INTRODUCTION:

Secnidazole, 1-(2-hydroxypropyl)- 2-methyl–5–nitroimidazole. (C₇H₁₁N₃O₃). It belongs to 6 Anti-infective drugs. It has anti-protozoal, anti-amebic and antigiardiasis action. It belongs to nitro-imidazole with amebicides as pharmacological group.

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 methods^1-3.HPLC^4-9, 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.

MATERIAL AND METHODS:

Shimadzu UV-1800 was used with 10 mm matched quartz cell to measure absorbance of solution. A Shimadzu analytical balance with 0.01 mg was used.

Chemical and reagents:

Reference standard of secnidazole was obtained with certificate analysis. All spectral absorbance measurements were made on Shimadzu UV-1800 with 10 mm matched cell.

Preparation of standard solution:

About 10 mg of standard secnidazole was weighed accurately and transferred in 100 ml of volumetric flask. About 30 ml of distilled water was added and sonicated for 15 minutes. The volume was adjusted up to the mark with distilled water to give concentration as 100 μg /ml.

Estimation from tablets:

Twenty tablets were weighed accurately and average weight of each tablet was determined. Powder equivalent to 10 mg of secnidazole was weighed and transferred in 100 ml of volumetric flask. A 30 ml of distilled water was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with distilled water to give concentration as 100 μg /ml. Such solution was used for analysis.

Experimental:

Method A: Zero order method

For the selection of analytical wavelength, 10 μg /ml solution of secnidazole was scanned in the spectrum mode from 370 nm to 200 nm by using distilled water as blank. The zero order spectrum was obtained by UV probe 2.42 software. From the spectrum, the absorbance of the spectrum was measured at 320 nm (Fig. 1).

Fig. 1. Overlay spectra of Zero order spectrum of secnidazole (20 μg/ml) showing absorbance at 320 nm

Into series of 10 ml graduated flask, varying amount of standard solutions of secnidazole was pipette out and volume was adjusted with distilled water as solvent. Solutions were scanned between 350 nm to 200 nm in spectrum mode. Absorbance of the resulting solutions were measured at between 320 nm by using distilled water as blank. The calibration curve was prepared in the concentration range of 2.5 to 15 μg/ml. (Fig. 2)

Fig. 2. Calibration curve for secnidazole at 320 nm by zero order Spectroscopy

Method B: Area under curve (AUC) method:

Area under curve method involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelengths such as λ₁ and λ₂. The area under curve between λ₁ and λ₂were calculated by UV probe 2.42 software. In this method, 10 μg/ml solution of secnidazole was scanned in the spectrum mode from 370 nm to 250 nm. From zero order spectrum the AUC calculation was done. The AUC spectrum was measured between 315 nm to 325 nm (Fig.3).

Fig. 3. Area under curve spectrum of secnidazole ( 20 μg/ml) showing area from 315 nm to 325 nm.

Into series of 10 ml graduated flask, varying amount of standard solutions of secnidazole was pipette out and volume was adjusted with distilled water. Solutions were scanned between 370 nm to 250 nm in spectrum mode. The AUC calculations were done and the calibration curve for secnidazole was plotted in the concentration range of 2.5 to 15 μg/ml (Fig. 4).

Fig. 4. Calibration curve for drotaverine hydrochloride by area under curve spectroscopy

Results of analysis are given in table 1.

Table 1: Values of results of optical and regression of drug

Parameter	Zero order method	Area under curve (AUC) method
Detection Wavelength (nm)	320	315-325
Beer Law Limits (µg/ml)	2.5-15	2.5-15
Correlation coefficient(r²)	0.9999	0.9999
Regression equation (y=b+ac)
Slope (a)	0.052	0.007
Intercept (b)	-0.002	-0.001

Validation:

Accuracy:

Accuracy of the proposed methods was carried as on the basis of recovery studies. It is performed by the standard addition method. Recovery studies were performed by adding standard drug at different levels to the pre-analyzed tablets powder solution and the proposed method was followed. From the amount of the drug estimated, the percentage recovery was calculated. The results of the analysis are shown in table (2, 3).

Table 2: Results of recovery of secnidazole for zero order method

Amount of Sample Added in (µg/ml)	Amount of Standard Added in (µg/ml)	Total amount recovered	Percentage recovery (%)	Standard deviation	Percentage of relative standard deviation (C.O.V.)
2.5	0	2.502747	100.11	0.0234	0.9336
2.5	2.5	4.964286	99.286	0.0259	0.5211
2.5	5	7.414835	98.864	0.0456	0.6148
2.5	7.5	10.00275	100.03	0.0259	0.2586
				Mean=0.0302	Mean=0.582

Table 3: Results of recovery of secnidazole for area under curve (AUC) method

Amount of Sample Added in (µg/ml)	Amount of Standard Added in (µg/ml)	Total amount recovered	Percentage recovery (%)	Standard deviation	Percentage of relative standard deviation (C.O.V.)
2.5	0	2.502357	100.0943	0.038979	1.55768
2.5	2.5	5.005792	100.1158	0.035632	0.711807
2.5	5	7.501882	100.0251	0.025238	0.336423
2.5	7.5	10.00381	100.0381	0.036924	0.369102
				Mean=0.034193	Mean=0.743753

Precision:

The method precision was established by carrying out the analysis of homogenous powder blend of tablets. The assay was carried out of drug by using proposed analytical method in six replicates. The values of relative standard deviation lie well within the limits indicated the sample repeatability of the method. The results obtained are tabulated in table 4.

Table 4: Precision- method precision

Experiment no.	Secnidazole taken in μg/ ml	Secnidazole in μg/ ml
		Zero order method	Area under curve method
1	10	10.019	10.026
2	10	9.9615	10.013
3	10	9.9808	9.934
4	10	10.038	10.014
5	10	10.019	10.052
6	10	10.000	9.986
	Standard deviation	0.028146	0.040479
	%RSD	0.281377	0.404624

Inter-day and intra-day precision:

An accurately weighed quantity of tablets powder equivalent to 5 mg of secnidazole was transferred to 100 ml of volumetric flask. A 30 ml of distilled water was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with methanol to give concentration as 50 μg /ml. Such solution was used for analysis.

For zero order method:

Solution was scanned between 370 nm to 250 nm in spectrum mode. Absorbance of the resulting solution was measured at 320 nm by using distilled water as blank. The absorbance of final solution was read after 0 hr., 3 hrs. and 6 hrs. in 10 mm cell at 320 nm for zero order (method A). Similarly the amplitude of the same solution was read on 1^st, 2^nd and 5^th day. The amount of secnidazole was estimated by comparison with standard at 320 nm for zero order, table 5.

For area under curve method:

Solution was scanned between 370 nm to 250 nm in spectrum mode. The area under curve of resulting solutions was measured at between 315 nm to 325 nm by using distilled water as blank. The area under curve of final solutions was read after 0 hr., 3 hrs. and 6 hrs. in 10 mm cell at 315 nm to 325 nm (method B). Similarly area under curve of the same solution was read on 1^st, 2^nd and 5^th day. The amount of secnidazole was estimated by comparison with standard at 315 nm to 325 nm, table 5.

Table 5: Summary of validation parameter for intra-day and inter-day

Sr. no.	Parameters	Zero order method	Area under curve (AUC) method
(A)	Intra-day precision (n=3) Amount found ± % RSD	100.015 0.1722	100.063 % 0.03419
(B)	Inter-day precision (n=3) Amount found ± % RSD	100.03 0.2586	100.038% 0.3692
(c)	Ruggedness Analyst to analyst(n=3) %RSD	0.1459	0.03647

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The limit of detection (LOD) is defined as the lowest concentration of an analyte that an analytical process can reliably differentiate from back-ground levels. In this study, LOD and LOQ were based on the standard deviation of the response and the slope of the corresponding curve using the following equations-

LOD = 3.3 σ/S and LOQ = 10 σ/S

Where σ is the standard deviation of the signal to noise ratio of the sample and S is the slope of the related calibrations graphs.

The limit of quantification (LOQ) is defined as the lowest concentration of the standard curve that can be measured with an acceptable accuracy, precision and variability. The values of LOD and LOQ are given in table 6.

Table 6: Values of results of LOD and LOQ

Parameters	Zero order method	Area under curve (AUC) method
Limit of Detection (μg/ml)	0.0760	0.0876
Limit of Quantification (μg/ml)	0.2303	0.2657

Ruggedness:

The ruggedness of the method is defined as degree of reproducibility of results obtained by analysis of secnidazole sample under variety of normal test conditions such as different laboratories, different analysts and different lots of reagents. Quantitative determination of secnidazole was conducted spectrophotometrically on one laboratory. It was again tested in another laboratory using different instrument by different analyst. The assays obtained in two different laboratories were well in agreement. It proved ruggedness of the proposed methods.

RESULT AND DISCUSSION:

The zero order and area under curve UV-spectroscopic methods are useful for routine analysis of secnidazole in bulk drug and formulation. The method was validated according to International Conference on Harmonization guidelines for validation of analytical procedures. Secnidazole has the absorbance maxima at 320 nm (method A) and in the AUC spectrum method areas were measured between 315 nm to 325 nm (method B). The polynomial regression data for the calibration plots showed good linear relationship in the concentration range of 2.5 to 15 μg/ml for zero order spectroscopy and area under curve methods and given in table1. Recovery studies were carried out by adding the pure drug to the previously analyzed tablet powder sample and shown in table 2, 3. The percentage recovery value indicates non interference from excipients used in formulation. The reproducibility and accuracy of the method were found to be good, which was evidenced by low standard deviation.

The most striking features of two methods are its simplicity and rapidity, not requiring tedious sample solutions preparations which are needed for other instrumental methods. From the results obtained it can be concluded that the proposed methods are fully validated and found to be simple, sensitive, accurate, precise, reproducible, rugged and robust and relatively inexpensive. So, the developed methods can be easily applied for the routine quality control analysis of secnidazole in pharmaceutical formulation.

ACKNOWLEDGMENT:

Authors express sincere thanks to the Principal, Dr. Tushar M. Desai of D. G. Ruparel College, Mumbai.

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 18.12.2018 Modified on 22.01.2019

Research J. Pharm. and Tech. 2019; 12(4):1753-1756.

DOI: 10.5958/0974-360X.2019.00293.2