Department of Quality Assurance, PRES College of Pharmacy (Women’s), Chincholi, Nashik-422102.
The HPLC method was developed and validated for simultaneous estimation of Indacaterol maleate. The method was developed after the several trials at the composition of Methanol: Phosphate Buffer 6.8 (75:25), flow rate 1.0 ml/min and 260 nm wavelength of detection and retention time was at 4.3 min. The calibration curve was plotted, and regression equation of Indacaterol maleate was found to be y = 38835x + 278291 with correlation coefficient (r2) of 0.9993. From the Accuracy study % recovery was found in the range of 99.97-100.4% which is in the limits according to the ICH guidelines. Intraday and Interday precision assures that % RSD was within the limits of ICH guidelines i.e NMT 2. Limit of detection and limit of Quantitation of Indacaterol is 0.17 µg/ml - 0.52 µg/ml respectively. Robustness was studied by deliberate variation i.e., change in Flow rate and change in Wavelength which was within 2 % of RSD and also the ruggedness study gives results within the limits of 2% in which variation in Analyst was studied. The stress degradation studies demonstrated susceptibility of the drug to acid, base, neutral hydrolysis, oxidative, thermal and photolytic stress conditions. The major degradation of drug was found to be in alkali and acidic stress condition. The result showed that the proposed chromatographic method was suitable for the accurate, precise and rapid simultaneous determination of Indacaterol maleate in its bulk form and pharmaceutical dosage form.
Indacaterol is an inhaled long-acting beta-2 adrenergic agonist used to relax bronchial smooth muscle and improve symptoms and airflow obstruction caused by Chronic Obstructive Pulmonary Disease (COPD) and moderate to severe asthma.
Fig 1: Structure of Indacaterol maleate
Indacaterol is a novel, ultra-long-acting, rapid onset ?(2)-adrenoceptor agonist developed for Novartis for the once-daily management of asthma and chronic obstructive pulmonary disease. It was approved by the European Medicines Agency (EMA) on 30 November 2009 and by the FDA on 1 July 2011. It is marketed in Europe as Onbrez and in America as Arcapta Neohaler. Indacaterol is provided as its maleate salt form. Indacaterol is also a chiral molecule but only the pure R-enantiomer is dispensed.
MATERIALS AND METHOD:
Instruments:
The chromatographic method was performed on Shimadzu HPLC system coordinated with a variable wavelength UV detector and a Rheodyne injector outfitted with 20µl fixed circle. An Reverse phase Phenomenex C18 (250mm x 4.6ID, Particle size: 5 micron) was utilized. Model - UV-3000 - M spectrophotometer and Wenser High Precision Balance Model: PGB 100 electronic equilibrium were utilized for Spectrophotometric judgments and gauging purposes individually.
Reagents and chemicals
Indacaterol maleate was procured from PharmaTech Solutions. HPLC grade Acetonitrile and water were acquired from Merck specialities private restricted, Mumbai.
Chromatographic conditions
Phenomenex C18 (250mm x 4.6ID, Particle size: 5 micron) was utilized for the chromatographic method at wavelength of 260 nm. Methanol: Phosphate Buffer (75:25) pH 6.8 was chosen as mobile phase for elution and same solvent was utilized in the preparation of standard and sample solutions. The elution was checked by infusing the 20µl and the flow rate was changed in accordance with 1.0 ml/min.
Preparation of Standard Stock solutions
Accurately 10.0 mg weighed quantity of Indacaterol was transferred to 10.0 mL volumetric flask. That was dissolved by adding 5.0 mL mobile phase and then the drug solution was diluted up to the mark with mobile phase to get the stock solution of 1000 µg/mL of Indacaterol. The working standard solutions of these drugs were obtained by appropriate dilution of the respective stock solution with mobile phase.
Preparation of Mobile Phase
Prepare mobile phase by taking methanol and phosphate buffer in various proportion Methanol: Phosphate Buffer 6.8 (75:25). Mobile phase was filtered through 0.45µm membrane filter and degassed by sonication for 20 min.
Selection of mobile phase
Pure drug solutions of Indacaterol (10µg/mL) were injected into the RP-HPLC system and run in different solvent systems. Different mobile phases systems like phosphate buffer, ACN and methanol were initially tried in the isocratic mode in order to determine the best conditions.
HPLC Method Development
Optimisation of RP-HPLC method
The HPLC method was developed for the simultaneous estimations of Indacaterol maleate. Different mobile phases were gone after for the method optimisation, however satisfactory retention times, hypothetical plates and good resolution were seen with Methanol: Phosphate Buffer (75:25) pH 6.8 using Phenomenex C18 (250mm x 4.6ID, Particle size: 5 micron) by isocratic method.
Table 1: Optimized Chromatographic Conditions
Validation of RP-HPLC method
Validation of the optimized RP-HPLC method was performed in accordance with the ICH Q2 (R) guidelines.
Linearity
Test solutions of different concentration were injected separately, and the chromatograms were recorded. A series of test preparations of Indacaterol (10-50 µg/ml) were prepared by taking 0.1-0.5 ml from the stock solution in five 10 ml volumetric flask and final volume make up to the mark with mobile phase. A 20 µl volume of each concentration was injected into HPLC, three times under the optimized chromatographic conditions.
Accuracy
Samples are prepared normally covering 50 % to 150 % of the nominal sample preparation concentration. These samples are analyzed and the recoveries of each are calculated.
Precision
Intraday precision study was carried out by preparing test solution of same concentration and analyzing it at three different times in a day. The same procedure was followed for two different days to determine interday precision. The result was reported as %RSD.
Limit of Quantitation (LOQ) & Limit of Detection (LOD)
The LOD and LOQ were analysed from the slope(s) of the calibration curve and the standard deviation (SD) of the peak areas using the formula LOD = 3.3 s/s and LOQ = 10 s/s.
Robustness
Robustness was calculated by changing the chromatographic conditions like compositions of mobile phase, detection wavelength, flow rate etc. and the % RSD should be reported. In the optimised conditions small changes were allowed and the extent to which the method was robust was determined. A deviation of ± 2 nm in the detection wavelength and ± 0.1 ml/min in the flow rate, were tried individually. Solutions of 100% test concentration with the specified n changes in the optimised conditions were injected to the system in triplicate.
Ruggedness:
Ruggedness is the study to determine effect of external parameters on the method. To evaluate ruggedness of the developed method, parameters were deliberately varied. These parameters included variation of system, different analyst, Atmospheric changes. Test solution prepared as per the test method and injected 3 concentrations of test solution into HPLC system with flow rate 1.0 ml/min by 2 different analysts.
Assay of marketed formulation
20 Capsules of marketed formulation (Onbrex 300mcg) were taken. Average weight of capsule sample was weighed and transferred to 100 mL volumetric flask & diluent was added to make up the volume. Sonicate for 10 min with occasional swirling. The above solution was filtered through 0.45?m membrane filter, The prepared stock solution is of 100 µg/ml of Indacaterol maleate. For Analysis 1.0 ml solution was withdrawn and diluted upto 10 ml and injected into system.
System suitability
System suitability parameters were measured to verify the system, method and column performance. Standard solution of Indacaterol maleate was injected into the system for five times and system suitability parameters were checked.
Table 2: Forced Degradation Conditions according to ICH guidelines
RESULT AND DISCUSSION
Linearity:
It was clarified from the analytical method linearity as the ability of the method to obtain test results that are directly proportional to the analyte concentration, within a speci?c range. The peak area obtained from the HPLC chromatograph was plotted against corresponding concentrations to obtain the calibration graph. Indacaterol maleate was found to be linear in the concentration range of 5-25?g/ml and Enalapril is in the range of 5-25?g/ml.
Table 3: Summary of results of Linearity
Fig 2: Calibration curve for Indacaterol maleate
Fig 3: Chromatograph of Indacaterol maleate
Accuracy
The accuracy of the method determines the closeness of results obtained by that method to the true value. From the results of accuracy testing, it was showed that the method is accurate within the acceptable limits. The % RSD is calculated for the Indacaterol maleate and all the results are within limits. Acceptable accuracy was within the range and not more than 2.0% RSD.
Table 4: Statistical validation for accuracy of Indacaterol maleate
Precision
Intraday and interday precision assures the repeatability of test results. The % RSD found was below 2 for Indacaterol maleate.
Table 5: Data for intraday precision of Indacaterol maleate
Table 6: Data for interday precision of Indacaterol maleate
Robustness
Robustness was studied by different deliberate variations in the chromatographic conditions i.e. Change in flow rate and wavelength. From robustness study % RSD was found to be within limit of 2 % for the Indacaterol maleate. Hence it is robust and complies as per ICH guidelines.
Table 7: Data for Robustness study of Indacaterol maleate
Ruggedness
Ruggedness was studied by different analyst. From robustness study % RSD was found to be within limit of 2 % for the Indacaterol maleate. Hence it is complying as per ICH guidelines.
Table 8: Data for ruggedness study of Indacaterol maleate
Specificity
Excipients and impurities were not interacting with the standard drugs. Hence the method is specific.
Table 9: Data for specificity study of Indacaterol maleate
% Assay of Marketed formulation
The % Assay of Onbrex 300mcg marketed formulation of Novartis was calculated.
Table 10. Data of % Assay of marketed formulation
System Suitability Parameters:
System suitability parameters were measured to verify the system, method and column performance. Standard solution of Indacaterol maleate was injected into the system for five times and system suitability parameters were checked.
Table 11: System suitability parameter
Degradation Studies
Stress testing of the drug substance can help to identify the likely degradation products, the stability and specificity of the analytical procedure. Degradation studies were performed on solutions containing Indacaterol maleate (30µg/ml).
Table 12: Results of Forced Degradation Studies for Indacaterol maleate
Fig. 4: Water Stressed Chromatogram
Fig. 5: Acid Stressed Chromatogram
Fig. 6: Alkali Stressed Chromatogram
Fig. 7: Peroxide Stressed Chromatogram
Fig. 8: Thermal Stressed Chromatogram
Fig. 9: Photolytic Stressed Chromatogram
SUMMARY
The Indacaterol maleate was found to be linear in the concentration range of 10-50 µg/ml. From Accuracy study % recovery of Indacaterol maleate was found in the range of 99.97-100.4% which is in the limits accordingly the ICH guidelines. Intraday and Interday precision assures that % RSD was within limits of ICH guidelines i.e., NMT 2 for both Indacaterol maleate. Limit of detection and limit of Quantitation of Indacaterol maleate is 0.17 µg/ml - 0.52 µg/ml. Robustness was studied by deliberate variation i.e., change in Flow rate and change in Wavelength which was within 2 % of RSD as per ICH guidelines. The ruggedness study gives results within the limits of 2% in which variation in Analyst was studied. The % assay of Onbrex 300mcg was found to be Indacaterol maleate (99.84%).
CONCLUSION
The proposed chromatographic method was found to be simple, precise, accurate, rapid and specific for determination of Indacaterol maleate from pure and its dosage forms. The mobile phase used for method development is very simple to prepare and economical also. The sample recoveries in the formulation were showing good results. This method is economical and run time is relatively short which enables rapid analysis among all the developed methods and hence, this method can be easily and conveniently adopted for in-vitro dissolution and routine analysis of Indacaterol maleate in pharmaceutical dosage form.
Degradation studies were performed on solutions containing Indacaterol maleate. The force degradation study was employed on five conditions i.e., Acidic, Alkaline, Oxidative, Thermal and Photolytic degradation. The degradation products produced during the stability study were well separated from the pure drug signifying the specificity of developed procedure. The major degradation of drug was found to be in Acid and Alkali stress condition.
ACKNOWLEDGEMENT
The authors are very much thankful to Dr. Charushila Bhangale, Principal of Pres’s College of Pharmacy, For Women, Nashik for his support and also thank to the management for providing the necessary facilities to carry out this research work.
REFERENCE
Trupti Bhalekar*, Kalpana Sable, Jaya Mehetre, Kiran Dhamak, Stability Indicating Rp-HPLC Method Development And Validation For The Estimation Of Indacaterol Maleate In Bulk And Tablet Dosage Form, Int. J. of Pharm. Sci., 2024, Vol 2, Issue 8, 2607-2617. https://doi.org/10.5281/zenodo.13221100
10.5281/zenodo.13221100
