Received Date: January 02, 2017; Accepted Date: February 28, 2017; Published Date: March 02, 2017
Citation: Debnath SK, Saisivam S, Debnath M (2017) Validated UV-Spectrophotometric Method for the Ethionamide Estimation in Bulk, Tablet and Nanoparticles. Int J Drug Dev & Res 9:20-23
The objective of present study was to develop a simple, specific and economic UV-spectrophotometric method for the quantitative determination of Ethionamide in bulk and in pharmaceutical formulations. Stock solution (1000 μg/ml) of Ethionamide was prepared in pH 7.4 phosphate buffer and sonicated for 20 min followed by determination of maximum absorbance. Stock solution (1000 μg/ml) was further diluted with pH 7.4 phosphate and obtained the standard curve. The methods were validated according to ICH guidelines of Q2B (2005) in terms of linearity, sensitivity, precision and accuracy for each analyte. Ethionamide shows the maximum absorbance at 288 nm in phosphate buffer (pH 7.4). I t follows Beer’s law in the concentration range of 6-18 μg/ml (r2=1). The detection limit (DL) and quantitation limit (QL) were 0.076 and 0.2301 μg/ml respectively. Accuracy and precision were found to be satisfactory. The developed methods were validated according to ICH guidelines. All the validation parameters were found to be satisfactory accordance with the standard values. The proposed method can be used for routine practice for the determination of Ethionamide in assay of bulk drug and pharmaceutical formulations.
Ethionamide; Method validation; Nanoparticles; PLGA
Tuberculosis continues to be a major worldwide epidemic with approximately one-third of the world population infected with Mycrobacterium tuberculosis . According to the WHO South-East Asia Region (SEAR) report in 2015, one fourth of the world population accounts for 38% morbidity and 39% mortality of the global burden of tuberculosis, with an estimated 4.5 million prevalent and 3.4 million incident cases and 440 000 deaths in 2013 . The lipid-rich mycobacterial cell wall is impermeable to many agents. Mycobacterial species are intracellular pathogens, and organisms residing within macrophages are inaccessible to drugs that penetrate these cells poorly. Finally, mycobacteria are notorious for their ability to develop resistance . This increasing drug resistance in tuberculosis represents a continuous challenge to the clinician and researcher since tuberculosis first detected . In the treatment of tuberculosis, first line drugs are normally used with large and repeated dose for several months. Due to patient’s non-compliance, these drugs fail- results drug resistance, multi drug resistance and extreme multi drug resistant. In that situation, 2nd line anti tubercular drug is used. Ethionamide (ETH) belongs to this category, has good clinical efficacy against Mycobacterium tuberculosis, but poor tolerable because of considerable gastrointestinal adverse effect, such as nausea, vomiting, anorexia, a metallic taste and abdominal pain. ETH is almost fully metabolized by the liver, being only approximately 5% of the antibiotic excreted in an unaltered form in the urine . Although it acts on similar fashion like Isoniazide, the side effects prohibit this drug from being used as a first line therapy . ETH is administered repeatedly due to its half-life of 2-3 hr . A drug and drug product are stable when its physical, chemical, therapeutics and toxicological properties are unchanged according to official monographs . Some HPLC estimation methods [1,9-11] and column chromatographic-mass spectrometric technique , Voltammetric determination , catalytic kinetic spectrophotometric method  are reported in the literature. But most of the cases either the method neither validated nor provide insufficient information for estimation of Ethionamide. Some of the cases used hazed chemicals.
So, efforts were made to develop an economical, simple, secure UV-spectrophotometric method using pH 7.4 Phosphate buffer. The developed method was validated and checked its suitability in the estimation of ETH entrapment in its nanoparticles form.
Ethionamide was procured from Shiro Pharma Chem. Pvt. Ltd., Navi Mumbai. Ethomid (Ethionamide tablet IP 250 mg, Macleods Pharmaceuticals Ltd.) was purchased from local market. PLGA (Resomer RG 755 S) was obtained as gift sample from Evonik Industries, Darmstadt, Germany. All other ingredients used were of AR grade. In every stage HPLC grade water was used.
The UV method was performed on SHIMADZU (Model: UV- 1800) double beam spectrophotometer with UV-probe software version 2.31. The absorption spectra of all the solution were carried out in the range of 200-400 nm.
.Preparation of stock solutions and test solutions and wavelength of maximum absorbance (λmax) determination
Stock solution (1000 μg/ml) of Ethionamide was prepared in pH 7.4 phosphate buffer and sonicated for 20 min. The absorption maximum was determine using different concentration solution by scanning at 200 to 400 nm in UV-Double been spectrophotometer (Table 1).
|Working λmax||288 nm|
|Beer’s law limit||6-18 µg/ml|
|Regression equation||y=0.052x+0.000 (with/without zero interpretation)|
|Regression coefficient (r2)||1|
|Slop of linear curve||0.052|
|SD of slope||0.0006|
|SD of intercept||0.0012|
Table 1: Summary of the UV method validation.
Preparation of working standard solution for calibration curve
The stock solution (1000 μg/ml) was further diluted with pH 7.4 phosphate. The diluted solution was vortexed and then used for further analysis. The calibration curve was plotted between concentrations and absorbances (Table 2).
|9||0.471 ± 0.001|
|12||0.629 ± 0.001|
|15||0.780 ± 0.001|
|18||0.937 ± 0.004|
Table 2: Linear curve of ethionamide at pH 7.4.
The methods were validated according to ICH guidelines of Q2B (2005) in terms of linearity, sensitivity, precision and accuracy for each analyte.
Linearity and range
Five point linear curve was prepared by plotting concentration (μg/ ml) vs absorbance and correlation co-efficient was calculated (Figure 1). The limit of detection (LOD) is defined as is the lowest concentration of a substance that an analytical process can reliably distinguished from the absence of that substance. The limit of quantification (LOQ) is defined as the lowest concentration of the standard curve that can be measured with acceptable accuracy, precision and variability (ICH guideline Q2B) (Table 1). The LOD and LOQ were also calculated as .
Analysis of tablet formulation
Twenty marketed tablets of Ethionamide (Ethomid, Macleods Pharmaceuticals Ltd.) were weighed and ground to fine powder; amount equal to 10 mg of Ethionamide was taken in 100 ml of volumetric flask and the volume was made up to mark with pH 7.4 phosphate buffer. The flask was sonicated farther for 20 min to solubilize the drug present in tablet powder. After sonication, filtration was done through Whatman Filter Paper No. 41. Filtrate was collected and further diluted with pH 7.4 phosphate buffer to get the final concentrations of both drugs in the working range. The absorbance of final diluted solution was observed at 288 nm (Table 3).
|Prepared conc.||Absorbance||Conc. recovered (μg/ml)||Dilution factor||Amount recovered in mg||% Label claim||Mean ± SD
SD Standard deviation, RSD Relative standard deviation, N=Average determination.
Table 3: Estimation of ethionamide.
Accuracy is the percent of recovered amount by assay from a known added amount. For the measurement of accuracy data from nine determinations over three concentration levels  was carried out as lower, intermediate and higher concentration from stock solutions and analyzed. The accuracy of the proposed methods was assessed by recovery studies at three different levels i.e., 80%, 100% and 120% . Result of recovery studies are reported in Table 4.
|Conc. Level||Sample No||Drug||Formulation||Amount Added (μg/ml)||Abs||Amt Recovered||% recovery||Mean
% Recovered± SD (N=3)
|80%||1||10ml of 15μg/ml||10 ml of 12μg/ml||13.5||0.7||13.46||99.72||99.76 ± 0.22||0.22|
|100%||1||10 ml of 15μg/ml||15||0.776||14.92||99.49||99.79 ± 0.27||0.27|
|120%||1||10 ml of 18μg/ml||16.5||0.858||16.50||100||99.85 ± 0.18||0.18|
Mean Recovery=99.80 ± 0.53
Table 4: Recovery studies on marketed formulations.
Repeatability was calculated by taking different levels of concentrations, prepared from pure drug stock solution and analyzed. Intermediate precision was calculated by taking the variations of interday and intra-day response. Respective concentrations from stock solution in triplicates were prepared three different times in a day and studied for intraday (n=9) and inter-day variation (n=15). The relative standard deviation (%RSD) of the estimated concentrations from the regression equation was taken as precision (Table 5).
|Intra Day||Inter Day|
|Repeatability ± SD (N=9)||% RSD||Repeatability ± SD (N=3)||% RSD||Repeatability ± SD (N=3)||% RSD||Repeatability ± SD (N=15)||% RSD|
Table 5: Results of validation (Mean ± SD).
Preparation of ethionamide nanoparticles
PLGA nanoparticles were prepared by solvent evaporation technique. PLGA incorporated dichloromethane solution of PTH was prepared and further transferred to 10 ml PVA solution for 10 min vortexing. A micro-emulsion was formed when sonicated for 5 min over the ice bath. The prepared emulsion kept on magnetic stirrer at room temperature for 3 hr to evaporate Dichloromethane. The nanoparticles were recovered by ultracentrifugation at 18000 rpm for 25 minutes followed by one wash with distilled water. Supernatant contained the un-entrapped ETH was estimated with this validated method and also checked the influences of other ingredients on the λmax.
UV-Spectrophotometric methods were developed for Ethionamide which can be conveniently employed for routine analysis in pharmaceutical dosage forms and will eliminate unnecessary tedious sample preparations. The standard error, standard deviation, and coefficient of variance were obtained for Ethionamide was satisfactorily low. Ethionamide shows λmax at 288 (Table 1). 5 Point calibration curve data was constructed in the range of 6 to 18 μg/ml, where the Beer’s law was obeyed (Table 2). The correlation co-efficient was found 1, which is within the limit (r2>0.990). The limit of detection (LOD) and limit of quantification (LOQ) for Ethionamide is found to be 0.076 μg/ml and 0.2301 μg/ml respectively, indicating that the proposed UV method is highly sensitive (Table 1). Assay value of Ethionamide in tablet formulations obtained as average of 99.47 ± 0.12 with relative standard deviation of 0.123% (Table 3). Assay values of formulation were same as label claim; this indicate that no interference of excipients in the determination of Ethionamide by the proposed method (Table 4). The standard deviation, coefficient of variance and standard error were obtained for Ethionamide was significantly low. Result of precision at different levels was found to be within acceptable limits (RSD<2) (Table 5). Indirect method was employed to estimate ETH entrapment. Percentage drug entrapment was found to be 88.80%, which also confirmed later on through direct method. Although different chemical was used in the formulation of ETH nanoparticles, no significant shifting of λmax was detected (Figure 2). So, this method can be used to estimate the ETH in bulk and pharmaceutical formulation without the influence of solvent effect.
The result and the validation parameters signify that the proposed method is simple, rapid, accurate and precise UV- spectrophotometric method without any hazard chemical. This method can be used for route analysis of Ethionamide either in bulk or in the pharmaceutical dosage forms even in the formulation of nanoparticles. There was no interference of commonly used chemicals or solvents in the estimation of Ethionamide.
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