Pure samples
Xylometazoline hydrochloride (99.7% purity) and ipratropium bromide (95.8% purity) working standards were used in the preparation of standard solution for quantitation of formulation products.
Formulation
Otrivin plus (manufactured by GlaxoSmithKline Pharmaceuticals Limited) was purchased from the local market. In-house nasal spray sample was used for all the parameters of the validation study.
Chemicals, reagents, and equipment
The liquid chromatographic system (Make-Shimadzu, Japan) LC-2010CHT, with VU/visible detector was used for development and validation. Intermediate precision and selectivity study was performed on Waters e 2695 with PDA detector (Model-Waters 2998). The reagents and chemicals used for preparation of mobile phase such as sodium di-hydrogen phosphate dihydrate (Rankem, India), 1-pentane sulphonic acid sodium salt (Merck, USA), orthophosphoric acid (Rankem, India), acetonitrile (Finar, India) were of HPLC grade. The HPLC run of xylometazoline and ipratropium bromide was carried out by optimized chromatographic conditions on a Inertsil ODS, 250 × 4.6 mm, 5 μm column. The analytical method development and validation was carried out on a nasal spray sample prepared in-house. The placebo sample was prepared by including all the components except ipratropium bromide and xylometazoline HCL
Preparation of mobile phase
In total, 1.56 g of sodium di-hydrogen phosphate dihydrate and 0.5 g of 1-pentane sulphonic acid sodium salt was weighed and transferred into a glass bottle containing 1 L of HPLC grade water. The solution was sonicated for 15 min and the pH adjusted to 4.7±0.1 using orthophosphoric acid. The solution was filtered through a 0.45-μm syringe filter (MDI, India) and used as mobile phase A, while acetonitrile was used as the mobile phase B. Water was used as a diluent for the preparation of standard, sample, and placebo solutions.
Optimization of chromatographic conditions
For better separation, and to achieve good column efficiency as per USP system suitability criteria, the method parameters were optimized using Inertsil ODS 3V, 250 × 4.6 mm, 5 μm HPLC column. The column temperature was set at 45 °C. The mobile phase flow was adjusted at 1.0 mL/min. Equal volume of (10 μl) sample, placebo, and standard samples were injected. The ipratropium bromide peak was eluted at about 5 min and xylometazoline HCL at about 12 min. The PDA detector was used during the method development for selection of wavelength and checking the peak purity of the samples subjected to force degradation. Both the compounds showed maximum absorbance at 210.0 nm.
Gradient program
|
Time (minutes)
|
Mobile phase A
|
Mobile phase B
|
|---|
|
0.01
|
70
|
30
|
|
5.00
|
70
|
30
|
|
12.00
|
40
|
60
|
|
14.00
|
50
|
50
|
|
15.00
|
50
|
50
|
|
17.0
|
70
|
30
|
|
22.0
|
70
|
30
|
Standard stock preparation
Xylometazoline hydrochloride standard stock solution preparation
Fifty milligrams of xylometazoline hydrochloride working standard was weighed and transferred into a 50-mL volumetric flask, 30 mL of diluent was added and sonicated to dissolve it completely. The volume was made up to 50 mL with the diluent and mixed well before use.
Ipratropium bromide standard stock solution
Sixty milligrams of ipratropium bromide working standard was weighed and transferred into a 50-mL volumetric flask. Nearly 30 mL of diluent was added and the mixture was sonicated to dissolve the contents completely. The volume was made up to 50 mL with the diluent, mixed well, and used for further analysis.
Preparation of standard solution
Five milliliters each of xylometazoline hydrochloride and ipratropium bromide from standard stock solutions was measured and transferred in to a 50-mL volumetric flask. The volume was made up with diluent and mixed well.
Sample preparation
About 2 g of the sample was weighed and transferred to a 10-mL volumetric flask, 7 mL diluent was added and sonicated for 10 min. The volume was made up to 10 mL with the diluent and mixed well. The sample was filtered through 0.45-μm PVDF filter and used for injection into HPLC system.
System suitability
The system suitability is a significant parameter in the method development and validation study of any drug dosage form. The system suitability is checked by injecting the six replicate standard solutions into the HPLC system. The limit for relative standard deviation (%RSD) of area under the curve of six replicate standard injections was kept at not more than 2.0%. The limit for column efficiency was kept at not less than 2000 theoretical plates. The limit for tailing factor was not more than 2.0 for both the analyte peaks.
Calculations
Equal volume of both the sample and standard solutions were injected into the HPLC system, and area under the curve for each analyte peak was recorded. The amount of xylometazoline hydrochloride and ipratropium bromide in % w/v was calculated.
Analytical method validation
The developed method was validated in terms of, accuracy, precision specificity, linearity, range, robustness, and stability of analytical solution as per the ICH guidelines.
Accuracy
The accuracy of an analytical method reflects the closeness of agreement between the values that is acceptable either as a conventional true or an accepted reference value, and the value observed by the technique. Standard solutions were spiked in to placebo at various concentration levels, i.e., 50, 100, and 150% of working concentration and analyzed according to the described method. The mean % recovery for the analytes at each concentration level should be in the range of 98-102% and % RSD of % recovery for the analytes at every level must not be greater than 2.0% as per ICH guidelines.
Precision
The precision of an analytical process defines the goodness of agreement among the series of measurements acquired from many identical samples. As per ICH validation guidelines, system precision, method precision, and intermediate precision were evaluated. The standard solution was injected in six replicates as described in analytical method and the % RSD was determined. The % RSD for peak area of both the analytes for six identical injections of standard solutions was set at not more than 2.0%.
Repeatability
The method precision was carried out by preparing six samples of a single batch. The % assay of the six samples was calculated. The precision of the method was examined by calculating the % RSD of the results.
Intermediate precision
Intermediate precision expresses ability of method to produce reliable results under laboratory conditions, viz., different days, analysts, system, and column. Six samples were prepared as per the test procedure by using the same batch of formulation and injected. The % assays of these samples were examined and the ruggedness of the method was estimated by calculating the % RSD of the results.
Specificity
Selectivity
Diluent, individual standard solutions for each analyte, placebo, and samples were prepared as mentioned in the analytical method section.
Force degradation
To prove the stability-indicating nature of the developed analytical method, the forced degradation study was carried out at different stress conditions like acid, base, oxidative, and thermal degradation.
For acid degradation, 1 mL of 0.1 N hydrochloric was added in the sample, and kept at 80 °C for 8 h. The sample was neutralized with the same quantity of 0.1N NaOH solution. For degradation under basic conditions, 0.1 mL of 0.1 N NaOH solution was added in the sample, and after 5 min, the sample was neutralized with 0.1N HCl solution. During the thermal degradation study, the sample was heated at 80 °C for 48 h in an oven and the sample analyzed. For oxidative degradation, 1 mL of 30% hydrogen peroxide was added to a sample solution. The sample was kept at room temperature for 24 h and analyzed.
Linearity
The linearity of the developed method was validated by analyzing the mixture of standard solution concentration in the range of 4-150% of working concentration. The solutions were injected in triplicate into the HPLC system, and the area under the curve of analyte peaks was recorded. The correlation co-efficient between concentration and peak area, slope, and intercept was evaluated.
Range
The developed method was checked for upper and lower amount of both the analytes in the sample for which it has been demonstrated that the method has suitable level of precision, linearity and accuracy. This study was covered under the linearity study.
Robustness
Robustness of an analytical method measures the capacity to stay unaffected by minor changes in the method parameters. In this study, parameters like variation in detection wavelength, flow rate, column oven temperature, mobile phase organic composition, and mobile phase buffer pH were studied.
Stability of analytical solution
The system suitability solution and sample solution was prepared on day zero of experiment and stored at room temperature. The solution was analyzed on subsequent days for 3 days. The standard solution used was prepared freshly for the investigation and the assay results were calculated for sample solution to evaluate the stability sample of solution. The solution is considered stable, if the cumulative % RSD of the stored sample and standard solution is not more than 2.0.
