One titrimetric and two spectrophotometric methods are described for the determination of ketotifen fumarate (KTF) in bulk drug and in tablets using cerium(IV) as the oxidimetric agent. In titrimetry (method A), the drug was treated with a measured excess of cerium(IV) in H2SO4 medium and after a standing time of 10 min, the surplus oxidant was determined by back titration with iron(II). The spectrophotometric procedures involve addition of a known excess of cerium(IV) to KTF in acid medium followed by the determination of unreacted oxidant by reacting with either
Ketotifen fumarate (KTF) (Figure
Structure of KTF.
The drug is official in British Pharmacopoeia [
To the best of our knowledge, no visual titrimetric method has ever been reported for KTF. Visual titrimetry and visible spectrophotometry may serve as a useful alternatives to many of the aforesaid sophisticated techniques because of their cost-effectiveness, ease of operation, sensitivity, fair accuracy, precision, and wide applicability.
Several visible spectrophotometric methods based on colour reactions involving the amino or thiophene of the ketotifen molecule can be found in the literature. El-Kousy and Bebawy [
There are three reports on the use of ion-pair reaction for the assay of KTF. An extractive spectrophotometric procedure was based ion-pair reaction using Azocarmine G as ion-pair reagent at pH 1.5. The ion-pair complex extracted into CHCl3 was measured at 540 nm [
The reported spectrophotometric methods suffer from some other disadvantage such as narrow linear range, poor sensitivity, dependence on critical experimental variables, tedious and time-consuming extraction steps, heating step, and/or use of expensive reagent or large amounts of organic solvents as indicated in Table
Comparison of the performance characteristics of the proposed methods with the existing visible spectrophotometric methods.
Serial |
Reagent(s) used | Methodology |
|
Linear range (µg/mL) |
Remarks | Reference |
---|---|---|---|---|---|---|
1 | (a) Molybdenum thiocyanate |
Methylene chloride extractable ion-pair complex measured | 469.5 |
5–37.5 |
Employs unstable oxidant, narrow linear range | [ |
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2 | (a) F-C reagent |
Measurement of the absorbance of blue colored chromogen |
720 |
4–28 |
Multistep, unstable oxidant, strict pH control, and tedious extraction procedure | [ |
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3 | (a) Bromophenol blue |
Chloroform extractable 1 : 1 ion-pair complex was measured | — | 0 | Required close pH control and narrow linear range and involved tedious time consuming extraction steps | [ |
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4 | Bromocresol green | Chloroform extractable 1 : 1 ion-pair complex was measured | 423 | 5.15–61.91 | Required close pH control and narrow linear range and involved tedious time consuming extraction steps | [ |
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5 | Ce(IV)- |
Oxidation form of |
460 |
0.4–8.0 |
Simple, highly sensitive, extraction free, and use of stable cerium(IV) solution | This work |
Ce(IV)- |
Oxidation form of |
470 | 0.4–10 |
From the foregoing paragraphs, it is clear that cerium(IV) despite its strong oxidizing power, versatility, and high stability in solution has not been applied for the assay of ketotifen. This paper describes for the first time the application of acidic cerium(IV) to the titrimetric and spectrophotometric determination of KTF using
A Systronics model 106 digital spectrophotometer (Systronics, Ahmedabad, Gujarat, India) with 1 cm path length matched quartz cells was used to record the absorbance values.
All chemicals used were of analytical reagent grade. Double distilled water was used throughout the investigation. Pharmaceutical grade KTF (99.78 percent pure) was procured from Cipla India, Ltd., Mumbai, India, as a gift and used as received. Asthafen-1 (Torrent pharmaceuticals, Sikkim, India) and Ketasma-1 (Sun pharmaceuticals, Sikkim, India) tablets were purchased from local commercial market.
Cerium(IV)
A 10 mL aliquot of the KTF solution containing 2–18 mg of KTF was transferred into a 100 mL conical flask. To this, 10 mL of 0.005 M cerium(IV) sulphate was added using a pipette, and the contents were mixed well and the flask set aside for 10 min. Finally, the unreacted oxidant was titrated with 0.005 M FAS solution using one drop of ferroin indicator. Simultaneously, a blank titration was performed, and the amount of the drug in the measured aliquot was calculated from the amount of cerium(IV) reacted.
The amount of KTF in the aliquot was calculated using the formula
Different aliquots
Varying aliquots
A standard graph was prepared by plotting absorbance against concentration, and the unknown concentration was read from the graph or computed from the regression equation derived using Beer’s law data.
Two hundred tablets were weighed and finely powdered. An accurately weighed quantity of the tablet powder equivalent to 100 mg KTF was transferred into a 50 mL calibrated flask and about 30 mL water. The content of the flask was shaken for 20 min, and finally the volume was completed to the mark with water. The content was mixed well and filtered through a Whatman No. 42 filter paper. First, 5 mL portion of the filtrate was discarded, and a suitable aliquot of the filtrate (2 mg mL−1 KTF) was then subjected to titrimetric analysis (method A). This tablet extract was diluted stepwise to get 20
Tablet extract equivalent to 20
A placebo blank containing starch (30 mg), acacia (35 mg), hydroxyl cellulose (35 mg), sodium citrate (40 mg), talc (30 mg), magnesium stearate (45 mg), and sodium alginate (35 mg) was prepared by combining all components to form a homogeneous mixture. A 50 mg of the placebo blank was accurately weighed and its solution was prepared as described under “tablets” and then subjected to analysis by following the general procedure.
A synthetic mixture was prepared by adding an accurately weighed 100 mg of KTF to about 150 mg of the placebo mentioned above. The extraction procedure applied for tablets was applied to prepare 2 mg mL−1 KTF solution. This was diluted to get 20
KTF is reported to undergo oxidation with NBS [
KTF was found to react with Ce(IV) sulphate in sulphuric acid medium. The titrimetric method (method A) involves oxidation of KTF by a known excess of Ce(IV) in sulphuric acid medium with the formation of ketotifen sulphoxide and the unreacted oxidant was determined by back titration with FAS. H2SO4 medium was favored over HCl and HClO4 medium since the reaction was found to yield a regular stoichiometry in the concentration range studied. Reproducible and regular stoichiometry was obtained when 0.65–1.29 M H2SO4 concentration was maintained. Hence, 5 mL of 5 M H2SO4 solution in a total volume of 25 mL (1 M H2SO4 overall) was found to be the most suitable concentration for the quantitative reaction between KTF and Ce(IV). Under the optimized reaction condition, there was found to be a definite reaction stoichiometry of 1 : 2 between KTF and Ce(IV) within the range of 2–18 mg of KTF.
In spectrophotometry (method B), the unreacted Ce(IV) was treated with
In methods B and C, the increasing concentrations of drug added to a fixed concentration of Ce(IV) yield decreasing concentrations of Ce(IV), and this concomitant decrease in concentration results in decreasing absorbance values of its reaction product with
The addition of
Absorption spectra, method B, (a, b, c, and d are absorption spectra of blank, 2, 4, and 8, 20
In method C, the orange coloured product of Ce(IV) with
Absorption spectra, method C, (a, b, c, and d are absorption spectra of, blank, 2, 6, and 8
Perchloric acid (4 M) medium was found ideal for rapid and quantitative reaction between KTF and Ce(IV) and to obtain maximum and constant absorbance values due to Ce(IV)-
In method B, to fix the optimum concentration of Ce(IV), different concentrations of oxidant were reacted with a fixed concentration of
Under the described experimental conditions, the reaction between KTF and Ce(IV) was complete within 10 min at room temperature (
In method C, with fixed concentrations of KTF, Ce(IV), ODS, and acid, the absorbance was measured by different intervals of time which showed that reaction time is five minutes, and the coloured product is stable for 15 min.
In order to select proper solvent for dilution, different solvents were tried. The highest absorbance values were obtained when 4 M HClO4 was used as diluting solvent, and substitution of 4 M HClO4 by other solvents (methanol, water, 6 M HClO4) resulted in decrease in the absorbance values in method B. In method C,
The present methods were validated for linearity, selectivity, precision, accuracy, robustness, ruggedness, and recovery. Over the range investigated (2–18 mg), a fixed stoichiometry of 1 : 1 [KTF : Ce(IV)] was obtained in titrimetry which served as the basis for calculations. In spectrophotometry, the calibration graphs were found to be linear from 0.4–8.0
The calibration graphs are given by the equation
Sensitivity and regression parameters.
Parameter | Method B | Method C |
---|---|---|
|
460 | 470 |
Colour stability, min | 60 | 15 |
Linear range, |
0.4–8.0 | 0.4–10 |
Molar absorptivity ( |
4.0 × 104 | 3.7 × 104 |
Sandell sensitivity*, |
0.0105 | 0.0136 |
Limit of detection (LOD), |
0.39 | 0.43 |
Limit of quantification (LOQ), |
1.19 | 1.32 |
Regression equation, |
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Intercept ( |
0.8744 | 0.8082 |
Slope ( |
−0.0914 | −0.0815 |
Standard deviation of |
2.2 × 10−2 | 6.6 × 10−2 |
Standard deviation of |
4.9 × 10−3 | 7.3 × 10−3 |
Regression coefficient ( |
−0.9998 | −0.9990 |
The repeatability of the proposed methods was determined by performing five replicate determinations. The intra-day and inter-day variation in the analysis of KTF was measured at three different levels. The accuracy of an analytical method expresses the closeness between the reference value and the found value. Accuracy was evaluated as percentage relative error between the measured and taken amounts/concentrations. The results of this study are compiled in Table
Results of intraday and interday accuracy and precision study.
Method |
KTF taken |
Intraday accuracy and precision ( |
Interday accuracy and precision ( |
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KTF found |
RE % | RSD % | KTF found |
RE % | RSD % | ||
A | 5.00 | 5.06 | 1.20 | 1.80 | 5.12 | 2.42 | 2.90 |
10.0 | 10.12 | 1.62 | 1.40 | 10.15 | 1.51 | 2.43 | |
15.0 | 14.80 | 1.34 | 0.94 | 14.72 | 1.58 | 1.92 | |
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B | 2.0 | 2.02 | 1.23 | 1.72 | 2.03 | 1.62 | 1.18 |
4.0 | 3.96 | 1.62 | 0.87 | 3.94 | 1.26 | 1.58 | |
6.0 | 6.08 | 1.33 | 1.46 | 6.17 | 1.90 | 0.88 | |
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C | 4.0 | 3.94 | 0.73 | 1.43 | 3.95 | 1.14 | 2.12 |
6.0 | 6.06 | 1.06 | 1.09 | 6.11 | 1.84 | 1.79 | |
8.0 | 7.90 | 1.21 | 1.18 | 7.88 | 1.47 | 1.09 |
In method A, KTF taken/found, is in mg and it is µg mL−1 in method B and C.
RE: relative error (%); RSD: relative standard deviation (%).
To evaluate the robustness of the methods, two important experimental variables, namely, standing time and volume of H2SO4, were slightly varied, and the capacity of all the methods was found to remain unaffected by small deliberate variations. The results of this study are presented in Table
Method robustness and ruggedness expressed as intermediate precision.
Method |
KTF taken |
Robustness | Ruggedness | ||
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H2SO4 volume, mL |
Reaction time, min |
Ineranalysis (% RSD), |
Intercuvettes/burettes (% RSD), |
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A | 5.0 | 0.88 | 1.63 | 0.81 | 0.85 |
10.0 | 1.33 | 1.83 | 1.93 | 1.23 | |
15.0 | 1.03 | 0.59 | 0.89 | 0.96 | |
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B | 2.0 | 2.60 | 2.11 | 2.11 | 2.38 |
4.0 | 2.71 | 1.36 | 2.63 | 1.82 | |
6.0 | 1.79 | 1.80 | 1.58 | 2.83 | |
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C | 4.0 | 2.66 | 2.70 | 2.01 | 2.71 |
6.0 | 2.45 | 1.83 | 2.79 | 1.99 | |
8.0 | 1.72 | 1.27 | 1.69 | 2.96 |
In titrimetry, standing times were 8, 10, and 12 min.
In spectrometric methods B and C, reaction time, 10 ± 1 min; volume of H2SO4 2 ± 0.2 mL and 1 ± 0.2 mL varied, respectively.
In the analysis of placebo blank, there was no measurable consumption of Ce(IV) in titrimetry and the same absorbance value as obtained for the reagent blank was recorded in method B and method C, suggesting the noninterference by the inactive ingredients added to prepare the placebo.
In method A, 5 mL of the resulting solution prepared by using synthetic mixture was assayed titrimetrically
Commercial KTF tablets were analyzed using the developed methods and also a reference method [
Results of analysis of tablets by the proposed methods and statistical comparison with the reference method.
Tablets analysed | Label claim, mg/tablet | Found* (percent of label claim ± SD) | |||
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Reference method | Proposed methods | ||||
Method A | Method B | Method C | |||
Asthafen | 1 | 100.8 ± 0.91 | 101.4 ± 0.89 | 102.8 ± 1.91 | 102.1 ± 1.75 |
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Ketasma | 1 | 102.3 ± 1.02 | 100.6 ± 0.98 | 102.8 ± 1.36 | 101.3 ± 0.88 |
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Tabulated
Tabulated
To further ascertain the accuracy and reliability of the methods, recovery experiments were performed via standard-addition procedure. Preanalysed tablet powder was spiked with pure KTF at three different levels and the total was found by the proposed methods. Each determination was repeated three times. The percent recovery of pure KTF added (Table
Results of recovery study via standard addition method with tablet.
Method | Tablet studied | KTF in tablet |
Pure KTF added |
Total found |
Pure KTF* |
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A |
Ketasma-1 | 6.03 | 3.0 | 9.24 | 102.3 ± 1.98 |
6.03 | 6.0 | 12.22 | 101.6 ± 1.25 | ||
6.03 | 9.0 | 15.29 | 101.3 ± 2.08 | ||
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B |
Ketasma-1 | 3.03 | 1.5 | 4.54 | 101.1 ± 1.26 |
3.03 | 3.0 | 6.09 | 102.1 ± 1.98 | ||
3.03 | 4.5 | 7.59 | 101.4 ± 1.34 | ||
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C |
Ketasma-1 | 2.05 | 1.0 | 3.06 | 101. 1 ± 1.63 |
2.05 | 2.0 | 4.03 | 99.36 ± 1.28 | ||
2.05 | 3.0 | 5.12 | 102.4 ± 1.53 |
Three methods have been developed for determination of ketotifen fumarate in bulk drug and in its tablets and validated as per the current ICH guidelines. The present visual titrimetric method is simple and economical compared to the reported coulometric method [
The authors declare that they have no conflict of interests with the company name used in the paper.
The authors thank Cipla India Ltd., Bangalore, India, for providing pure ketotifen fumarate. The authors are grateful to the authorities of the University of Mysore, Mysore, India, for permission and facilities.