Development of Analytical method by RP-HPLC Technique for determination of Secnidazole in API and Pharmaceutical Dosage Form
Rajan V. Rele*, Sandip P. Patil
Central Research Laboratory, D. G. Ruparel College, Matunga, Mumbai 400016
*Corresponding Author E-mail: drvinraj@gmail.com
ABSTRACT:
A novel reverse phase liquid chromatographic method was developed and validated for estimation of anti-protozoal, anti-amebic and antigiardiasis drug, secnidazole in its dosage form i.e. tablets. The reverse phase HPLC analysis was carried out on isocratic system. The column was Peerless basic C18 (50mm x 4.6mm, 3µm) with ambient temperature. The mobile phase consisted of buffer: methanol in proportion 80:20 % (v/v). The flow rate was maintained at 1 ml / min. The detection was carried out at wavelength 310 nm. The method was validated as per ICH guidelines for system suitability, linearity, accuracy and precision. The linear ranges were 50-150 µg/ml for secnidazole. The accuracy and precision were found to be well within the acceptable limit. The method was successfully applied for determination secnidazole of in dosage form with good recoveries.
KEYWORDS: Secnidazole, Tri ethyl amine, Phosphoric acid, Methanol, HPLC.
INTRODUCTION:
It is used mainly to treat intestinal ameobiasis, fiardiasis, trichomoniasis and bacterial vaginosis. All such disease symptoms are treated with a single once only dose of such drug.
According to the literature review several methods has been developed for drug, like spectroscopy methods1-3. HPLC4-9, HPTLC10. The proposed aim of the study was to develop simple, accurate, specific and precise UV spectrophotometric method for the estimation of drug in the bulk and pharmaceutical formulation.
Structure of secnidazole
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 0.01 mg was used.
Materials and reagents:
Reference standard of secnidazole were obtained from reputed firms with certificate of analysis. Analytical grade of methanol, phosphoric acid and tri ethyl amine were used from Merck and the HPLC grade water was obtained by using Millipore water system.
Procedures:
Standard stock solution:
About 10 mg of standard secnidazole was weighed accurately and transferred in 10 ml volumetric flask. About 5 ml of diluent (buffer: methanol (80:20 % 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 10 ml 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 10 mg of secnidazole was weighed accurately and transferred in 10 ml volumetric flask. About 5 ml of diluent (buffer: methanol (80:20 % v/v) was added and sonicated for 5 minutes. The volume was adjusted up to mark with diluent to give concentration as 1000 μg /ml. The working sample solution was prepared by diluting 1 ml of 1000 μg/ ml solution to 10 ml with diluent to give 100 μg/ ml. A 10 µl 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 Peerless Basic C18 (50mm x 4.6mm x 3μm) column offered more advantage over other columns. The mobile phase was a mixture of buffer and methanol (80:20 % v/v). The buffer was 1 ml of tri-ethyl amine dissolved in 1000 ml of HPLC grade water, with pH 3.0. It was adjusted with dilute phosphoric acid. The flow rate of the mobile phase was adjusted to 1 ml /min. The detection was carried out at wavelength 310 nm. The injection volume of the standard and sample solution was set at 10.0 µl. The elution and resolution parameters of drug were recorded at the wavelength 310 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.
Figure.1: chromatogram of standard secnidazole
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 for alprazolam. (n = 6).
|
Retention time |
Symmetry Factor |
Area |
% Area |
|
3.940minutes |
1.24 |
8958675 |
100.00 |
Linearity:
The linearity of the method was determined for secnidazoleat 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 = The correlation coefficient (r2) was 0.999 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 |
test |
wt in mg |
area |
quantity added in μg /ml |
quantity recovered in μg /ml |
% recovery |
mean recovery |
|
80% |
1 |
10.28 |
7124292 |
16.64 |
16.60 |
99.76 |
99.88 |
|
2 |
2.07 |
7109995 |
16.64 |
16.57 |
99.56 |
||
|
3 |
2.06 |
7164126 |
16.64 |
16.69 |
100.31 |
||
|
100% |
1 |
2.09 |
8967867 |
20.8 |
20.90 |
100.46 |
100.60 |
|
2 |
2.11 |
8980628 |
20.8 |
20.92 |
100.60 |
||
|
3 |
2.08 |
8992914 |
20.8 |
20.95 |
100.74 |
||
|
150% |
1 |
2.07 |
10755068 |
24.96 |
25.06 |
100.40 |
100.24 |
|
2 |
2.09 |
10743063 |
24.96 |
25.03 |
100.29 |
||
|
3 |
2.10 |
10716878 |
24.96 |
24.97 |
100.04 |
* Average of triplicate analysis
Precession:
The method precision was established by carrying out the analysis of alprazolam. 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 |
Found Area |
% assay |
|
Solution-1 |
10.22 |
8958675 |
99.77 |
|
Solution-2 |
10.28 |
8974143 |
100.53 |
|
Solution-3 |
10.24 |
8972377 |
100.12 |
|
Solution-4 |
10.19 |
8944261 |
99.32 |
|
Solution-5 |
10.26 |
8954114 |
100.11 |
|
Solution-6 |
10.27 |
8963488 |
100.31 |
|
|
Mean Assay |
100.02 |
|
|
|
SD |
0.429 |
|
|
|
RSD |
0.428 |
|
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 mobile phase composition, variation ± 0.2 units
In wavelength, variation ± 5 units
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 secnidazole its formulation. Twenty tablets of secnidazole were used. A portion equivalent to 10 mg of secnidazole was weighed accurately. It was dissolved in 10 ml of diluent to obtain final concentration 1000µg/ml. The working sample solution was prepared by diluting 10ml of 1000μg/ml solution to 100 ml with diluent to give 100μg/ml.
10 µl of this solution was injected under specified conditions. The analyte peaks were identified by comparison with respective standard and sample chromatogram were recorded. (Fig. no.3).
Fig 3: chromatogram of secnidazole (sample)
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 secnidazole was 3.96 min. The linearity was in the range of 50-150μg/ml. The regression equation of the linearity was given as Y=10787x+200000 where X is concentration of secnidazole in μg/ml. and Y is corresponding peak area. The coefficient of co-relation was 0.996. The result shows that an excellent correlation between peak area and concentration of secnidazole in the range indicated. The relative standard deviation for method precision was 0.74 (limit % RSD < 2.0%). The mean recovery of the secnidazole was 99.89 %. The high percentage recovery indicates that the proposed method is highly accurate.
The use of 0.1 % tri ethyl amine (pH 3.0) and methanol (80:20 % (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 0.664 and percent relative standard deviation 0.74. (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 secnidazole from active pharmaceutical ingredient and marketed formulation due to its simplicity and non interference of other peaks and relatively short retention time as 3.96. 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.
ACKNOWLEDGEMENT:
Authors express sincere thanks to the Principal, Dr. T.M. Desai, D. G. Ruparel college for providing laboratory facilities.
REFERENCES:
1. Márcia Regina Marcílio, Alexia Lorenzi Raiser, Ligia Paula fumagalli Rudy Bonfilio, Carla Regina , Righetti, Elton Brito Ribeiro, Dênia Mendes de Sousa valladão. Determination and validation of secnidazole in tablets by uv spectrophotometric, Biosci. J., Uberlândia, 33( 5); 2017: 1351-1361.
2. K. Senthil kumar, B. Manasa, E. Nagamani1, G. Manoj Varma1, Errolla Mahesh, B. Sudhakar and TV. Narayana, Spectrophotometric determination of secnidazole using folin ciocalteu’s and sodium carbonate, international journal of research in pharmacy and chemistry, 2(3); 2012:809-815.
3. Senthil Kumar Krishnan, G. Pavan Kumar, P. Ameerbasha, B. Chidananda, BL Monudeep, K. Sashikiran and B. Sudhakar, Spectrophotometric determination of secnidazole using hydroxylamine and sodium carbonate, international journal of pharmaceutical and chemical sciences, 2 (1);2013:136-141.
4. Priyanka P. Suryawanshi, Pathan Azhar Khan, Manadar Abhyankar, Sandeep S. Sonawane, Paraag Gide, A Validated Stability – Indicating Liquid Chromatographic Method for Secnidazole, Journal of Innovations in Pharmaceuticals and Biological Sciences, 2 (1); 2015:, 45-52.
5. Nasiruddin Ahmad Farooqui, A. Anton Smith, H.K. Sharma and R. Manavalan, Analytical Method Development and Validation of Secnidazole Tablets by RP-HPLC, Journal of Pharmaceutical. Science and Research. 2 (7); 2010: 412-416.
6. Tanzina Sharmin, Mariyam Akter and Mohammad Salim Hossain, Analytical method development and validation of Secnidazole in the tablet dosage form by RP-HPLC method, International Current Pharmaceutical Journal, ,5(4); 2016: 41-44.
7. Ramesh Adepu, Dr. Leela Mohan Kumar Pallapothu and Dr. A. Ashok Kumar, A novel method for the determination of secnidazole in human plasma by using liquid chromatography-electro sprays ionization tandem mass spectrometry, European Journal of Biomedical and Pharmaceutical Sciences, 4( 11); 2017: 587-592.
8. Ali Gamal Ahmed Al-Kaf, EL-Rashed. Ahmed Gad kariem, Ali Abdo Saif Ahmed Algaradi1 and Talal Alssmani, Development and Validation of an RP-HPLC Method for Estimation of Secnidazole and Its Degradation Products in Tablets, Global Journal of Pharmacy & Pharmaceutical Sciences, 1(1);2016: 1-9.
9. K. Sivarami Reddy, B. Ramachandra & N.V.S. Naidu, Validation of RP-HPLC method for the estimation of secnidazole in bulk and its pharmaceutical dosage forms International Journal of Scientific & Engineering Research, 5( 8); 2014:1250-1261.
10. P. S. Jain, Tulsidas R. Lohar, Nikita N. Kale and S. J. Surana, Development and Validation of Stability-Indicating HPTLC Method for Estimation of Secnidazole in Bulk Drug and Pharmaceutical Dosage Form, Journal of Advanced Drug Delivery, 1(4); 2014: 144-156.
Received on 31.03.2019 Modified on 20.05.2019
Accepted on 28.06.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2019; 12(10):4799-4802.
DOI: 10.5958/0974-360X.2019.00829.1