Development and Validation of Stability Indicating Reverse Phase High Performace Liquid Chromatography Method for the Determination of Umeclidinium and Vilanterol in Pharmaceutical Dosage Form

A Sensitive, fast, linear and accurate liquid chromatography technique was developed for the simultaneous determination of Umeclidinium and Vilanterol in Powder dosage form. The estimation was carried out using Phenomenex C18 column (150 × 4.6 mm, 5μ) with ammonium acetate: acetonitrile taken in the ratio 60:40 as mobile phase and pumped at a flow rate of 0.9 ml/min at 300C. Detection wavelength selected was 245 nm. Retention times of Umeclidinium and Vilanterol were found to be 2.219 min and 2.794 min. The method was validated in terms of linearity, precision, accuracy, limit of detection, limit of quantification as per International council for harmonization guidelines. Degradation studies performed indicated the stability of the drug. All of Original Research Article Hamsa et al.; JPRI, 33(42A): 208-219, 2021; Article no.JPRI.72858 209 these analytical validation parameters were evaluated, and the percent relative standard deviations were calculated, indicating the method's suitability for determination of Umeclidinium and Vilanterol in pharmaceutical dosage form.


Preparation of diluent
Based up on the solubility of the drugs, diluent was selected, Acetonitrile and Water taken in 50:50 ratio.

Preparation of standard stock solutions
VI (2.5mg) and UMEC (6.25mg) were accurately weighed and added to a 10ml volumetric flask, added with 3/4th of the diluents, then sonicated for 10 minutes. Standard stock solution was prepared by filling flasks with diluents and labeled them as such. (VI (250 µg/ml), UMEC (625 µg/ml)).

Preparation of sample stock solutions
In a 50 ml volumetric flask, the contents of nasal spray administered by 50 actuations (25 and 62.5 µg each) were collected. Then 20ml acetonitrile was added, and it was sonicated for 25 minutes before being filled to the mark, yielding 1250 and 3125µg/ml. It was centrifuged for 20 minutes. Finally, the supernatant was collected and filtered through 0.45 µm filters (Millipore, Milford and PVDF).

Preparation of sample working solution
2ml of the sample stock solution was pumped into a 10ml volumetric flask, which was then filled with diluent. (VI (25 µg/ml), UMEC (62.5 µg/ml)).

System suitability parameters
The parameters, peak tailing, resolution and USP plate count were defined by preparing standard solutions of VI (25ppm) and UMEC (62.5ppm) and the solutions were injected six times.

Specificity
In the presence of elements, specificity refers to the ability to measure the analyte unambiguously. In this approach we do not consider interfering peaks of these drugs in blank and placebo at retention times. So, the method is said to be accurate.

Precision
The method was checked for both intra-day and inter-day precision.

Linearity
Appropriate aliquots of UMEC/VI standard stock solutions were placed in separate 10 ml volumetric flasks and diluted up to the mark with mobile phases to achieve final concentrations of 15.625-93.75µg/ml for UMEC and 6.25-37.5µg/ml for VI. The solutions were then injected into the system and chromatograms were registered.

Accuracy
Recovery trials were used to determine the method's accuracy. For this experiment, a standard addition approach was used. A known amount of UMEC and VI were introduced, equivalent to 50%, 100%, 150% of the drug's label claim, respectively. Each addition sets were performed three times. The accuracy was calculated as percentage of recovered analytes.

Robustness
Tiny subtle improvements were made in methods such as flow rate, mobile phase ratio and temperature, but the effects have not been recognized and were within the limits of ICH Guidelines. Robustness conditions such as Flow minus (0.8ml/min), Flow plus (1.0ml/min), mobile phase minus, mobile phase plus, temperature minus (25°C) and temperature plus (35°C) were established and samples were double-injected. System suitability parameters were not much affected and all parameters were passed. Percentage RSD was within the limit.

LOD
The lowest amount of analyte that can be detected is referred to as the LOD. LOD values are derived from calibration curve using standard deviation (SD) and slope of calibration curve. The formula for determining LOD is, LOD = 3.3 × avg SD/slope

LOQ
The smallest quantity of analyte that can be measured is referred as LOQ. LOQ is measured from calibration curve using standard deviation (SD) and slope of calibration curve. The formula for determining LOQ is, LOQ = 10 × avg SD/slope

Degradation Studies
Degradation studies were carried out by treating samples with acidic, alkaline, oxidative, hydrolytic, thermal condition and photolytic application. Acidic and alkaline degradation were carried using 2N HCl and 2N NaOH respectively. Oxidative degradation was performed using 20% H2O2 solution. Acidic, alkaline and oxidative degradation studies were kept at 60º C for 30 min. the stress testing within hydrolytic degradation were studied by refluxing the sample in H2O for 1hr at 60ºC. To study dry heat degradation, the standard drug solution was placed in the oven at 105 °C for 1 hr. The drug's photolytic degradation stability was also studied by exposing the 250μg/ml VI and 625μg/ml UMEC solution to UV Light by placing the beaker in the UV chamber for 1day or 200wt hours/m2 in the photo stability chamber. The resulting solution were diluted 10 µl were injected into the system and the sample stability was evaluated by the chromatograms.

System suitability parameter
All system suitability parameters, peak tailing, resolution and USP plate count were within range according to ICH rules. The resolution was found to be 4.66. System suitability parameter and acceptance limit are shown in Table 1.

Specificity
No intrusive peaks at retention time of main drug were observed after injecting blank and placebo, so the method was said to specific. Chromatograms are depicted in Figs. 3-5.

System precision
System precisions of the drug UMEC/VI were obtained using six injections from the flask of standard working solution. Percentage RSD was found to be 1.0 and 0.9 for UMEC/VI. Average mean, standard deviation, %RSD, area were depicted in Table 2. Since the %RSD value is below the limit, the system precision was passed.

Repeatability (intraday precision)
Multiple samples were taken from a sample stock solution, and six working sample solutions of the same concentration were produced. Each injection from each working sample solution was administered, and the results were collected and expressed in Table 3. Percentage RSD was found to be 0.8 and 0.6 for UMEC/VI.

Interday Precision
Multiple samples were taken from a sample stock solution and six working sample solutions of the same concentration were prepared. Each injection from each working sample solution was administered on next day of preparation, and the results were collected and expressed in Table 4. Percentage RSD was found to be 1.6 and 1.1 for UMEC/VI.

Linearity
The method was linear over the range 15.625 -93.75µg/ml and 6.25 -37.5µg/ml for UMEC/VI. The calibration curve was constructed by plotting the response factor against a concentration of drugs (Fig. 6, Fig. 7). The slope and intercept value for calibration curve was y = 16315x + 10672, R² = 0.9992 for UMEC and y = 12261x + 4146.2, R² = 0.999 for VI. The results show an excellent correlation between the response factor and the concentration of drugs. Data depicted in Table 5.

Accuracy
The approach's accuracy was tested three times using the conventional addition technique, each time in triplicate. The proportion of standard drug recovered by the recovery study was then used to calculate the accuracy. Table 6 shows the mean UMEC/VI recovery rates from the combined formulation. Accuracy is measured in terms of % Recovery. Percentage Recovery was obtained as 100.09% and 99.94% for UMEC/VI.

Robustness
Robustness of the method was determined by changing three HPLC variables, like flow rate (0.8 and 1.0 ml min-1), mobile phase ratio (65B: 35A and 55B:45A) and column temperature (25 and 35˚C). The effects of these changes on the% RSD of both the substances (UMEC/VI) were determined. The value of percentage standard deviation was within the range, as shown in Table 7, which indicates that the developed method is robust and none of the variables possess significant effects on the method performance. Table 8 shows the minimum detection limit and minimum quantitation limit of UMEC/VI. LOD of UMEC/VI were found to be 0.33 and 0.15. LOQ of UMEC/VI were found to be 1.01 and 0.45, respectively.

Assay
The label containing Anoro Ellipta specifies UMEC 62.5 mcg, VI 25mcg. Assay with dosage type was performed. The mean percentage of assays obtained for UMEC and VI were 99.64 and 100.79 percent respectively. Assay data is depicted in Table 9 and Table 10.

Degradation Studies
Acidic, alkaline, oxidative, thermal, photolytic and hydrolytic degradation studies were performed on UMEC/VI. Since the percentage degradation was under acceptance criteria, it represents the stability-indicating properties of the method. Percentage degradation was demonstrated in Table 11. The chromatograms of the sample are shown in Figs. 8 -13.

CONSENT AND ETHICAL APPROVAL
It is not applicable.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.