ambroxol was between 99.42% and 101.18%, and the mean recovery of Doxofylline was between 99.37% and 100.28%, which was satisfactory.Table 3
Results of recovery study of Ambroxol.
Concentration level
Standard: amount added in sample solution inμg/mL
Amount recovered inμg/mL
% 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 7.5μg/mL.Table 4
Results of recovery study of Doxofylline.
Concentration level
Standard: amount added in sample solution inμg/mL
Amount recovered inμg/mL
% 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 100μg/mL.Table 5
Statistical data for Ambroxol and Doxofylline by HPLC method.
Parameter
Ambroxol
Doxofylline
Linear range (μg/mL)
3.75–11.25
50–150
Slope
57.06
34.24
Limit of detection (μg/mL)
0.59
1.80
Limit of quantitation (μg/mL)
4.97
15.06


#### 3.2.6. Limit of Detection (LOD)/Limit of Quantitation (LOQ)

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:(1)LOQ=10×NB,LOD=3×NB,
where N is the standard deviation (SD) of the peak areas (triplicate injections) of the drug and B is the slope of the corresponding calibration curve.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 0.597μg/mL and 1.809 μg/mL, respectively, and the LOQ value 4.970 μg/mL and 15.061 μg/mL, respectively.


#### 3.2.7. Robustness

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 Tables6 and 7. The result shown that during all variance conditions, assay value of the test preparation solution was not affected and it was in accordance with that of actual. System suitability parameters were also found satisfactory; hence the analytical method would be concluded as robust.Table 6
Robustness data for Ambroxol.
Factors
% RSD
Tailing factor (AS)
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
14.672Table 7
Robustness data for Doxofylline.
Factors
% RSD
Tailing factor (AS)
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


#### 3.2.8. System Suitability

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 (RT), theoretical plates (N), peak resolution (R), peak asymmetry (T), and repeatability were evaluated using five replicate injections of the drugs. Acceptance criteria for system suitability are as follows: asymmetry should not be more than 2.0, resolution should not be more than 2.0, theoretical plate should not be less than 6800, and % RSD of peak area should not be more than 2.0 as per the results of present study, and the outcomes of validation studies were shown as the good results during all validation parameters. The result of system suitability study of the developed assay method was shown in Table 8.Table 8
System suitability test parameter.
System suitability parameters
Ambroxol
Doxofylline
Retention times (RT) (min)
3.510
7.247
Theoretical plates (N)
7318
7293
Resolution (RS)
14.850
Tailing factor (AS)
1.455
1.413The 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.


## 3.1. Method Development and Optimization

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 1 and 2). The acidic pH range was found suitable for solubility, resolution, stability, theoretical plates, and peak shape of both components. Best results were obtained with 0.05 M potassium dihydrogen orthophosphate pH 4.5 with phosphoric acid solution that improved the peak shapes of Ambroxol and Doxofylline. For the selection of organic constituent of mobile phase, acetonitrile was chosen to reduce the longer retention time and to attain good peak shape. Therefore, final mobile phase composition consisting of a mixture of buffer-pH 4.5 (0.05 M KH2PO4): acetonitrile (60 : 40), set at a flow rate of 1.0 mL/min was selected for the chromatographic analysis. Optimized mobile phase proportion was providing good resolution between Ambroxol and Doxofylline.Figure 1
Chemical structure of Ambroxol.Figure 2
Chemical structure of Doxofylline.Figure 3
HPLC chromatogram of Ambroxol and Doxofylline standards.Figure 4
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.


## 3.2. Method Validation

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 [16–21] (Table 5).


### 3.2.1. Specificity

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.


### 3.2.2. Linearity and Range

For linearity, five-point calibration curve was obtained in a concentration range from 3.75 to 11.25μg/mL for Ambroxol and 50–150 μg/mL for Doxofylline. The response of the drug was found to be linear in the investigation concentration range, and the linear regression equation for Ambroxol was y=57.06x-12.67 with correlation coefficient 0.997 (Figure 5) and for Doxofylline was y=34.24x-60.39 with correlation coefficient 0.998 (Figure 6) where x is the concentration in μg/mL and y is the peak area in absorbance unit.Figure 5
Linearity study for Ambroxol.Figure 6
Linearity study for Doxofylline.


### 3.2.3. Precision (Repeatability and Reproducibility)

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 Table1 for intraday and interday precision study for both Ambroxol and Doxofylline. The RSD values for intraday precision study and interday precision study were <2.0% for Ambroxol and Doxofylline that confirm the method was precise.Table 1
Precision data for Ambroxol and Doxofylline.
Drug
Concentration (μg/mL)
Intraday (n=3) average ± SD
C.V.
Interday (n=3) average ± SD
C.V.
Ambroxol
3.75
209.47
±
1.71
0.82
209.88
±
2.29
1.09
7.50
419.91
±
4.43
1.05
421.32
±
4.84
1.15
11.25
629.12
±
6.11
0.97
627.44
±
5.84
0.93
Doxofylline
50
1689.00
±
16.78
0.99
1691.66
±
14.54
0.86
100
3384.19
±
40.70
1.20
3396.37
±
35.26
1.04
150
5067.86
±
38.96
0.77
5055.92
±
55.21
1.090


### 3.2.4. Accuracy (% Assay)

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 (Table2).Table 2
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


### 3.2.5. Recovery Studies

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 11.25μg/mL) and Doxofylline (50, 100, and 150 μg/mL) were added to a diluent preparation, and the amount of Ambroxol and Doxofylline recovered was calculated. For each concentration, three sets were prepared and injected in duplicate. % recovery was calculated at each level and recorded as shown in Tables 3 and 4. The mean recovery of Ambroxol was between 99.42% and 101.18%, and the mean recovery of Doxofylline was between 99.37% and 100.28%, which was satisfactory.Table 3
Results of recovery study of Ambroxol.
Concentration level
Standard: amount added in sample solution inμg/mL
Amount recovered inμg/mL
% 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 7.5μg/mL.Table 4
Results of recovery study of Doxofylline.
Concentration level
Standard: amount added in sample solution inμg/mL
Amount recovered inμg/mL
% 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 100μg/mL.Table 5
Statistical data for Ambroxol and Doxofylline by HPLC method.
Parameter
Ambroxol
Doxofylline
Linear range (μg/mL)
3.75–11.25
50–150
Slope
57.06
34.24
Limit of detection (μg/mL)
0.59
1.80
Limit of quantitation (μg/mL)
4.97
15.06


### 3.2.6. Limit of Detection (LOD)/Limit of Quantitation (LOQ)

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:(1)LOQ=10×NB,LOD=3×NB,
where N is the standard deviation (SD) of the peak areas (triplicate injections) of the drug and B is the slope of the corresponding calibration curve.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 0.597μg/mL and 1.809 μg/mL, respectively, and the LOQ value 4.970 μg/mL and 15.061 μg/mL, respectively.


### 3.2.7. Robustness

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 Tables6 and 7. The result shown that during all variance conditions, assay value of the test preparation solution was not affected and it was in accordance with that of actual. System suitability parameters were also found satisfactory; hence the analytical method would be concluded as robust.Table 6
Robustness data for Ambroxol.
Factors
% RSD
Tailing factor (AS)
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
14.672Table 7
Robustness data for Doxofylline.
Factors
% RSD
Tailing factor (AS)
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


### 3.2.8. System Suitability

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 (RT), theoretical plates (N), peak resolution (R), peak asymmetry (T), and repeatability were evaluated using five replicate injections of the drugs. Acceptance criteria for system suitability are as follows: asymmetry should not be more than 2.0, resolution should not be more than 2.0, theoretical plate should not be less than 6800, and % RSD of peak area should not be more than 2.0 as per the results of present study, and the outcomes of validation studies were shown as the good results during all validation parameters. The result of system suitability study of the developed assay method was shown in Table 8.Table 8
System suitability test parameter.
System suitability parameters
Ambroxol
Doxofylline
Retention times (RT) (min)
3.510
7.247
Theoretical plates (N)
7318
7293
Resolution (RS)
14.850
Tailing factor (AS)
1.455
1.413The 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.


## 3.2.1. Specificity

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.


## 3.2.2. Linearity and Range

For linearity, five-point calibration curve was obtained in a concentration range from 3.75 to 11.25μg/mL for Ambroxol and 50–150 μg/mL for Doxofylline. The response of the drug was found to be linear in the investigation concentration range, and the linear regression equation for Ambroxol was y=57.06x-12.67 with correlation coefficient 0.997 (Figure 5) and for Doxofylline was y=34.24x-60.39 with correlation coefficient 0.998 (Figure 6) where x is the concentration in μg/mL and y is the peak area in absorbance unit.Figure 5
Linearity study for Ambroxol.Figure 6
Linearity study for Doxofylline.


## 3.2.3. Precision (Repeatability and Reproducibility)

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 Table1 for intraday and interday precision study for both Ambroxol and Doxofylline. The RSD values for intraday precision study and interday precision study were <2.0% for Ambroxol and Doxofylline that confirm the method was precise.Table 1
Precision data for Ambroxol and Doxofylline.
Drug
Concentration (μg/mL)
Intraday (n=3) average ± SD
C.V.
Interday (n=3) average ± SD
C.V.
Ambroxol
3.75
209.47
±
1.71
0.82
209.88
±
2.29
1.09
7.50
419.91
±
4.43
1.05
421.32
±
4.84
1.15
11.25
629.12
±
6.11
0.97
627.44
±
5.84
0.93
Doxofylline
50
1689.00
±
16.78
0.99
1691.66
±
14.54
0.86
100
3384.19
±
40.70
1.20
3396.37
±
35.26
1.04
150
5067.86
±
38.96
0.77
5055.92
±
55.21
1.090


## 3.2.4. Accuracy (% Assay)

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 (Table2).Table 2
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


## 3.2.5. Recovery Studies

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 11.25μg/mL) and Doxofylline (50, 100, and 150 μg/mL) were added to a diluent preparation, and the amount of Ambroxol and Doxofylline recovered was calculated. For each concentration, three sets were prepared and injected in duplicate. % recovery was calculated at each level and recorded as shown in Tables 3 and 4. The mean recovery of Ambroxol was between 99.42% and 101.18%, and the mean recovery of Doxofylline was between 99.37% and 100.28%, which was satisfactory.Table 3
Results of recovery study of Ambroxol.
Concentration level
Standard: amount added in sample solution inμg/mL
Amount recovered inμg/mL
% 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 7.5μg/mL.Table 4
Results of recovery study of Doxofylline.
Concentration level
Standard: amount added in sample solution inμg/mL
Amount recovered inμg/mL
% 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 100μg/mL.Table 5
Statistical data for Ambroxol and Doxofylline by HPLC method.
Parameter
Ambroxol
Doxofylline
Linear range (μg/mL)
3.75–11.25
50–150
Slope
57.06
34.24
Limit of detection (μg/mL)
0.59
1.80
Limit of quantitation (μg/mL)
4.97
15.06


## 3.2.6. Limit of Detection (LOD)/Limit of Quantitation (LOQ)

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:(1)LOQ=10×NB,LOD=3×NB,
where N is the standard deviation (SD) of the peak areas (triplicate injections) of the drug and B is the slope of the corresponding calibration curve.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 0.597μg/mL and 1.809 μg/mL, respectively, and the LOQ value 4.970 μg/mL and 15.061 μg/mL, respectively.


## 3.2.7. Robustness

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 Tables6 and 7. The result shown that during all variance conditions, assay value of the test preparation solution was not affected and it was in accordance with that of actual. System suitability parameters were also found satisfactory; hence the analytical method would be concluded as robust.Table 6
Robustness data for Ambroxol.
Factors
% RSD
Tailing factor (AS)
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
14.672Table 7
Robustness data for Doxofylline.
Factors
% RSD
Tailing factor (AS)
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


## 3.2.8. System Suitability

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 (RT), theoretical plates (N), peak resolution (R), peak asymmetry (T), and repeatability were evaluated using five replicate injections of the drugs. Acceptance criteria for system suitability are as follows: asymmetry should not be more than 2.0, resolution should not be more than 2.0, theoretical plate should not be less than 6800, and % RSD of peak area should not be more than 2.0 as per the results of present study, and the outcomes of validation studies were shown as the good results during all validation parameters. The result of system suitability study of the developed assay method was shown in Table 8.Table 8
System suitability test parameter.
System suitability parameters
Ambroxol
Doxofylline
Retention times (RT) (min)
3.510
7.247
Theoretical plates (N)
7318
7293
Resolution (RS)
14.850
Tailing factor (AS)
1.455
1.413The 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.



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*Source: 834240-2013-07-25.xml*