DETERMINATION AND VALIDATION OF SECNIDAZOLE IN TABLETS BY UV SPECTROPHOTOMETRIC

Secnidazole, a 5-nitroimidazole, is a drug used in the treatment against protozoa, and several bacterial infections. This study purpose was to develop and validate a UV spectrophotometric method to determine secnidazole in pharmaceutical tablet dosage forms once there is no method reported in the pharmacopoeia yet. The quantification was performed using methanol as solvent at 325 nm (maximum wavelength) and three kinds of products marketed in Brazil (reference, generic and similar tablets) containing 1g of secnidazole. The method obeyed Beer’s law in the concentration range of 4 20 μgmL respectively. The method was validated according to the International Conference on Harmonization (ICH) and Brazil National Health Surveillance Agency (ANVISA) guidelines, showing accuracy, precision, selectivity, robustness and linearity. Tests such as weight range, friability, disintegration, hardness and dissolution were carried out to check tablets’ quality and all the trials showed to be in accordance with the general test guidelines of the Brazilian Pharmacopoeia. The dissolution test was carried out and the developed method was applied. The method developed is suitable for the estimation of secnidazole in tablets without any interference from the excipients and can be used for routine in quality control. Still, it's a simple, fast and low cost method.

In Brazil, the manufactured drugs are classified as reference drugs, generic and similar medicines (BERTOLDI; BARROS; HALLAL, 2005) and may have different excipients, since since they have to presente pharmaceutical bioequivalence.Due to this, it is important to have a method for determining secnidazole tablet wherein the excipients do not interfere with the analysis.As the secnidazole has no monograph for tablets in the official compendiums, the aim of this study was developed to validate and apply a method of UV spectrophotometry cheap, simple and useful for the quantitative determination of secnidazole in preparation of commercial pharmaceutical tablets.

Reagents and excipients
Secnidazole reference substance was supplied by Pharmanostra (China/Hong Kong) with assigned purity of 98,0%.Secnidazole reference product (Secnidal ® tablets, Sanofi-Aventis) and secnidazole generic tablets from two different brands (codified as product A and product B) all contained 1g of the active component were purchased from a local market.For ethical reasons, the brands of these products will not be reported.All studies were performed before the expiration of the validity period of the similar samples.

Identification
Fourier transform infrared spectroscopy (FTIR) was used to identify secnidazole in three different tablet formulations using an Affinity-1 Fourier Transform infrared spectrophotometer (Shimadzu ® ).Spectra were recorded at room temperature in the 3500-500 cm -1 range.After recording a background spectrum, potassium bromide pellets containing about 1-2% of secnidazole were prepared and immediately analyzed on FTIR spectrophotometer.In each sample, 20 scans were recorded with resolution of 4 cm -1 .

Weight variation
This test was performed according to the procedure described on general chapters of the Brazilian Pharmacopoeia.Twenty tablets of each pharmaceutical product (reference, A and B) were weighed individually using an analytical balance.The average weight, standard deviation and variation coefficient were then calculated.For tablets with average weight of 1g, the requirements of the Brazilian Pharmacopoeia are met if the weights of no more than two of the tablets differ from the average weight by more than 5,0 % and no tablet differs in weight by more than double that percentage (BRAZIL, 2010).

Friability
This test was performed according to the method described on general chapters of the Brazilian Pharmacopoeia by using a tablet friability apparatus (friabilator).Ten pre-weighed tablets of each pharmaceutical product (reference, A and B) were placed in the friabilator and then rotated at 25 rpm for 4 min.The tablets were accurately reweighed after removing any loss from the samples.The percentage of weight loss was calculated as follows: % Friability = (Loss in weight / Initial weight) × 100.The Brazilian Pharmacopoeia establishes a mean weight loss of not more than 1.5% (BRAZIL, 2010).

Disintegration test
The disintegration time of tablets was performed using a disintegration apparatus as specified in general chapters of the Brazilian Pharmacopoeia (2010).Six tablets of each pharmaceutical product (reference, A and B) were placed in each compartment of the disintegration apparatus basket and then acrylic disks were added.
The basket was attached to the device and subjected to vertical movements using water as immersion fluid at 37° C ± 2 ° C until complete disintegration of the tablets.The requirement of the Brazilian Pharmacopoeia is met if all tablets tested are completely disintegrated in less than 30 minutes (BRAZIL, 2010).

Hardness
The hardness was determined over 10 tablets as described in general chapters of the Brazilian Pharmacopoeia by using a hardness tester.A minimum hardness of 3 kgf was adopted as the acceptance criteria (BRAZIL, 2010).

Drug content
There isn't a method for determination secnidazole in tablets in main official compendiums.However, drug content was estimated using a spectrophotometric method which was properly developed and validated as recommended in international guidelines (ICH 2005).
Twenty tablets of each pharmaceutical product (reference, A and B) were totally powdered and the weight equivalent of the one average weight was transferred into a 100 mL volumetric flask, 40 mL of methanol was added, and the flask was sonicated for 5 min.The volume of the flask was made up with methanol obtaining the concentration of 200 µgmL -1 .From standard solutions, aliquots of 100 to 1000 µL were transferred to 10mL flasks and the volume completed with methanol.Samples were determined in spectrophotometer at a wavelength of 325 nm.
The adopted acceptance criterion was not lower than 80.0 percent and neither higher than 120.0 percent of the labeled amount (BRAZIL, 2003).

Dissolution test
In vitro dissolution test was carried out using a discriminative dissolution method described in general methods of the Brazilian Pharmacopeia (2010).The dissolution conditions were: hydrochloride acid 0,1M (900 mL) and a paddle stirring apparatus at rate of 100 rpm.
Ten milliliters of dissolution medium (controlled at 37.0 ± 0.5 °C) were sampled after 60 min, and the samples were analyzed by the spectrophotometric method.Six samples were assayed for each product.According to the Brazilian Pharmacopoeia (2010), the requirements are met in the first acceptance criteria if the quantities of active ingredient dissolved from each one of six dosage units is not less than Q + 5% and the requirements are met in the second acceptance criteria if the average of the quantities of active ingredient dissolved from twelve dosage units is equal to or greater than Q, and no unit is less than Q -15%.

Validation
The quantification method was validated according to the criteria established by the Resolution n° 899 of May 29 th , 2003, National  Health Surveillance Agency (BRAZIL, 2003), International Conference on Harmonization (ICH) (ICH 2005), through the parameters of selectivity, linearity, precision, accuracy, robustness, detection limit and quantification limit.

Specificity
Specificity was evaluated by analyzing solutions containing all the components of the different tablets, excepting the secnidazole.The excipients were prepared in methanol and subject to the some analytical conditions the same analytical conditions of the samples.The system response was examined for the presence of interference or overlaps with Secnidazole responders at 325nm.

Linearity
The analytical curves were obtained with eight concentrations of reference solution in the range of 4-20 µgmL -1 for the spectrophotometric method.All trials were was prepared in triplicate.The linearity was evaluated by linear regression analysis by the lest square regression method, which was used to calculate the correlation coefficient and slope of the regression line.

Precision
The precision was determined by repeatability (intra-day) and intermediate precision (inter-day).Repeatability was evaluated by analyzing secnidazole work standard solutions at the same concentration and during the same day.Intermediate precision was studied comparing the assay in three different days by two analysts, six replicates at a concentration of 12 µgmL -1 .The data were analyzed at 325 nm.The percentages of relative standard (% R.S.D.) of the analytical response were calculated.

Accuracy
The accuracy was determined by the recovery of a known amount of standard secnidazole solution added to samples of product reference, product A and product B. Different levels of secnidazole standard concentrations were used: lower concentration (4 µgmL -1 ), intermediate concentration (8 µgmL -1 ) and higher concentration (12 µgmL -1 ).The samples were prepared in triplicate, analyzed by the proposed method and the recovery percentages were determined to evaluate the accuracy.

Detection limit (LOD) and quantitation limit (LOQ)
The LOD and LOQ were calculated based on the standard deviation of the response and the slope using three independent analytical curves, as defined by ICH guidelines (ICH, 2005).It is always useful to demonstrate that the analyses are being conducted in a region which is above the LOQ value.It was obtained the LOD and LOQ form equations: LOD = 3 (SD/a); LOQ = 10 (SD/a), where SD is the standard deviation of the intercept with the axis in three calibration curves and a is the slope of the line.

Robustness
Robustness tests examine the effect operational parameters have on the analysis result.
For the determination of the method it was used reagents from different suppliers and operators.

RESULTS AND DISCUSSION
Secnidazole is soluble in water, but with dependent solubility according to pH, as it increases in media pH lower than 7, the solubility of the substance is increased (RIVERA et al, 2000).It was observed that until pH 2,0 the secondary nitrogen ring is protonated, and the molecule is more soluble.From pH 2,1 this nitrogen deprotonates and the solubility decreases (increase log D), and according to ChemSketch software (ACD labs®) the molecule of secnidazole has the same solubility from pH 4,4 to pH 12,8.The result is showed in Figure 2 and agree with the results obtained by Rivera (2000).

Identify
The FTIR results clearly showed that the absorptions bands of all samples are coincident with secnidazole reference standard.These results confirmed the unequivocal identification of C 7 H 11 N 3 0 3 on analyzed samples (Figure 3).

Weight variation
The Table 1 lists the individual weights of twenty tablets of each pharmaceutical product (reference, A and B) with their average weight, standard deviation and coefficient of variation.The Table 1 showed that all tested units are within the limit established by the Brazilian Pharmacopeia (2010).In addition, none tablet weighed more or less than double the established percentage of 5%.In addition, the coefficient of variation of all tested products was less than 2,5%.

Friability
The results of friability of the analyzed products are shown in Table 2.The results in Table 2 showed that all samples lost less than 0.5% weight when subjected to friability test.Therefore, all samples are within the limit permitted by pharmacopeia establishing a maximum of 1,5% (BRAZIL, 2010).

Disintegration time
The results of disintegration time of the analyzed products showed that the time for complete tablet disintegration in all cases was less than 5 minutes, in accordance with the Brazilian Pharmacopoeia that requires maximum time of thirty minutes for disintegration test (BRAZIL, 2010).

Hardness
The results of Hardness of the analyzed products are shown in Table 3.It is seen that all that all tested units showed hardness higher, therefore, all samples complied with Brazilian Pharmacopoeia requirements for this test that established a maximum of 3,0 kgf (BRAZIL, 2010).

Drug content
The results of drug content of the analyzed products are shown in Table 4.The label percentage claim found for the procedure is in accordance with Anvisa and the ICH which set the limit between 80 and 120% for drugs that are not described in monographs (BRAZIL, 2003;ICH, 2005).Rivera et al (2000) determined secnidazole in aqueous solutions in the range of 5,0 to 15µgmL -1 and Sonpetkar et al (2012) used the spectrophotometric method for estimation of secnidazole using as solvent a mixture water: methanol (30:70) with concentrations of 1,0 to 4,0µgmL -1 .When these procedures were taken, the precision and accuracy were lost, probably due to the excipients used in these formulations and the values of absorbance declined in ten minutes.Because of this, the use of methanol by itself was important to determinate secnidazole in these tablets.

CONCLUSIONS
The quality tests for tablets of the products reference, A and B were considered satisfactory in relation to their identification.average weight, friability, disintegration test, hardness.
A UV spectrophotometric method were developed to determine secnidazole in tablets.This method can be applied to the drug content of secnidazole tablets and dissolution test.
The proposed method is simple, quick, accurate, precise, robust and showed adequate detection and quantification limits.Moreover, the method can be easily applied in routine quality control laboratories.

Figure 2 .
Figure 2. Log D (log ρ) in function of pH calculated for secnidazole molecule using the software ChemSketch.

Figure 3 .
Figure 3. Identification of standard the standard secnidazole and products (reference, A and B).

Table 1 .
Individual weight, average weight, standard deviation and coefficient of variation of tablets claimed to contain 1g of secnidazole.

Table 2 .
Friability of tablets claimed to contain 1g of secnidazole.

Table 3 .
Hardness of tablets claimed to contain 1g of secnidazole

Table 4 .
Drug content of tablets claimed to contain 1g of secnidazole.

Table 5 .
Determination and validation of secnidazole… MARCÍLIO, M. R. et al.Dissolution test of product reference, A and B.

Table 6 .
Precision of the propose method for tablets secnidazole 1g.