Int J Curr Pharm Res, Vol 12, Issue 3, 69-73Original Article
UV-SPECTROPHOTOMETRIC METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF IPRATROPIUM BROMIDE IN API AND PHARMACEUTICAL DOSAGE FORM
ANJALI P. KOKANE*, VARSHA S. TEGELI, BHAGYASHRI S. SHINDE
Department of Quality Assurance D. S. T. S. Mandal’s College of Pharmacy, Solapur, Maharashtra, India
Email: anjalikokane333@gmail.com
Received: 26 Jan 2020, Revised and Accepted: 24 Mar 2020
ABSTRACT
Objective: The current work intended towards the developed and validated by using Simple, rapid, sensitive, precise and specific method UV Spectrophotometric method for the estimation of Ipratropium bromide in API and pharmaceutical formulation.
Methods: Water used as a solvent and the absorbance of the drug was measured at the absorbance’s maxima of Ipratropium bromide λmax is 214 nm.
Result: Calibration curve plotted in concentration range 20-120µg/ml exhibit the linearity relationship with line equation y=0.0062x+0.3161 and r2=0.995. The Accuracy was found to be 99.5-100.1%, the precision % RSD= 0.12888-0.30533, and the LOD and LOQ is 8.78266-28.5881. The method was found to comply with all the validation parameters as per ICH guidelines indicating the sensitivity of the method towards analyte.
Conclusion: The method can be used satisfactory for the routine analysis of Ipratropium Bromide present in API and Pharmaceutical dosage form.
Keywords: Ipratropium bromide, UV spectrophotometer, Method validation
© 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijcpr.2020v12i3. 38309. Journal homepage: https://innovareacademics.in/journals/index.php/ijcpr
INTRODUCTION
Ipratropium bromide is a muscarinic cholinergic antagonist and is used in asthma. Ipratropium bromide has bronchial smooth muscle relaxant properties due to its action on muscarinic receptor. Ipratropium bromide is a bromide salt form of Ipratropium, synthetic derivative of alkaloid atropine with anticholinergic properties. It is used in treating symptoms of asthma, cold, chronic obstructive pulmonary disease due to chronic bronchitis. Ipratropium bromide is short-acting anticholinergic drug in asthma.
Chemical name is (1R,3R,5S,8R)-3-[(3-hydroxy-2-phenyl propanoyl) oxy]-8-methyl-8 (propan-2-yl) 8-azabicyclo [3.2.1] octan-8-ium-bromide.
Fig. 1: Chemical structure of Ipratropium bromide
Literature survey reveals that the Ipratropium bromide has been estimated by RP-HPLC1, LC/MS, HPTLC with fluorescence detection.
The aim of work is to develop UV spectrophotometry and RP-HPLC [2-6] method for the estimation of Ipratropium bromide in pharmaceutical formulation.
MATERIALS AND METHODS
Instrument
For weighing a calibrated weighing balance (Shimadzu AY220) of 1 mg, sensitivity was used. A systronics UV visible double beam spectrophotometer 2201 was used with 1 cm matched quartz cell. All the glassware were made of borosilicate and were calibrated.
Chemicals
API-Ipratropium bromide pure API was gifted by Vamsi Pharmaceutical Ltd. Solapur, Maharashtra.
UV spectroscopic method
Solvent selection
Ipratropium bromide is soluble in water so the water was used as the solvent.
Preparation of standard stock solution
The standard stock solution of Ipratropium bromide was prepared by transferring accurately weighed 10 mg of Ipratropium bromide into 10 ml volumetric flask containing 5 ml of water, dissolve properly. The volume was made up to the mark by using water to give a concentration of 1000 µg/ml, from this 4 ml of the solution was transferred to 20 ml of volumetric flask and made up the volume with water to give a concentration of 200 µg/ml which is a standard solution and it is further diluted with water to get concentration range 20-120µg/ml.
Determination of absorption maxima
The standard stock solution of 200µg/ml was scanned in the range of 200-400 nm to determine the wavelength of maximum absorption. The drug showed maximum absorption at 214 nm.
Preparation of calibration curve
For the preparation of calibration curve solutions of concentration 20-120 µg/ml were prepared by pipetting out 1, 2, 3, 4, 5, 6, ml of 200 µg/ml solution into 10 ml volumetric flask and made up the volume up to the mark with water. The absorption of each solution was measured at 214 nm against water as blank. Calibration curve of the Ipratropium bromide was plotted by taking the absorption obtained on the Y-axis and concentration of the solution on the X-axis. The curve showed linearity in the range of 20-120 µg/ml with a correlation coefficient of 0.9954.
Fig. 2: Calibration curve of ipratropium bromide (IPRA)
Method validation
The developed method was validated as per ICH guidelines for the following parameters.
Linearity
1, 2, 3, 4, 5, 6 ml of standard Ipratropium bromide solution was transferred into a series of 10 ml volumetric flask. The volume was made up to the mark with water to obtain the concentration of 20, 40, 60, 80, 100, 120 µg/ml. The absorption of these solutions was recorded and the graph of absorption against concentration was plotted. The correlation coefficient (r²) of the least squares linear regression of Ipratropium bromide was calculated.
Range
The range of the analytical method was decided from the interval between the upper and lower level of the calibration curve by plotting the curve.
Accuracy
The recovery study was carried out by the standard addition method by adding the known amount of Ipratropium bromide to the pre-analyzed sample at three different concentration levels that are 80%, 100%, 120% of assay concentration and percent recovery were calculated.
% Recovery =observed value/true value*100
Precision
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same sample under the prescribed condition. The precision of the method was determined in term of repeatability, intra-day precision and inter-day precision (Intermediate precision). Intra-day precision was determined by analyzing the drug at the concentration (60µg/ml) and each concentration for these two times on the same day. Inter-day precision was determined similarly, but the analysis is carried out daily for the two consecutive days.
Repeatability: Repeatability of the method was determined by analyzing six samples of the same concentration of the drug 60µg/ml. The absorbance of each was measured.
Robustness
The robustness of the developed method is its capacity to remain unaffected by small changes in altered concentration. To determine the robustness of the method, the wavelength was studied at±5 nm.
Ruggedness
Ruggedness was determined by carrying out the analysis by two different analysts and the respective absorbance was noted and the results were indicated as % RSD.
Limit of detection
Detection limit was determined based on the standard deviation of absorbance of same concentration that is a standard stock solution of Ipratropium bromide (60 µg/ml) and LOD was calculated by LOD=3.3(SD/S).
Where, SD-standard deviation; S-slope of the curve
Limit of quantification
Quantification limit was determined based on the standard deviation of the peak area of same concentration that is a standard solution of Ipratropium bromide (60µg/ml) was prepared six times and LOQ= 10(SD/S).
Where, SD-standard deviation; S–slope of the deviation.
RESULTS
Preliminary analysis of drug
Observation and Results of Preliminary Analysis of Ipratropium bromide.
Linearity
The linearity of this method was determined at range from 20-120 µg/ml for the Ipratropium Bromide the regression equation was found to be 0.0062x+0.3161, R2=0.995
Accuracy
The accuracy of the analytical method for Ipratropium Bromide was determined at 80, 100, 120% level of the standard solution. Absorbance was measured at 214 nm and the results were expressed in the term of % recovery.
Precision
The precision (measurement of intra-day, inter-day and repeatability) result showed good reproducibility with the relative standard deviation (%RSD) below 0.12888-0.30533 and this indicates that the method was highly precise.
Table 1: Preliminary analysis of drug
| Test | Observation | Result |
| Description | White powder | Complied |
| Solubility | Soluble in water, methanol | Complied |
Fig. 3: Determination of wavelength of maximum absorption was found to be 214 nm
Table 2: Linearity
| S. No. | Conc. (µg/ml) | Absorbance |
| 1 | 20 | 0.434 |
| 2 | 40 | 0.553 |
| 3 | 60 | 0.695 |
| 4 | 80 | 0.838 |
| 5 | 100 | 0.919 |
| 6 | 120 | 1.105 |
The linearity for Ipratropium bromide was found to be linear in the range of 20-120 µg/ml with R2=0.995 and the strainght line equation as y=0.0062x+0.3161
Fig. 4: The accuracy of the analytical method for Ipratropium Bromide was determined at 80, 100, 120% level of the standard solution. Absorbance was measured at 214 nm and the results were expressed in the term of % recovery
Table 3: Accuracy
| S. No. | % level | Spike amount (µg/ml) | Spiked amount (wrt. sample) | Abs. | Amount recovered | % RSD of % recovery |
| 1 | 80 | 79.840 | 79.8 | 0.735 | 99.5 | 1.0 |
| 2 | 100 | 99.800 | 99.8 | 0.919 | 99.5 | 0.3 |
| 3 | 120 | 119.760 | 119.8 | 1.105 | 100.1 | 0.4 |
Table 4: Intra-day morning precision
| S. No. | Concentration (µg/ml) | Absorbance | SD | % RSD |
| 1. | 60 | 0.694 | 0.00089443 | 0.12888 |
| 2. | 60 | 0.695 | ||
| 3. | 60 | 0.694 | ||
| 4. | 60 | 0.693 | ||
| 5. | 60 | 0.693 | ||
| 6. | 60 | 0.695 |
Table 5: Inter-day precision
| S. No. | Concentration | Absorbance | %SD | %RSD |
| 1. | 60 | 0.683 | 0.00209762 | 0.30533009 |
| 2. | 60 | 0.689 | ||
| 3. | 60 | 0.688 | ||
| 4. | 60 | 0.687 | ||
| 5. | 60 | 0.687 | ||
| 6. | 60 | 0.688 |
Limit of detection
For the limit of detection
Limit of quantification
For Limit of Quantification
Robustness
For Robustness
Ruggedness
Ruggedness was determined by carrying out the analysis by two different analyst and the respective absorbance was noted and the results were indicated as %RSD.
DISCUSSION
Preliminary analysis of Ipratropium bromide such as description, solubility was performed.
UV spectrophotometry for Ipratropium bromide
Selection of wavelength
Ipratropium bromide showed the maximum absorbance at 214 nm. Hence, analysis is carried out at 214 nm. Ipratropium bromide showed absorption maxima at 214 nm and obeyed Beer’s law in the concentration range of 20-120 µg/ml with the correlation coefficient 0.995which is within acceptance criteria. The accuracy was from 99.5 and 100.1% at 214max 200-400 nm, respectively. The %RSD less than showed the method is precise. The limit of detection (LOD) was found to be 8.78266 and the limit of qualification (LOQ) to be 28.5881, respectively.
Summary and conclusion
A simple, precise, rapid and accurate UV method for determination of Ipratropium bromide from pure and its tablet formulation has been developed and validated. The proposed method can be used for the routine determination of Ipratropium bromide in bulk and pharmaceutical dosage formulation.
Table 6: Limit of detection
| LOD(µg/ml) | 8.78266 |
Table: 7 Limit of quantification
| LOQ(µg/ml) | 28.5881 |
Table 8: For robustness
| S. No. | Wavelength | Absorbance | SD | %RSD |
| 1. | 214 | 0.434 | ||
| 0.436 | 0.002 | 0.4587 | ||
| 0.438 | ||||
| Avg. =0.436 | ||||
| 2. | 216 | 0.432 | ||
| 0.437 | 0.004 | 0.9262 | ||
| 0.440 | ||||
| Avg.=0.436 |
Table 9: Ruggedness
Analyst 1
| Concentration | Absorbance | Statistical analysis |
| 60 | 0.683 | Avg. =0.684 |
| 60 | 0.684 | SD =0.001 |
| 60 | 0.685 | %RSD =0.1461 |
Analyst 2
| Concentration | Absorbance | Statistical analysis |
| 60 | 0.684 | Avg. =0.685 |
| 60 | 0.685 | SD =0.001 |
| 60 | 0.686 | %RSD =0.1459 |
Table 10: Summary of UV spectrophotometry method of Ipratropium bromide
| S. No. | Parameter | Values(214 nm) |
| 1. | Beers law limit (µg/ml) | 20-120 |
| 2. | Absorption maxima (nm) | 214 nm |
| 3. | Correlation coefficient (r²) | 0.995 |
| 4. | Standard regression equation | 0.0062x+0.3161 |
| 5. | Accuracy (%recovery) | 99.5-100.1% |
| 6. | Precision (%RSD) | 0.12888-0.30533 |
| Inter-day | 0.12888 | |
| Intra-day | 0.30533 | |
| 7. | LOD | 8.78266 |
| 8. | LOQ | 28.5881 |
| 9. | Robustness (%RSD) | 0.4587 and 0.9262 |
| 10 | Ruggedness (%RSD) | 0.1461 and 0.1459 |
The development of a validated method for the determination of Ipratropium bromide at λ max has shown a similar result; therefore, analysis can be done either at 214 nm with the same accuracy and precision.
FUNDING
Nil
AUTHORS CONTRIBUTIONS
All the authors have contributed equally.
CONFLICT OF INTERESTS
Declare none
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