Application of RP-HPLC Technique for Development of Analytical Method for Validation of Albuterol Sulphate from Bulk Drug and Dosage Form
Rajan V. Rele, Pratamesh P. Tiwatane, Sandip P. Patil
Central Research Laboratory, D. G. Ruparel College, Matunga, Mumbai 400016.
*Corresponding Author E-mail: drvinraj@gmail.com
ABSTRACT:
Instrumentation:
The HPLC system Merck-Hitachi equipped with separation module and UV detector (L-7400) was used. The chromatogram was recorded and peaks are quantified by means EZChrom Elite software. A Shimadzu analytical balance with 0.01mg was used.
Materials and reagents:
Reference standard of Albuterol sulphate was obtained from reputed firms with certificate of analysis. Analytical grade of phosphoric acid and tri ethyl amine and HPLC grade of methanol were used from Merck and the HPLC grade water was obtained by using Millipore water system.
Procedures:
Standard stock solution:
About 10mg of standard albuterol sulphate was weighed accurately and transferred in 10ml volumetric flask. About 5ml of diluent (buffer: methanol (75:25% v/v)) was added and sonicated for 5 minutes. The volume was adjusted to the mark with diluents to give concentration as 1000μg/ml. The working standard solution was prepared by diluting 1 ml of 1000μg/ml solution to 10ml with diluent to get concentration 100μg/ml.
Sample preparation:
Twenty tablets were weighed accurately and average weight of each tablet was determined. The powder equivalent to 10mg of Albuterol sulphate was weighed accurately and transferred in 10ml volumetric flask.
Figure.1: chromatogram of standard Albuterol sulphate
About 5ml of diluent (buffer: methanol (75:25% v/v) was added and sonicated for 5 minutes. The volume was adjusted up to mark with diluent to give concentration as 1000μg/ml. The working sample solution was prepared by diluting 1ml of 1000μg/ml solution to 10ml with diluent to give 100μg/ml. A 10µl was injected for analysis.
Method Development:
Chromatographic condition:
Different columns containing octyl and octadecyl silane stationary phase were tried for separation and resolutions. It was found that Hpersil BDS C18 (150mm x 4.6mm x 5μm) column offered more advantage over other columns. The mobile phase was a mixture of buffer and methanol (75:25% v/v). The buffer was 1ml of tri-ethyl amine dissolved in 1000 ml of HPLC grade water. The pH 3.5 was adjusted with dilute phosphoric acid. The flow rate of the mobile phase was adjusted to 1ml /min. The detection was carried out at wavelength 225 nm. The injection volume of the standard and sample solution was set at 20.0µl. The elution and resolution parameters of drug were recorded at the wavelength 225 nm and its response optimization was compared with adequate sensitivity. It produced well shaped peaks for the drug assay. A chromatogram of the drug assayed is depicted in fig. 1.
Method validation:
System suitability:
System performance parameters of developed HPLC method were determined by injecting standard solutions. Parameters such retention time, area, % area and asymmetry were shown in Table-1. It indicated good performance of the system.
Table – 1 : System performance parameters . (n = 6).
|
Retention time |
Symmetry factor |
Area |
% Area |
|
2.86 minutes |
1.55 |
10159623 |
100.00 |
Linearity:
The linearity of the method was determined for albuterol sulphate from six concentrations level ranging from 50 to 150 µg/ml. The calibration curve was constructed by plotting response factor against concentration of the drugs. The regression equation was given as y = 11267 x + 37546. The correlation coefficient (r2) was 0.9999 and concentration range indicated above. The results of the same are tabulated in the table 2.
Table 2: Linearity – regression analysis data
|
Parameters |
Values |
|
Correlation Coefficient (r) |
0.9999 |
|
Intercept (y) |
37546 |
|
Slope (m) |
11267 |
Accuracy:
The accuracy of the method was determined by recovery experiments. The recovery studies were carried out and percentage recovery was calculated and presented in Table 3.
Table 3 : Accuracy - %Recovery
|
level |
Sample No. |
weight in mg |
area |
quantity added in μg/ml |
quantity recovered in μg/ml |
% recovery |
mean recovery |
|
80% |
1 |
10.28 |
8072623 |
81.68 |
81.36 |
99.61 |
99.69 |
|
2 |
10.25 |
8077348 |
81.68 |
81.41 |
99.67 |
||
|
3 |
10.18 |
8086433 |
81.68 |
81.50 |
99.78 |
||
|
100% |
1 |
10.26 |
10141809 |
102.1 |
102.22 |
100.12 |
100.08 |
|
2 |
10.19 |
10131216 |
102.1 |
102.11 |
100.01 |
||
|
3 |
10.20 |
10141809 |
102.1 |
102.22 |
100.12 |
||
|
150% |
1 |
10.24 |
15109687 |
153.15 |
152.29 |
99.44 |
99.48 |
|
2 |
10.18 |
15114913 |
153.15 |
152.34 |
99.47 |
||
|
3 |
10.22 |
15124433 |
153.15 |
152.44 |
99.54 |
||
|
Mean recovery of all level |
99.75 |
||||||
* Average of triplicate analysis
Fig 2: chromatogram of albuterol sulphate (sample)
Precession:
The method precision was established by carrying out the analysis of albuterol sulphate. The assay was carried out of the drug using analytical method in six replicates. The value of relative standard deviation lies well with the limits (0.10%). The results of the same are tabulated in the table 4.
Table 4: Precision – method precision.
|
Test |
Weight of test sample used in mg |
Found Area |
% assay |
|
Solution-1 |
10.21 |
10159623 |
99.61 |
|
Solution-2 |
10.25 |
10169406 |
100.10 |
|
Solution-3 |
10.26 |
10173775 |
100.24 |
|
Solution-4 |
10.23 |
10182125 |
100.03 |
|
Solution-5 |
10.27 |
10186301 |
100.46 |
|
Solution-6 |
10.31 |
10133098 |
100.32 |
|
|
Mean Assay |
100.13 |
|
|
|
SD |
0.296 |
|
|
|
RSD |
0.296 |
|
Stability of solution:
The stability studies of the solutions under study were established by keeping the solutions at room temperature for 24 hours. The results indicated no significant change in the assay results of the same solutions. It confirmed the stability of the drug in the solvents used for the analysis.
Robustness:
Robustness study of the method was performed by making slight changes in the chromatographic conditions.
In flow rate, variation ± 0.2°C.
In wavelength, variation ± 5units
In variation mobile phase composition ± 0.2unit
The chromatograms demonstrated have no marked changes that developed HPLC method was robust.
Method Application:
The validated high performance liquid chromatographic method was applied for determination of albuterol sulphate its formulation. Twenty tablets of albuterol sulphate were used. A portion equivalent to 10mg of Albuterol sulphate was weighed accurately. It was dissolved in 10ml of diluent to obtain final concentration 1000µg/ml. The working sample solution was prepared by diluting 10ml of 1000μg/ml solution to 100ml with diluent to give 100μg/ml.
10µl of this solution was injected under specified conditions. The analyte peaks were identified by comparison with respective standard and sample chromatogram were recorded. (Fig.2).
The assay results expressed as mg/tablets are shown in Table-3. It indicated the amount of each drug in the product meet the requirement.
RESULTS AND DISCUSSION:
In the proposed method, the retention time of albuterol sulphate was 12.86min. The linearity was in the range of 50-150μg/ml. The regression equation of the linearity was given as Y= 11267x - 37546 where X is concentration of albuterol sulphate in μg/ml. and Y is corresponding peak area. The coefficient of co-relation was 0.9999. The result shows that an excellent correlation between peak area and concentration of albuterol sulphate in the range indicated. The relative standard deviation for method precision was 0.296 (limit %RSD < 2.0%). The mean recovery of the albuterol sulphate was 100.13%. The high percentage recovery indicates that the proposed method is highly accurate.
The use of 0.1% tri ethyl amine (pH 3.5) and methanol 75:25% (v/v) gave peak with good resolution. The robustness studies indicated that there was no effect other parameters on the drug study. No interfering peaks were found in the chromatogram of the formulation within the run time indicted that excipents used in the formulation did not interfere the estimation of drug.
The reproducibility, repeatability and accuracy of the proposed method were found to be satisfactory which is evidenced by low values of standard deviation. (Table no. 4) The accuracy and reproducibility of the proposed method was confirmed by recovery experiments, performed by adding known amount of the drug to the pre-analyzed active pharmaceutical ingredient and reanalyzing the mixture by proposed method. (Table no. 3) The percent recovery obtained indicates non- interference from the excipients used in the formulations. The methods reported in literature method indicate large retention time. Hence more time will be required for validation of drug and its formulation as well it requires more amount of organic solvent.
This can be successfully used for validation of drug as well as for determining stability of drug in various conditions as per ICH guidelines.
Thus the proposed RP-HPLC method is used for validation of albuterol sulphate from active pharmaceutical ingredient and marketed formulation due to its simplicity and non-interference of other peaks and relatively short retention time as 2.86 minutes. It is more precise, accurate, linear, robust, simple and rapid method. Hence the proposed RP-HPLC method is strongly recommended for the quality control of the raw material, active pharmaceutical ingredient and pharmaceutical formulation per ICH guidelines.
ACKNOWLEDGMENT:
Authors express sincere thanks to the principal, of D.G. Ruparel College, Mumbai for encouragement and providing laboratory facilities.
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Received on 05.01.2022 Modified on 03.02.2022
Accepted on 23.02.2022 ©AJRC All right reserved
Asian J. Research Chem. 2022; 15(2):155-158.