2.4. General Procedures

A 10 mL aliquot of the drug solution containing 1.0–8.0 mg of ETM was placed in a 100 mL titration flask and acidified with 5 mL of 2 M H₂SO₄. Ten milliliters of 0.01 M cerium (IV) solution was pipetted into the flask and the contents were mixed well. After a standing time of 5 min, the residual oxidant was titrated with ferrous ammonium sulphate (FAS) solution using a drop of ferroin indicator. A blank titration was performed, and the amount in the aliquot was computed from the amount of cerium (IV) that reacted with ETM.

Different aliquots (0.0,0.25,0.5,…, 2.5 mL) of 20 μg/mL ETM solution were accurately transferred into a series of 10 mL calibrated flasks. To each flask 3 mL of 5 M H₂SO₄ was added, followed by 1 mL of 100 μg/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 H₂SO₄. 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.

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 H₂SO₄ was added, and the content was shaken for 20 min; the volume was diluted to the mark with 0.1 M H₂SO₄, 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 μg/mL level for assay by spectrophotometry.

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 “n” 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 ks/b , where k = 3 for LOD and 10 for LOQ, s is the standard deviation of seven blank absorbance readings, and b 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 H₂SO₄. 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.