International Journal of Drug Development and Research

Keywords

Ethionamide; Method validation; Nanoparticles; PLGA

Introduction

Tuberculosis continues to be a major worldwide epidemic with approximately one-third of the world population infected with Mycrobacterium tuberculosis [1]. 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 [2]. 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 [3]. This increasing drug resistance in tuberculosis represents a continuous challenge to the clinician and researcher since tuberculosis first detected [4]. 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 [5]. Although it acts on similar fashion like Isoniazide, the side effects prohibit this drug from being used as a first line therapy [6]. ETH is administered repeatedly due to its half-life of 2-3 hr [7]. A drug and drug product are stable when its physical, chemical, therapeutics and toxicological properties are unchanged according to official monographs [8]. Some HPLC estimation methods [1,9-11] and column chromatographic-mass spectrometric technique [7], Voltammetric determination [5], catalytic kinetic spectrophotometric method [12] 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.

Materials and Methods

Materials used

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.

Instrumentation

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).

Parameters	UV Method
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
LOD	0.076
LOQ	0.2301

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).

Concentration (µg/ml)	Absorbance
6	0.310 ±0.003
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.

Method validation

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 [13].

 

Figure 1: Calibration curve of ethionamide at pH 7.4.

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 (N=3)	RSD
8	0.413	7.942	1000	7.942	99.28	99.20±0.14	0.140
0.412	7.923	1000	7.923	99.04
0.413	7.9423	1000	7.942	99.28
12	0.621	11.942	1000	11.942	99.52	99.36±0.16	0.161
0.619	11.904	1000	11.904	99.20
0.62	11.923	1000	11.923	99.36
16	0.831	15.981	1000	15.981	99.88	99.84±0.07	0.070
0.831	15.981	1000	15.981	99.88
0.83	15.962	1000	15.962	99.76
Mean	99.47±0.12	0.123

SD Standard deviation, RSD Relative standard deviation, N=Average determination.

Table 3: Estimation of ethionamide.

Accuracy

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 [14] 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% [15]. 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)	% RSD
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
2	0.699	13.44	99.57
3	0.702	13.5o	100
100%	1	10 ml of 15μg/ml	15	0.776	14.92	99.49	99.79 ± 0.27	0.27
2	0.78	15	100
3	0.779	14.98	99.87
120%	1	10 ml of 18μg/ml	16.5	0.858	16.50	100	99.85 ± 0.18	0.18
2	0.855	16.44	99.65
3	0.857	16.48	99.88

Mean Recovery=99.80 ± 0.53

Table 4: Recovery studies on marketed formulations.

Precision

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).

Conc (μg/ml)	Intra Day	Inter Day
DAY-1	DAY-2	DAY-3
Repeatability ± SD (N=9)	% RSD	Repeatability ± SD (N=3)	% RSD	Repeatability ± SD (N=3)	% RSD	Repeatability ± SD (N=15)	% RSD
5	4.99±0.017	0.334	4.93±0.017	0.344	4.79±0.032	0.668	4.91±0.083	1.703
10	9.99±0.013	0.128	9.95±0.014	0.137	9.79±0.029	0.295	9.91±0.089	0.899
15	14.97±0.017	0.0167	14.88±0.029	0.194	14.75±0.055	0.371	14.87±0.099	0.666

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.

Results and Discussion

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.

 

Figure 2: λmax determination of ethionamide nanoparticles.

Conclusion

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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References

1. Vannelli TA, Dykman A, Montellano O, Paul R (2002) Enzyme catalysis and regulation: The antituberculosis drug ethionamide is activated by a flavoprotein monooxygenase. J BiolChem 277: 12824-12829.
2. World Health Organization (2015) Tuberculosis Control in the South-East Asia Region. Annual TB Report.
3. Desai D, Shah M (2015) A Review: Validated Analytical Methods Developed on Antitubercular Drug. Rifampicin 5: 254-265.
4. Medina G, Chapman PT (1965) Treatment with Ethionamide in various drug combination. Drug Resist PulmTuberc 47: 146-152.
5. Ferraz BR, Leite FR, Batista BL, Malagutti AR (2016) Voltammetric determination of ethionamide in pharmaceutical formulations and human urine using a boron-doped diamond electrode. Journal of the Brazilian Chemical Society 27: 677-684.
6. Hallbauer UM, Schaaf HS (2011) Ethionamide-induced hypothyroidism in children. South Afr J Epidemiol Infect 26:161-163.
7. Conte JE, Golden JA, Quitty MMC, Kipps J, Lin ET, et al. (2000) Effects of AIDS and gender on steady-state plasma and intrapulmonary Ethionamide concentrations. Antimicrob Agents Chemother44: 1337-1341.
8. Rehman M, Yousuf RI, Shoaib MH (2014)A stability-indicating high performance liquid chromatographic assay for the simultaneous determination of pyridoxine, ethionamide, and moxifloxacin in fixed dose combination tablets. Chromatography Research International,pp: 1-8.
9. Ramachandran G (2014) Simple and rapid high pressure liquid chromatography method for estimation of ethionamide in plasma 4: 1-5.
10. Ahmad M, Madni AU, Usman M (2009) In-vitro release and pharmacokinetics of anti-tubercle drug ethionamide in healthy male subjects. J Bioanal Biomed 1: 46-49.
11. Auclair B, Nix DE, Adam RD, James GT, Peloquin CA (2001) Pharmacokinetics of ethionamide administered under fasting conditions or with orange juice, food, or antacids. Antimicrob Agents Chemother 45: 810-814.
12. Walash MI, Metwally MS, Abdelal AA (2004) Spectrophotometric and kinetic determination of some sulphur containing drugs in bulk and drug formulations. Bull Korean ChemSoc 25: 517-524.
13. Debnath SK,Saisivam S, Dash DK, Debnath M (2015) Development and validation of UV-spectrophotometric methods for quantitative estimation of Prothionamide in pure and pharmaceutical dosage forms. IntCurr Pharm J 4: 402-404.
14. Kavathia A, Misra M (2013) Development and validation of RP-HPLC and UV-spectrophotometric methods for rapid simultaneous estimation of amlodipine and benazepril in pure and fixed dose combination. Arabian Journal of Chemistry,pp: 1-8.
15. Jain R, Jain N, Kumar D, Kumar V (2013) Novel UV spectrophotometer methods for quantitative estimation of metronidazole and furazolidone using mixed hydrotropysolubilization. Arabian Journal of Chemistry,pp: 1-6.