Application of Cerium (IV) as an Oxidimetric Agent for the Determination of Ethionamide in Pharmaceutical Formulations

Two simple methods are described for the determination of ethionamide (ETM) in bulk drug and tablets using cerium (IV) sulphate as the oxidimetric agent. In both methods, the sample solution is treated with a measured excess of cerium (IV) solution in H2SO4 medium, and after a fixed standing time, the residual oxidant is determined either by back titration with standard iron (II) solution to a ferroin end point in titrimetry or by reacting with o-dianisidine followed by measurement of the absorbance of the orange-red coloured product at 470 nm in spectrophotometry. In titrimetry, the reaction proceeded with a stoichiometry of 1 : 2 (ETM : Ce (IV)) and the amount of cerium (IV) consumed by ETM was related to the latter's amount, and the method was applicable over 1.0–8.0 mg of drug. In spectrophotometry, Beer's law was obeyed over the concentration range of 0.5–5.0 μg/mL ETM with a molar absorptivity value of 2.66 × 104 L/(mol·cm). The limits of detection (LOD) and quantification (LOQ) calculated according to ICH guidelines were 0.013 and 0.043 μg/mL, respectively. The proposed titrimetric and spectrophotometric methods were found to yield reliable results when applied to bulk drug and tablets analysis, and hence they can be applied in quality control laboratories.


Introduction
Ethionamide (ETM), chemically known as 2-ethylthioisonicotinamide, is a second-line orally administered drug that is used for the treatment of multidrug resistant tuberculosis [1]. The drug has been in use since 1960s, because it is cheap, easily available, relatively nontoxic, and efficacious [2]. ETM is a structural analog of isoniazid [3,4] and is found to inhibit mycolic acid biosynthesis [5] with good bioavailability [6].
Employing AgS-ion-selective electrode as the sensor, Obtemperanskaya et al. [22] have reported a micro method by titration of the drug solution with 0.01 M AgNO 3 . The titrant used in the previously reported method [20] is unstable and requires daily standardization whereas the method employing membrane electrode [22] is tedious and time-consuming. It is desirable that the methods used in routine analysis should be simple and rapid with minimum experimental operations. Though ETM is prone to oxidation, a stable and strong oxidant such as cerium (IV) did not figure among the several titrimetric or spectrophotometric reagents that have been employed earlier for the assay of ETM. The reported spectrophotometric methods suffer from some disadvantages such as need for longer contact time, pH adjustment, multistep reactions, extraction step, and dependence on critical experimental variables.
In this paper, we describe two simple, rapid, and sensitive methods for the determination of ETM in pharmaceuticals using cerium (IV) as the oxidant. The methods are based on the oxidation of ETM by a measured excess of cerium (IV) in H 2 SO 4 medium followed by the determination of the unreacted oxidant either by titration with iron (II) visually (titrimetry) or by reacting it with ortho-dianisidine and measuring the absorbance of the orange-red coloured product at 470 nm (spectrophotometry). The two methods were found to be fairly accurate and precise in addition to being more sensitive compared to the previously reported methods.

Chemicals and Reagents.
All chemicals used were of analytical reagent grade. Double distilled water was used throughout the investigation.

Cerium (IV) Solution (0.01 M).
An approximately 0.01 M cerium (IV) solution was prepared by dissolving the required quantity of cerium (IV) sulphate (Loba Chemie, Mumbai, India) in 0.5 M H 2 SO 4 with the aid heat and filtered using glass wool; the solution was standardized [23] with pure ferrous ammonium sulphate (Loba Chemie, Mumbai, India) and used in titrimetry. The stock standard solution was diluted appropriately with 0.5 M H 2 SO 4 to get 100 g/mL cerium (IV) for use in spectrophotometry.
Ferrous Ammonium Sulphate, FAS (0.01 M). It is prepared by dissolving the calculated amount of the chemical in water in the presence of few drops of dilute H 2 SO 4 and standardized using pure potassium dichromate [23].
Sulphuric Acid (5 M). Concentrated acid (98%; sp. gr. 1.82, Merck, Mumbai, India) was diluted appropriately with water to get 5 M acid and used in spectrophotometry, and the same solution was diluted to 2 M level for use in titrimetry.

Spectrophotometric
Assay. Different aliquots (0.0, 0.25, 0.5, . . . , 2.5 mL) of 20 g/mL ETM solution were accurately transferred into a series of 10 mL calibrated flasks. To each flask 3 mL of 5 M H 2 SO 4 was added, followed by 1 mL of 100 g/mL Ce(IV) solution. The contents were mixed well and the flasks were set aside for 10 min. Finally, 1 mL of 0.05% ODS solution was added to each flask, and the volume was brought to the mark with 5 M H 2 SO 4 . The absorbance of each solution was measured after 5 min at 470 nm against a water blank.
A standard graph was prepared by plotting the difference between blank absorbance and sample absorbance as a function of concentration of the drug, and the concentration of the unknown was computed using the regression equation derived from the absorbance-concentration data.

Procedure for Tablets.
Twenty tablets were weighed accurately and ground into a fine powder. A portion of the powder equivalent to 100 mg of ETM was weighed accurately and transferred into a 100 mL calibrated flask, 60 mL of 0.1 M H 2 SO 4 was added, and the content was shaken for 20 min; the volume was diluted to the mark with 0.1 M H 2 SO 4 , mixed well, and filtered using Whatman 42 filter paper. The filtrate (1 mg/mL in ETM) was used in assay by titrimetry, and the same solution was diluted to 20 g/mL level for assay by spectrophotometry.

Procedures for Method Validation.
The assay validation procedures were carried out according to the current ICH guidelines [24], which include linear range, limits of detection (LOD) and quantification (LOQ), precision, accuracy, robustness, ruggedness, and selectivity.
(1) Linear Range, LOD, and LOQ. In titrimetry, the range was determined by titrating different amounts of drug under optimized conditions and the " " value (number of moles of cerium (IV) reacting with each mole of ETM) was calculated. In spectrophotometry, the linearity was assessed by the calibration graph, which was constructed by plotting the absorbance versus concentration of ETM and the regression equation was calculated. The LOD and LOQ were calculated using the relation / , where = 3 for LOD and 10 for LOQ, is the standard deviation of seven blank absorbance readings, and is the slope of the calibration curve [25].
(2) Accuracy and Precision. The accuracy of the proposed methods was determined on the basis of the difference in mean calculated and amount/concentration taken (% deviation from the actual concentration, DFA); and the precision was determined by calculating the intraday and interday relative standard deviation. These were computed by analyzing standard solution of ETM at three levels seven times on the same day (intraday) and on five consecutive days (interday).
(3) Robustness and Ruggedness. Robustness was evaluated by assaying the standard solutions after slight but deliberate variations in the analytical conditions like contact time and volume of H 2 SO 4 . Ruggedness, on the other hand, was assessed by a study in which the determination was performed by three analysts and also by a single analyst using three different burettes (titrimetry) and cuvettes (spectrophotometry).
(4) Selectivity. The placebo blank and synthetic mixture were analyzed by the developed methods and the results compared with those obtained on standard drug solution. A placebo blank of the composition: 20 mg talc, 30 mg starch, 20 mg sucrose, 20 mg lactose, 10 mg gelatin, 20 mg sodium alginate, 30 mg magnesium stearate, and 20 mg methyl cellulose was prepared by homogeneous mixing in a mortar. Ten milligrams of placebo was placed in a 50 mL calibration flask and its extract was prepared as described under Section 2.4.3. To 50 mg of the placebo blank prepared above, 100 mg of pure ETM was added and mixed thoroughly and the mixture was quantitatively transferred into a 100 mL calibrated flask; and then steps described under Section 2.4.3 were followed.
(5) Application to Tablets. Tablet solution prepared as described earlier was subjected to analysis by applying the developed procedures by taking 5 mL aliquot (titrimetry) and 3 mL aliquot (spectrophotometry) in five replicates, and the measured analytical signal was used to calculate the percent of the label claim. For comparison, the tablet extract in glacial acetic acid was titrated potentiometrically with acetous perchloric acid [7].
(6) Recovery Test. Preanalyzed tablet powder was spiked with pure drug at three levels and the total quantity of the drug was calculated, and finally the percent recovery of the pure drug added was calculated.

Results and Discussion
Cerium (IV) sulphate is a chemical compound which is frequently used as an oxidizing agent in titrimetric methods. The orange colour of cerium (IV) ion is reduced to the colourless cerium (III) ion.
Cerium (IV) is a powerful oxidizing agent which finds immense applications in the analysis of several pharmaceuticals [26][27][28][29][30][31][32]. This property of the oxidant was used in the present assay. The drug (ETM) was allowed to react with cerium (IV) in H 2 SO 4 medium and gets oxidizing to its sulphoxide.

S
After an appropriate reaction time, the residual oxidant was determined by two approaches. In titrimetry, the unreacted oxidant was determined by titration with FAS using ferroin indicator.
The amount of cerium (IV) reacted was related to the amount of drug, and the drug-oxidant reaction followed a 1 : 2 stoichiometry which served as the basis of the calculations. In spectrophotometry, the unconsumed oxidant was determined by reacting with ODS as shown in (4)

ODS
Coloured species  The calibration graph is a plot of the difference in absorbance of the reagent blank and sample solution versus the concentration of ETM (Figure 2), and this served as basis for the quantification. The possible reaction pathways and basis of assays are shown in Scheme 1.
In spectrophotometric method, three blanks were prepared. The first blank which contained all reactants except ETM gave maximum absorbance. The second blank contained only Ce (IV) and H 2 SO 4 . The third blank contained optimum amounts of ODS and acid. Since the last two blanks had negligible absorbance at 470 nm, measurements were made against double distilled water. A contact time of 5 min was found optimum for the range (1-8 mg) studied with 0.01 M cerium (IV) solution. A fixed reaction stoichiometry of 1 : 2 (drug : oxidant) was found for the investigated range of ETM. Beyond these limits (<1 and >8 mg), slightly inconsistent reaction ratios were obtained.
The ability of cerium (IV) to oxidize ETM and also ODS to an orange-red coloured product was exploited for the indirect 0.1591 Standard deviation of ( ) 9 . 8 9 × 10 −2 Standard deviation of ( ) 2 . 1 8 × 10 −2 Regression coefficient ( ) 0.9989 * Limit of determination as the weight in g/mL of solution, which corresponds to an absorbance of = 0.001 measured in a cuvette of cross-sectional area 1 cm 2 and = 1 cm. * * = + , where is the absorbance, is the concentration in g/mL, is intercept, and is slope. spectrophotometric assay. A slightly higher concentration of H 2 SO 4 was required for the twin oxidation steps involved, and to stabilize the coloured product. The oxidation of drug took somewhat a longer time (10 min) as compared to titrimetry (5 min), and a further 5 min was required to stabilize the oxidation product of ODS, which was stable for the next 30 min thereafter.

Linearity, LOD, and LOQ of Spectrophotometric Method.
The absorbance-concentration plot was linear with a good correlation coefficient (0.9989) in the 0.5-5.0 g/mL range. Sensitivity parameters such as molar absorptivity ( ), Sandell's sensitivity, LOD, and LOQ along with the slope and intercept of the regression equation are compiled in Table 1. Low values of LOD and LOQ and high value of ( ) confirm the sensitivity of the method for the determination of ETM in bulk drug as well in drug product.  relative error (% RE), an indicator of accuracy values, were calculated to be ≤2% (intraday) and <2.1% (interday), as shown in Table 2.

Robustness and Ruggedness.
To evaluate the robustness, two experimental variables, namely, contact time and acid concentration, were altered slightly deliberately, and the influence of these changes was studied on the performance of the methods. The performance remained unaffected as shown by small values of % RSD (≤2.31). Determination of drug in solution at three levels was done by using three different burettes in titrimetric method and three cuvettes in spectrophotometric method and also by three persons using the same equipment. The person-to-person and equipmentto-equipment variations did not significantly affect the results as shown in Table 3.

Selectivity.
To determine the selectivity of the described methods, placebo and synthetic mixture analyses were performed. Replicate analyses of placebo blank gave a titer value almost equal to that blank titration in titrimetry and absorbance value very much the same as the reagent blank in spectrophotometry. When the synthetic mixture was subjected to analysis, at three amount/concentration levels by the proposed methods, the percent recoveries of pure drug ranged from 99.34 ± 1.12 to 101.7 ± 2.34 indicating noninterference from the inactive ingredients.

Application to Tablets.
Three brands of tablets of 250 mg strength were analyzed by the proposed methods and the results are presented in Table 4. The same tablets were also analyzed by the reference method [7] for comparison. The results revealed that there is a close agreement between the results obtained by the proposed methods and those of the reference method, besides the label claim. When the results were statistically evaluated by applying Student'stest for accuracy and variance ratio -test for precision, the calculated -and -values did not exceed the tabulated values at the 95% confidence level and four degrees of freedom, suggesting that the proposed methods and the reference method have similar accuracy and precision.
3.2.6. Accuracy by Recovery Study. Accuracy of the proposed methods was further confirmed by recovery study following the standard-addition procedure. The percent recovery values of pure drug added shown in Table 5 unambiguously demonstrate that inactive ingredients such as talc, gelatin, starch, In titrimetry, ETM taken/found is in mg and the same was in g/mL in spectrophotometry. * Contact time used: 4, 5, and 6 min in titrimetric method; 8, 10, and 12 min in spectrophotometric method. * * Volumes of H 2 SO 4 were 4, 5, and 6 mL (2 M) in titrimetric method and 2.5, 3.0, and 3.5 mL (5 M) in spectrophotometric method. (Tabulated -value at the 95% confidence level and for four degrees of freedom is 2.78).
(Tabulated -value at the 95% confidence level and for four degrees of freedom is 6.39).

Conclusions
The oxidation reaction between the ETM and cerium (IV) in acid medium was advantageously exploited for the development of two simple, rapid, cost-effective, and sensitive methods for the determination of ETM in pharmaceuticals. The methods use cheap and easily available chemicals and an inexpensive instrument which can be accessed in any industrial quality control laboratory. The methods employ a stable oxidant unlike the previously reported titrimetric and spectrophotometric methods. Titrimetry is applicable over a micro scale (<10 mg) compared to the reported titrimetric methods including the official method, which would require 300-500 mg per trial. The proposed spectrophotometric method has a molar absorptivity value of 2.66 × 10 4 L/(mol⋅cm) with a linear dynamic range of 0.5-5.0 g/mL and is one of the most sensitive methods ever developed for ETM (Table 6). Hence, the proposed methods can be Journal of Pharmaceutics 7