INTRODUCTION:

Secnidazole is a next-generation 5-nitroimidazole antimicrobial agent. It is approved for used in Europe, Asia, South America and Africa. Recently in the USA as a single-dose (2 gm) treatment of bacterial vaginosis (BV). (1) Bacterial vaginosis (BV) is one of the common vaginal infections among women. Bacterial vaginosis (BV) is one of the common causes of abnormal vaginal discharge which enhances the acquisition and transmission of a range of sexually transmitted infections. The usual treatment for BV is metronidazole; hence 30% of women have recurrence within 60 to 90 days after treatment. There are some studies which assessed the effect of secnidazole on BV. (2, 3)

It is have structural similarity with other 5-nitroimidazoles, but displays improved oral absorption and prolong elimination half-life than antimicrobial agents in this class. It is useful against many anaerobic Gram-positive and Gram-negative bacteria and protozoa. In September 2017, FDA granted approval to secnidazole under the market name Solosec as a single-dose oral treatment for BV. The antimicrobial therapy is only intended to treat or prevent infections that are proven or strongly suspected to be caused by susceptible bacteria. (4)

SECNIDAZOLE

Molecular formula	C₇H₁₁N₃O₃
Chemical name	1-(2-methyl-5-nitroimidazol-1-yl)propan-2-ol
Molecular weight	185.18 g/mol
Synonyms	Secnidazole Secnidazol Flagentyl Secnidazolum
Melting point	76.0 °C
Solubility	Water, Methanol, chloroform,
BCS Class	1

Mechanism of action:

Secnidazol enter inside micro-organisms by passive diffusion and activated by reduction of the 5-nitro group. This intracellular reduction occurs via the pyruvate-ferredoxin oxidoreductase complex and results in a concentration gradient across the cell membrane which, improve transport of the parent drug into the cell. Because the electron affinity of the 5-nitroimidazoles is greater than that of reduced ferredoxin, the drug disturbing the normal electron flow, which, in tum, enhances transport of the active form of drug into the cell. Because the electron affinity of the 5-nitroimidazoles is much more than that of reduced ferredoxin, the drug disturbing the normal electron flows.

DNA is the intracellular target of the 5-nitroimidazoles. Drug induced DNA damage results in strand breakage, loss of the helical structure and impaired template function. Furthermore, cytotoxicity may be greatest in micro-organisms with DNA containing a high percentage of adenine and thymine. (5,6)

Pharmacokinetic:

Secnidazole is completely absorbed after oral administration. It is absorbed very fast after administration. Plasma Drug conc. is straight for the therapeutic dose range of 0.5 to 2g. Protein Binding accounts for only about 15% of total plasma drug concentration and the Volume of distribution is low (49.2L). The concentration of secnidazole which Remains in the plasma for at least 48 hours after a single 2gm dose appears to be Well within the range corresponding to minimum inhibitory conc. (MIC) values Reported by most of researchers for in vitro sensitivity of B.fragilis, E. histolytica And T. vaginalis etc. The metabolism of secnidazole is not well known but, as compared with metronidazole, the drug may be undergoes oxidation in the liver. Secnidazole metabolite is found in urine as glucuronide conjugates.(7)

Experimental:

Instrumentation:

· HPLC system - Merck-Hitachi equipped with separation module

· UV detector - L-7400

· Software - EZChrom Elite.

· Analytical balance - Shimadzu (0.01mg)

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:

1) Standard stock solution:

Weighed accurately 10mg of standard secnidazole and transferred into 10 ml volumetric flask. Add 5ml of diluent (buffer: methanol (80:20% v/v)) and sonicated for 5 minutes. The final volume adjusts with diluents to give concentration 1000μg/ml. The working standard solution was prepared by diluting 1ml of 1000μg/ml solution to 10 ml with diluent to get concentration 100μg/ml.

2) Sample preparation:

Weighed accurately 20 tablets and calculate avg. weight of each tablet and make a powder of it. Weighed accurately powder equivalent to 10mg of secnidazole and add into 10ml volumetric flask. Add 5 ml of diluent and sonicated for 5 minutes. The volume was adjusted with diluent to give concentration 1000μg /ml. The preparation of working sample solution was done by diluting 1ml (1000μg/ml) solution into 10ml with diluent to get solution of 100μg/ ml. A 20µl was injected for analysis.

3) Chromatographic condition:

· Column/stationary phase - Peerless Basic C18 (50mm x 4.6mm x 3μm) column.

· Mobile phase - Buffer and methanol (80:20 % v/v). The buffer was 1 ml of triethyl amine dissolved in 1000ml of HPLC grade water, with pH 3.0 (phosphoric acid.).

· Flow rate - 1ml/min

· Detection wavelength 310nm.

· Injection volume of the standard and sample solution - 20.0µl

Fig - Standard Chromatogram of Secnidazole.

Method validation:

System suitability:

Performance of system parameters for developed HPLC method was determined by injecting standard solutions. Parameters like retention time, area, % area etc. were shown in Table-1. It indicated good performance of the system.

Accuracy:

Accuracy was performed in triplicate after using pure drug equivalent to 80, 100, and 120% of the standard concentration of Secnidazole (20µg/ml). Recovery was found in the range from 99.76-100.04%. The recovery of Secnidazole by proposed method is satisfactory as percent of relative standard deviation is not more than ± 2.0% and mean recovery between 99.98-100.24%. The results of the same are shown in the table 2.

Precision:

The precision of the developed HPLC method was expressed in terms of percent relative standard deviation (% RSD). At first, standard sample (20µg/ml) was run for six times. After confirmation of system suitability parameters (number of theoretical plates, tailing factors etc.), six samples were injected. Then % RSD was calculated as 0.428. % RSD values less than 2, revealed high precision of the method. The results of the same are tabulated in the table 3.

Linearity:

The linearity of the method was determined for secnidazole at six concentrations level ranging from 50 to 150 µg/ml. The calibration curve was made 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 3.

Robustness:

There are different ways to check the robustness of methods such as variations of pH in a mobile phase, variations in mobile phase composition, different columns (different lots and/or suppliers), flow rate, detection wavelength, temperature etc. In this study, standard deviation of peak areas was calculated by changing three parameters.

· Flow rate, variation ± 0.2˚C.

· Mobile phase composition, variation ± 0.2 units

· Wavelength, variation ± 5 units

The chromatograms demonstrated have no marked changes that developed HPLC method was robust. (8,9)

Observations

Table 1: System suitability parameters.

Retention time	3.940minutes
Symmetry Factor	1.24
Area	8958675
% Area	100.00

Table2: Accuracy -% Recovery

Level	80%			100%			150%
Test	1	2	3	1	2	3	1	2	3
Wt. in mg	10.28	2.07	2.06	2.09	2.11	2.08	2.07	2.09	2.10
Area	7124292	7109995	7164126	8967867	8980628	8992914	10755068	10743063	10716878
Quantity added in µg/ml	16.64	16.64	16.64	20.08	20.08	20.08	24.96	24.96	24.96
Quantity recovered in µg/ml	16.60	16.57	16.69	20.90	20.92	20.95	25.06	25.03	24.97
%recovery	99.76	99.56	100.31	100.46	100.60	100.74	100.40	100.29	100.04
Mean recovery	99.88			100.60			100.24

Table 3: Precision

test	Solution 1	Solution 2	Solution-3	Solution-4	Solution-5	Solution-6
Weight of sample used	10.22	10.28	10.24	10.19	10.26	10.27	Mean assay	SD	RSD
Found area	8958675	8974143	8972377	8944261	8954114	8963488
% assay	99.77	100.53	100.12	99.32	100.11	100.31	100.02	0.429	0.428

Table 4: Linearity

Parameters	Values
Correlation Coefficient (r)	0.9999
Intercept (y)	37546
Slope (m)	11267

RESULT:

In this method, the retention time of secnidazole was found to be 3.96 min. Secnidazole obeys the linearity in the range of 50-150μg/ml. The regression eq. of the linearity was found,

Y=10787_x + 200000

Where

X is conc. of secnidazole in μg/ml.

Y is 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 recovery of the secnidazole was 99.89% in newly developed method. The high percentage recovery indicates that the develop method had high accurate.

CONCLUSION:

The developed HPLC method for the estimation of Secnidazole was performed in mobile phase Buffer: methanol (80:20, v/v). This method is successfully validated as per ICH guidelines. It is indicate that this newly developed method was found to be accurate, simple, precise, and reproducible. This method can be easily applied for quality control analysis in industry. The retention time of this method will significantly reduce the analysis time and cost.

REFERENCE:

1. Paul Nyirjesy and Jane R Schwebke; Secnidazole: next-generation antimicrobial agent for bacterial vaginosis treatment; Future microbiology, Vol. 13, No. 5.

2. Mohamed A. Abd El Aziz, Foruzan Sharifipour, Parvin Abedi, Shayesteh Jahanfar and Helen Marie Judge; Secnidazole for treatment of bacterial vaginosis: a systematic review, BMC Women’s Health volume 19, Article number: 121 (2019)

3. Vishal Singh, Kritarth Nama; Secnidazole Oral Granules: A Novel Drug Formulation and an Effective Alternative in Treatment of Bacterial Vaginosis; International Journal of Innovative Research and Growth 2018;3(4).

4. National library of medicine, pubchem, secnidazol.

5. Edwards DI.; Nitroimidazole drugs - action and resistance mechanisms. I. Mechanisms of action; J Antimicrob Chemo-ther 1993; 31: 9-2.

6. Van Reken DE, Pearson RD. Antiparasitic Agents. In: Mandell GL, Douglas Jr RG, Bennett JE, editors; Principles and practice of infectious diseases; 3rd ed. New York: Churchill Livingstone, 1990: 398-427.

7. Jane c. Gillis and Lynda R. Wiseman; Secnidazole A Review of its Antimicrobial Activity, Pharmacokinetic Properties and Therapeutic Use in the Management of Protozoal Infections and Bacterial Vaginosis Drugs; Drug evaluation, 1996; 51 (4): 621-638

8. Rajan V. Rele, Sandip P. Patil; Development of Analytical method by RP-HPLC Technique for determination of Secnidazole in API and Pharmaceutical Dosage Form; Research J. Pharm. and Tech. 2019;12(10): -4799.

9. 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, March 2016, 5(4): 41-44.

Received on 17.02.2021 Modified on 09.03.2021

Research Journal of Science and Technology. 2021; 13(2):100-104.

DOI: 10.52711/2349-2988.2021.00015