The research work describes development of stability – indicating reverse phase – HPLC method for Secnidazole in the presence of its degradation products, generated from forced degradation studies. Secnidazole was subjected to forced degradation under acidic, basic, photolytic and oxidative conditions. Successful separation of drug from degradation products formed under forced degradation was achieved on a C 18 column (250 mm × 4.6 mm, 5 mcm) using methanol : water (60 : 40 v/v) as a mobile phase at a flow rate of 1mL/min. The detection was carried out at 310 nm. The method was validated for linearity, range, accuracy and precision.The developed method can be used to evaluate quality of regular production samples and also to test stability samples of secnidazole tablets. The intra and inter day results at each level were subjected to one way ANOVA and F values at each level were obtained as a ratio of between mean square and within mean square. The F values were found to be within limits. And the t-test value was obtained as 1.9725.
Keywords: Secnidazole; Secnidazole tablets; HPLC; forced degradation studies; method validation.
Secnidazole is chemically 1-(2- hydroxypropyl)-2- methyl-5- nitroimidazole (Figure 1). It is a relatively new antiprotozoal agent used in the treatment of amoebiasis and has also been © tried in trichomoniasis. [1] It is not official in any pharmacopoeia. Secnidazole is completely absorbed after oral administration. The adverse effects of secnidazole are abdominal pain, dizziness,
Several analytical procedures have been reported for the determination of secnidazole in pharmaceutical formulations, either alone or in combination with other drugs. These include spectrophotometric, [6-11] differential pulse polarographic, [12] supercritical fluid chromatographic, [13] GLC [14] and HPLC methods. [2,10] For stability testing, four different analytical methods, viz. derivative spectroscopy, HPLC, TLC densitometry and colorimetry were developed. But only few stabilityindicating HPLC methods for drug analysis has been reported.
The aim of present work is to develop an accurate, specific, and repeatable stability – indicating HPLC method for the estimation of secnidazole from tablets in presence of its possible degradation products. Further, the proposed method was validated as per ICH guideline Q2 (R1).
Pharmaceutical grade secnidazole was supplied as a gift sample by Meditab Speacialities Pvt. Ltd., Daman. Methanol used was of HPLC grade and other chemicals and solvents used in the studies were of AR grade and purchased from S.D. Fine Chemicals, Mumbai, India. Double distilled water was used throughout the experiment. The 0.45 μ Nylon filter papers were purchased from Pall India Pvt. Ltd, Mumbai (India).
HPLC system used consisted of pump PU – 2080 plus (JASCO, Tokyo, Japan with Rheodyne loop injector (7725 i) of capacity 25 mcL and detection was carried out by UV – 2077 detector. The data acquisition was done using Borwin chromatograohy software version 1.50. The chromatographic seperations were carried out on a HiQsil C18 column (250 mm × 4.6 mm, 5 mcm, Kya Technologies Corporation, JAPAN).
Mobile phase consisted of methanol : water (60 : 40 % v/v). The mixture was filtered through 0.45 micron membrane filter, ultrasonically degassed and used. Flow rate of 1mL/min with iscocratic elution and the column eluent being monitored at 310 nm at ambient temperatures. The injection volume was 20 mcL.
Standard secnidazole with a concentration of 1 mg/ml was used in all the degradation studies. The bulk drug was subjected to hydrolysis by refluxing the sample solution in hydrochloric acid (1N, 0.5N, and 0.1N), sodium hydroxide (1N, 0.5N, 0 0.1N, 0.01N) at temperature of 100 C. After completion of the degradation processes, the solutions were neutralized and diluted with mobile phase. For the oxidative studies, the standard secnidazole solution was subjected to stress conditions in 30 % and 3 % hydrogen peroxide at room temperature for different time intervals and then heated in water bath for 10 min to remove excess of hydrogen peroxide. Photo degradation studies were performed by exposing the drug powder to direct sunlight for different time interval. At the end of exposure, the drug samples were quenched, diluted with mobile phase so as to achieve a concentration of 10 mcg/mL with respect to the initial concentration of the drug and subjected to chromatographic analysis. In each case, suitable blanks and controls were used to preclude errors. Appearance of secondary peak and reduction in area of the drug peak was taken as an indication of degradation. [1]
Twenty tablets were weighed and finely powdered. A quantity of powder equivalent to 10 mg of secnidazole was transferred to a 100 ml volumetric flask and shaken with 70 ml of methanol for 10 min. The volume was made up to 100 ml with methanol. The solution was filtered. From the filtrate appropriate dilutions were made mobile phase. The tablet sample solution was injected and chromatogram was obtained. The peak area of the secnidazole was calculated. Using the regression equations and peak areas of the sample, the amount of secnidazole in the sample was calculated. The amount of secnidazole per tablet was thus found. [2]
Analytical method validation was carried out as per ICH method validation guideline Q2 (R1).
Standard stock solution of secnidazole was prepared in methanol and mixed to obtain final concentration of about 1 mg/mL.
From the above stock solution, dilutions were made with mobile phase to get seven calibration standards with concentrations of 2, 4, 6, 8, 10, 12, 14 mcg/mL. The calibration standards were analyzed in three replicates and the peak areas were plotted against corresponding drug concentrations and were subjected to least square linear regression to get an equation for line of best fit. The slope and intercept of the least square line were noted. [15]
The accuracy and precision were evaluated by fortifying a powder mixture of blank tablets with amounts of drug corresponding to 80, 100, 120% of label claimed and analyzing the resulting mixtures in three replicates over three days. The % recovery of added drug and % relative standards deviation (RSD) were taken as measures of accuracy and precision, respectively. Also, the results obtained were subjected to one way ANOVA and between – day mean square compared to the within – day mean square by F-test . [15]
The specificity of the proposed method was established by the complete separation of secnidazole in presence of its degradation products. Blank tablets of secnidazole were chromatographed before and after subjected to stress conditions. Absence of peaks in the blank runs at the retention time of drug and degradation products was taken as indication of specificity. [15]
Different mobile phases were tried and a best resolution was achieved with an initial run of a mixture of methanol and water in the ratio of 60:40 (v/v) to separate secnidazole from its degradation products. The optimum wavelength for detection was 310 nm at which better detector response was obtained. The retention time for secnidazole was observed to be 4.32 min.
Secnidazole was found to degrade under acidic, alkaline, oxidative, and photolytic conditions, while it was stable under dry heat and wet heat conditions. Table 1 summarizes the degradation behavior of secnidazole and the retention times of degradation products obtained under tested conditions. Figure 2 gives the representative chromatograms of stressed samples of secnidazole showing well resolved peaks of degradation products under various stress conditions.
In the calibration studies it was found that the calibration line was linear in the range of 2-14 mcg/ml (Figure 3). The response for the drug was linear and the calibration 2 equation was y = 48871x + 54281 with R = 0.998. The drug content was found to be 100% ±1.32 of the added amount indicating that the method can be used for analysis of secnidazole from tablet formulation. The t-test value was obtained as 1.9725.
The data obtained from precision and accuracy experiments are summarized in Table 2. Mean values of amount added and intraday % RSD values were very low indicating acceptable accuracy and precision of the method. The intra and inter day results at each level were subjected to one way ANOVA and F values at each level were obtained as a ratio of between mean square and within mean square. The F values were found to be found to be less than the tabulated F (2,6) at α=0.05 (Tabulated F=5.14). This indicated that there was no significant difference between intra and inters –day variability, suggesting good intermediate precision of the method.
The chromatograms of blank and placebo solutions showed no interfering peak at the retention time of the drug indicating
This study presents a simple and validated stability-indicating HPLC method for the estimation of secnidazole in the presence of its degradation products. The developed method is specific, accurate, precise and robust. All the peaks of the degradation products formed during forced degradation studies were well separated from the analyte peak. This demonstrates that the developed method was specific and stability-indicating. The developed method can be used for routine analysis of secnidazole tablets or for assay of secnidazole tablets from stability batches.
Table 4: Determinations of active ingredients in formulation Sr. No. Sample Label Claimed (mg/ml) 1 secnidazole 1000 2 secnidazole 1000 3 secnidazole 1000
Authors are thankful to the Mumbai Educational Trust’s Institute of Pharmacy, Bhujbal Knowledge City, Nashik for providing necessary analytical facilities and Meditab Specialities Pvt. Ltd., Daman for providing gift sample of secnidazole