INTRODUCTION:

Secnidazole (Fig.1) is a second-generation 5-nitroimidazole antimicrobial agent that is structurally related to other 5-nitroimidazoles. Chemically, Secnidazole is 1-(2-methyl-5-nitroimidazol-1-yl) propan-2-ol¹. It is an antimicrobial agent indicated for the treatment of bacterial vaginosis in adult women. It enters the bacterial cell as a prodrug without an antimicrobial activity. The drug is converted to an active form via reduction of nitro groups to radical anions by bacterial enzymes².

Fig. 1: Chemical structure of Secnidazole

Literature survey revealed that few analytical methods such as spectrophotometric^3-7, HPLC^8-19, and UPLC^20,21methods have been reported for the estimation of Secnidazole individually or in combination with other drugs. The author developed a rapid and new HPLC method and validated as per ICH guidelines²² for the estimation of Secnidazole in pure and pharmaceutical dosage forms.

MATERIALS AND METHODS:

Instrumentation:

To develop a high pressure liquid chromatographic method for quantitative estimation of Secnidazole using Waters HPLC system on Agilent C8 column (250 mm x 4.6 mm, 5μ) was used. The instrument is equipped with an auto sampler and DAD detector. A 20 μL rheodyne injector port was used for injecting the samples. Data was analyzed by using Empower2 software.

Chemicals and solvents:

The working standard of Secnidazole was provided as gift sample from Pharma Train, Hyderabad, India. The market formulation Ambiform tablets (Secnidazole 500 mg) were procured from local market. HPLC grade water and acetonitrile were purchased from E.Merck (India) Ltd, Mumbai, India. Potassium dihydrogen phosphate and orthophosphoric acid of AR grade were obtained from S.D. Fine Chemicals Ltd, Mumbai, India.

Chromatographic conditions:

A mixture of phosphate buffer and acetonitrile in the ratio of 85:15, V/V was found to be the most suitable mobile phase for ideal chromatographic separation of Secnidazole. The solvent mixture was filtered through a 0.45 μ membrane filter and sonicated before use. It was pumped through the column at a flow rate of 1.0 mL/min. Injection volume was 20 µL and the column was maintained at ambient temperature. The column was equilibrated by pumping the mobile phase through the column for at least 30 minutes prior to the injection of the drug solution. The detection of the drug was monitored at 228 nm. The run time was set at 12 min.

Preparation of phosphate buffer pH 2.5:

1.36 grams of potassium dihydrogen phosphate was weighed and transferred into a 1000 mL beaker, dissolved and diluted to 1000 mL with HPLC water. pH was adjusted to 2.5 with orthophosporic acid.

Preparation of mobile phase and diluent:

850 mL of the phosphate buffer was mixed with 150 mL of acetonitrile. The solution was degassed in an ultrasonic water bath for 5 minutes and filtered through 0.45 µm filter under vacuum. The same mobile phase was used as diluent.

Preparation of standard solution:

50 mg of Secnidazole was accurately weighed, transferred to 50 mL volumetric flask and is dissolved in 25 mL of the diluent. Sonicated the solution for few minutes to dissolve the drug completely. Then it is filtered through 0.45 μ filter and the volume is made up to 50 mL with diluent to get a concentration of 1000 µg/mL stock solution. Further pipetted 1 mL of the above stock solution into a 10 mL volumetric flask and diluted up to the mark with diluent to obtain required concentrations.

Preparation of sample solution:

Twenty commercial capsules contents were weighed to obtain the average tablet content weight and the contents were mixed. A sample of the mixed tablet content of the powder equivalent to 50 mg of Secnidazole was accurately weighed, transferred to 50 mL volumetric flask and is dissolved in 25 mL of the diluent. Sonicated the solution for few minutes to dissolve the drug completely. Then it is filtered through 0.45 μ filter and the volume is made up to 50 mL with diluent to get a concentration of 1000 µg/mL stock solution. Further pipetted 1 mL of the above stock solution into a 10 mL volumetric flask and diluted up to the mark with diluent to obtain required concentrations of Secnidazole in pharmaceutical dosage form.

Linearity:

Several aliquots of standard solution of Secnidazole was taken in different 10 mL volumetric flasks and diluted up to the mark with diluent such that the final concentrations of Secnidazole were in the range of 80 to 120 µg/mL. Evaluation of the drug was performed with UV detector at 228 nm, peak area was recorded for all the peaks. The correlation coefficient value of Secnidazole was 0.999. The results show that an excellent correlation exists between peak area and concentration of drug within the concentration range indicated.

Limit of detection and limit of quantification:

The limit of detection (LOD) is the smallest concentration that can be detected but not necessarily quantified as an exact value. LOD is calculated from the formula;

LOD = 3.3σ /S; Where, σ = standard deviation of the response, S = slope of calibration curve.

The limit of quantitation is the lowest amount of analyte in the sample that can be quantitatively determined with precision and accuracy. LOQ is calculated from formula;

LOQ = 10σ /S; Where, σ = standard deviation of the response, S = slope of calibration curve

The LOD for Secnidazole was found to be 0.924 µg/mL. The LOQ for Secnidazole was found to be 2.772 µg/mL.

System suitability:

System suitability parameters like retention time, resolution, theoretical plates and tailing factor were calculated and compared with standard values.

Accuracy:

The accuracy of the method was assessed by recovery study of Secnidazole in the dosage form at three concentration levels. A fixed amount of preanalyzed sample was taken and standard drug was added at 50%, 100% and 150% levels. Each level was repeated three times. The content of Secnidazole per capsule was calculated. The mean recovery of Secnidazole was found to be 100.30% that shows there is no interference from excipients and the lower values of RSD of assay indicate the method is accurate.

Precision:

The precision was determined for Secnidazole in terms of intra-day and inter-day precision.For intra-day precision evaluation, a standard solution of fixed concentration was injected at various time intervals and %RSD for Secnidazole was 0.79% (limit %RSD < 2.0%). In addition, the inter-day precision was studied by injecting the same concentration of standard solution on consecutive days and the %RSD for Secnidazole was 1.12% (limit %RSD < 2.0%).

Ruggedness and robustness:

The ruggedness of the method was determined by carrying out the experiment on different instruments by different operators using different columns of similar types. Robustness of the method was determined by making slight changes in the chromatographic conditions. It was observed that there were no marked changes in the chromatograms, which demonstrated that the HPLC method so developed is rugged and robust.

Assay:

20 µL of each standard and sample solution were injected and from the peak area of Secnidazole, amount of each drug in samples were computed. The result of assay undertaken yielded 99.60% of label claim of Secnidazole.

RESULTS AND DISCUSSION:

The HPLC procedure was optimized with a view to develop an accurate method in capsule dosage form using Agilent C8 column (250 x 4.6 mm, 5 μ) in isocratic mode with mobile phase composition of phosphate buffer: acetonitrile (85:15, V/V) and pH adjusted to 2.5 with orthophosphoric acid. The use of phosphate buffer and acetonitrile in the ratio of 85:15, V/V resulted in peak with good shape and resolution. The flow rate was 1.0 mL/min and the drug component was measured with UV detector at 228 nm. The results of optimized HPLC conditions were shown in Table 1. The method was linear in the range of 80 to 120 µg/mL for Secnidazole with correlation coefficient of 0.999. The linearity results were shown in Table 2 and the linearity curve was shown in Fig. 2. The % recovery was found to be 100.30% for Secnidazole, which indicate the method is accurate. The results of recovery studies were shown in Table 3. The %RSD for intra-day precision and inter-day precision for Secnidazole were found to be 0.79 and 1.12, which indicate the method is precise. The results of precision studies were shown in Table 4.

Table 1: Optimized chromatographic conditions of Secnidazole

Parameter	Condition
Mobile phase	Phosphate buffer: Acetonitrile (85:15, v/v)
pH	2.5
Diluent	Mobile phase
Column	Agilent C8 (250 X 4.6 mm, 5µ)
Column temperature	Ambient
Wave length	228
Injection volume	20 µL
Flow rate	1 mL/min
Run time	12 min.
Retention time	7.995 min.

Table 2: Linearity results of Secnidazole

Concentration in μg/mL	Area
80	1334400
90	1508196
100	1691902
110	1835027
120	1998270

Table 3: Recovery results of Secnidazole

Level	Amount added	Amount found	% Recovery	Mean recovery
50%	25	25.15	100.61	100.30
100%	50	50.22	100.44
150%	75	74.89	99.85

Table 4: Precision studies of Secnidazole

Concentration (µg/mL)	Intra-day precision (%RSD)	Inter-day precision (%RSD)
100	0.79	1.12

Fig. 2: Linearity curve of Secnidazole

The retention time of Secnidazole was 7.995 min. The number of theoretical plates was 5286 and tailing factor was 0.98 for Secnidazole, which indicates efficient performance of the column. The limit of detection and limit of quantification for Secnidazole were found to be 0.924 µg/mL and 2.772 µg/mL, which indicate the sensitivity of the method. The summary of system suitability parameters and validation parameters were shown in Table 5. Validated method was applied for the determination of Secnidazole in commercial formulations. The %assay was found to be 99.60% for Secnidazole and the assay results were shown in Table 6.

Table 5: Summary of system suitability and validation parameters of Secnidazole

Parameter	Results
Linearity range (μg/mL)	80-120
Correlation coefficient	0.992
Theoretical plates (N)	5286
Tailing factor	1.12
LOD (μg/mL)	0.924
LOQ (μg/mL)	2.772

Table 6: Assay results of Secnidazole

Formulation	Label claim	Amount found	%Assay
Ambiform	500 mg	498 mg	99.60%

Typical chromatogram of drug Secnidazole was shown in Fig. 3. No interfering peaks were found in the chromatogram of the formulation within the run time indicating that excipients used in the formulation did not interfere with the estimation of the drug by the proposed HPLC method.

CONCLUSION:

A simple, precise, selective and sensitive RP-HPLC method with UV detection for Secnidazole was developed and validated. This method will be useful for the easy and quick estimation of Secnidazole with almost no interferences in bulk and pharmaceutical dosage forms.

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Received on 01.12.2017 Modified on 20.12.2017

Asian J. Research Chem. 2018; 11(1):134-138.

DOI:10.5958/0974-4150.2018.00028.7