SECTION 7.0 DRUG PROFILE AND LITERATURE SURVEY...
Transcript of SECTION 7.0 DRUG PROFILE AND LITERATURE SURVEY...
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
368
SECTION 7.0
DRUG PROFILE AND LITERATURE SURVEY
7.0.1 DRUG PROFILE
Ketotifen fumarate (KTF) is chemically known as 4,9-Dihydro-4-(1-methyl-4-
piperidinylidene)-10 H-benzo[4,5]cyclohepta-[1,2-b] thiophen-10-one, belongs to the
group of cycloheptathiophenones [1]. Its molecular formula is C19H19NOS C4H4O4 and
molecular weight is 425.5 g mol-1. KTF has the following chemical structure: O
S
NCH3
HOOH
O
O
KTF is white amorphous solid powder soluble in water, hydrochloric acid,
sulphuric acid, acetonitrile, chloroform, dichloromethane and methanol. Ketotifen was
first synthesized in 1971 by Bourquin et al [1].
KTF is an antiallergic drug with stabilizing action on mast cells, analogous to that
of sodium cromoglycate, and anti-H1 effect [2]. Ketotifen is given orally as fumarate in
the prophylactic management of asthma and is used in the treatment of allergic conditions
such as rhinitis and conjunctivitis [3]. Ketotifen is a nonbronochodilator, antiallergic
properties and a specific anti-H1 effect [4].
The drug is official in British Pharmacopoeia [5], which describes a
potentiometric titration with perchloric acid in acetic anhydride-acetic acid medium.
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
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7.0.2 LITERATURE SURVEY OF ANALYTICAL METHODS FOR KETOTIFEN
FUMARATE
7.0.2.1 Titrimetric and UV-spectrophotometric methods
To the best of our knowledge, no visual titrimetric method has ever been reported
for KTF. Visual titrimetry and visible spectrophotometry may serve as an useful
alternatives to many sophisticated techniques because of their cost-effectiveness, ease of
operation, sensitivity, fair accuracy and precision and wide applicability. Potentiometric
titrations methods employing polymeric membrane sensors were also developed, where
the method involved potentiometric titration of the drug with sodium tetraphenylborate
[6, 7]. Coulometric titration of KTF in pure drug and in tablet forms using
biamperometric end-point detection was developed by Ciesielski [8].
Mohamed and Aboul-Enein [9] developed a UV-spectrophotometric method for
the determination KTF in bulk drug and capsules. The method was based on the
measurement of absorbance in acetate buffer (pH 5) at 300 nm. Beer’s law was obeyed
over a concentration range 2-300 µg mL-1. Another method involves the measurement of
absorbance of drug solution in 2 M HCl at 298 nm [10].
7.0.2.2 Visible spectrophotometric methods
Several visible spectrophotometric methods based on colour reactions involving
the amino or thiophene group of the ketotifen molecule can be found in the literature.
Kousy and Babawy [11] have developed three methods for the assay of KTF in bulk drug
and in tablets; the first method was based on extraction of drug-cobalt thiocyanate ternary
complex with methylene chloride and estimation by indirect atomic absorption method
via the determination of the cobalt content in the complex after extraction in 0.1 M
hydrochloric acid at 240.7 nm. The second method is based on the formation of orange
red ion-pair by the reaction of KTF and molybdenum thiocyanate with absorption
maxima at 469.5 nm in dichloromethane. Beer’s law was obeyed over a concentration
range of 5-35 g mL-1 KTF, whereas, the third method was a charge transfer reaction
between drug and 2,3 -dichloro-5,6 -dicyano-p-benzoquinone in acetonitrile medium and
the drug was quantified over a concentration range of 10-80 g mL-1. Sastry and Naidu
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
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[12] have determined the drug in tablets and syrup based on the reduction of Folin-
Ciocalteau reagent to a blue coloured chromogen measurable at 720 nm over a
concentration range of 4-28 g mL-1. In the same article, KTF was treated with a
measured excess of N-bromosuccimide (NBS), and the unreacted oxidant was reacted
with celestine blue, and the change in absorbance was measured at 540 nm. The third
method was based on the formation of colored species by the coupling of the diazotized
sulphanilamide, absorbance of the colored species was measured at 520 nm.
There are three reports on the use of ion-pair reaction for the assay of KTF. An
extractive spectrophotometric procedure 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 [12]. Sane et al., [13] have described four extractive spectrophotometric
procedures based on this reaction using, bromophenol blue, bromothymol green,
bromothymol blue and bromocresol purple as ion- pair reagents in acidic medium. The
drug has also been determined spectrophotometrically based on ion-pair complex
formation with bromocresol green [14] at pH 3.0 followed by extraction into chloroform
and measurement at 423 nm. Beer’s law is obeyed over the concentration range 5.15–
61.91 µg mL-1 KTF. The charge–transfer reaction of KTF with picric acid was developed
by Vachek [15]. The method involves extraction of the picric acid-drug complex into
chloroform and measurement of the extract at 405 nm.
7.0.2.3 Chromatographic methods
Many chromatographic methods were found in the literature for the assay of KTF
in pharmaceuticals. Bondesil CN (250mm ×4.6 mm) column consisting of CH3CN:
sodiumaacetate 0.01 M, pH 3.5 (75:25 v/v) as mobile phase at the flow rate of 2 mL min-1
and UV detection at 254 nm [16] was developed for the assay of KTF. Chen and Qu [17]
developed a HPLC method for the assay of KTF in tablets. The method was carried on a
column of KR100-5SIL (2.6 mm × 150 mm), with a mobile phase of ethanol-H3PO4-
ethylenediamine (50:50:0.02), at a flow rate of 0.2 mL min-1 and detection made at 302
nm. Nnane et al., [18] developed a method consists of a 5 µm Bondapak C18 column (30
cm × 3.9 mm i.d.) equipped with a 10 µm pellicular ODS guard column (5cm×2mm i.d.)
with 1mM-phosphate buffer of pH 7.4/methanol/acetonitrile/trimethylamine
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
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(29.8:45:25:0.2) as mobile phase (1 mL min-1) and detection at 299 nm. Another method
consists of a column (15 cm × 3.9 mm) of ODS (5 µm) with aq. 10% acetonitrile (1 mL
min−1) as mobile phase and detection at 220 nm. Calibration graphs were rectilinear up to
0.0358 mg mL−1 [19]. A method consisting of ion-pair extraction were analysed by
HPLC on a column (25 cm × 4 mm) packed with LiChrosorb-CN (10 µm) with various
mobile phases, chiefly hexane - CH2Cl2 - acetonitrile - propylamine (at various ratios)
and 254-nm detection [20]. . A RP-HPLC method on a Thermo C18 (250mm × 4.6 mm
id) column in isocratic mode with mobile phase of methanol:10 mM ammonium acetate
(30:70 %vol./vol. pH: 3.5) was used. The flow rate was 1mL min-1 and effluents were
monitored at 298nm [21]. Samreen [22] developed a RP-HPLC method and validated for
determination of KTF in a pharmaceutical formulation. The drug was chromatographed
on reversed-phase C18 column, using mixtures of phosphate buffer/acetonitrile. The
eluents were monitored at different wavelengths.
KTF was also assayed by HPTLC [23], LC/MS/MS [24] methods.
7.0.2.4 Other techniques
Ketotifen has been assayed by chemiluminescence spectrophotometry [25-
27].capillary electrophoresis [28], spectrofluorimetry [29], direct differential pulse
polarography and adsorptive-stripping-voltammetry [30]. Determination of KTF has
also been carried out based on
The literature survey presented in the foregoing paragraphs reveals that the ability
of KTF to undergo redox reaction has not been fully exploited for its titrimetric and
spectrophotometric assay. Based on these reactions, one titrimetric, four visible
spectrophotometric methods were developed and validated by the author. There is no
report on the stability-indicating UV-spectrophotometric method for the determination of
KTF, to fill this void; author developed two stability-indicating UV-spectrophotometric
methods. Besides, an UPLC method which is stability-indicating was also developed. The
details are presented in this Chapter (VII).
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
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SECTION 7.1
SENSITIVE AND ECO-FRIENDLY METHODS FOR THE ASSAY OF
KETOTIFEN USING CERIUM(IV) AND TWO REAGENTS
7.1.1 INTRODUCTION
The chemistry, importance and applications of Ce(IV) in pharmaceutical analysis
are described in Section 5.1.1.
From the literature presented in previous section, 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 Section describes for the first time, the application
of acidic cerium (IV) to the titrimetric (Method A) and spectrophotometric determination
of KTF using p- dimethyl amino benzaldehyde (Method B) and o- dianisidine (Method
C)as chromogenic agents.
7.1.2 EXPERIMENTAL
7.1.2.1 Apparatus
The instrument is the same that was described in Section 2.2.2.1.
7.1.2.2 Materials
All chemicals used were of analytical reagent grade. Double distilled water was
used throughout the investigation. Pharmaceutical grade KTF (99.78 % pure) was
procured from Cipla India, Ltd., Mumbai as a gift and used as received. Asthafen-
1(Torrent pharmaceuticals, Sikkim, India,) and Ketasama-1 (Sun pharmaceuticals,
Sikkim, India) tablets were purchased from local commercial market.
7.1.2.3 Reagents and solutions
Cerium(IV) sulphate (0.01 M) was prepared as described in Section 5.1.2.3.
It was used for titrimetric work and diluted to obtain working concentrations of
300 and 100 g mL-1 cerium(IV) for spectrophotometric methods B and C, respectively,
with the same solvent.
Perchloric acid (4M): Perchloric acid (4M) was prepared by diluting appropriate volume
of commercially available acid (70%; Merck, Mumbai, India) with water.
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Sulphuric acid (5M) and ferroin indicator were prepared as described under Section
5.1.2.3.
p-Dimethylamino benzaldehyde (p-DMAB) (0.5%):An accurately weighed 0.5 g of p-
DMAB (Merck, Mumbai, India) was transferred to a 100 mL volumetric flask, dissolved
in 4 M HClO4 and the volume was made upto the mark with the same solvent.
o-Dianisidine(ODS) (0.05%) :An accurately weighed 50 mg of o-dianisidine (Merck,
Mumbai, India) was transferred to a 100 mL volumetric flask, dissolved in ethanol and
the volume was made upto the mark with the same solvent.
Standard KTF solution
A stock standard solution equivalent to 2.0 mg mL-1 KTF was prepared by
dissolving 200 mg of pure drug with water in a 100 mL calibrated flask. This solution
was used in the titrimetric work and a working concentration of 20 µg mL-1 was prepared
by step wise dilution with water and used in method C. For method B, 200 µg mL-1 KTF
solution was prepared separately by dissolving accurately weighed 20 mg of pure drug in
4 M HClO4 and diluting to volume with the same solvent in a 100 mL standard flask, the
resulting solution (200 µg mL-1) was diluted to 20 µg mL-1 with 4 M HClO4 and used for
assay.
7.1.3 ASSAY PROCEDURES
Titrimetry (method A)
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 0.01 M cerium(IV) was added using a
pipette, the contents were mixed well and the flask set aside for 10 min. Finally, the
unreacted oxidant was titrated with 0.01 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,
Amount (mg) = VMwR/n
where, V= mL of Ce(IV) reacted with the drug.
Mw= relative molecular mass of KTF.
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
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C= molar concentration or strength of oxidant in mol L-1.
n= number of moles of Ce(IV) reacting with each mole of KTF.
Spectrophotometry using p-dimethylaminobenzaldehyde (method B)
Different aliquots (0.2, 0.5…….4 mL) of standard 20 µg mL-1 KTF solution in
4M HClO4 were transferred into a series of 10 mL calibrated flasks by means of a
microburette and the total volume in all the flasks was adjusted to 4 mL by adding 4 M
HClO4. To each flask, 2 mL of 5M H2SO4 and 1 mL of 300 µg mL-1 Ce(IV) solution
were added, and the content was mixed well. After a standing time of 10 min, 1 mL of
0.5% p-DMAB was added to each flask and the volume was made up to mark with 4 M
HClO4. After 5 min, the absorbance of the each coloured product was measured at 460
nm against water.
Spectrophotometry using o-dianisidine (method C)
Varying aliquots (0.20, 0.5, ……5.0 mL) of a standard 20 µg mL-1 KTF solution
were transferred into a series of 10 mL volumetric flasks using a microburette and the
total volume was brought to 5 mL by adding water. To each flask, were added 1 mL of
5M H2SO4 followed by 1 mL of 100 µg mL-1 Ce(IV) solution; and the content was
mixed well, kept aside for 10 min at ambient temperature. Finally, 1 mL of 0.05% o-
dianisidine was added to each flask, the volume was made up to mark with water, and
mixed well. The absorbance of each solution was measured at 470 nm against water
blank after 5 min.
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.
Procedure for tablets
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,
finally the volume was completed to the mark with same acid. The content was mixed
well, and filtered through a Whatman No. 42 filter paper. First 5 mL portion of the filtrate
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
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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 µg
mL-1 KTF with water, used in spectrophotometry (method C), by following the procedure
described earlier.
Tablet extract equivalent to 20 µg mL-1 KTF was prepared separately using 4 M
HClO4 and subjected to analysis in spectrophotometric method B by following the
general procedure.
Placebo blank synthetic mixture analyses
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 ‘Procedure for tablets’, and then subjected to analysis by
following the general procedure.
To about 150 mg of the placebo blank of the composition described above, 100
mg of KTF was added and homogenized, transferred to a 100 mL calibrated flask and the
solution was prepared as described under ‘Procedure for tablets’, and then subjected to
analysis by the procedure described above.
7.1.4 RESULTS AND DISCUSSION
The present work is based on the oxidation of the sulphur atom of the KTF
molecule by a measured excess of cerium (IV) in acid medium and the surplus oxidant
was determined by either titrimetry or spectrophotometry.
7.1.4.1Titrimetry
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 (Scheme 7.1.1) and the
unreacted oxidant was determined by back titration with FAS. H2SO4 medium was
favored over to HCl and HClO4 medium since the reaction was found to yield a regular
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
376
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.
H+Ce(IV)(Measured excess)+ Unreacted Ce(IV)
O
S
NCH3
+
O
S
NCH3
HO OHO
O
HO OHO
O
O
KTF + H+ Oxidation product of KTF
Ce(IV)(Known excess)
+ Unreacted Ce(IV)
Unreacted Ce(IV)
Titrated with standard FAS using ferroin indicator (method A)
Unreacted Ce(IV)
o-Dianisidinein ethanol
Orange coloured product measured at 470 nm (method C)
+Unreacted Ce(IV)
p-DMAB in HClO4 medium
Orange coloured product measured at 460 nm (method B)
+
Scheme 7.1.1 Tentative reaction pathway for reaction between KTF and Ce(IV) in acid medium.
7.1.4.2 Spectrophotometry
Absorption spectra
The addition of p-DMAB to Ce(IV) resulted in the formation of yellowish red
coloured product with maximum absorption at 460 nm against blank, the decrease in the
absorption intensity at 460 nm, caused by the presence of the drug, was directly
proportional to the amount of the drug reacted in method B shown in Figure 7.1.1. In
method C, the orange coloured product of Ce(IV) with o-dianisidine shows maximum
absorption at 470 nm against its corresponding reagent blank as shown in Figure
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
377
7.1.1,the decrease in the absorption intensity at 470 nm, caused by the presence of the
drug, was directly proportional to the amount of the drug .
Figure 7.1.1 Absorption spectra of: a, b,c and d (blank, 2, 4 and, 8 20 µg mL-1 KTF, respectively (Method B) e,f,g and h (blank, 2, 6 and 8 µg mL-1 KTF, respectively) (Method C).
The unreacted Ce(IV) was treated with p-DMAB in HClO4 medium to yield
formic acid and p-dimethylaminophenol, which upon further oxidation gave the
corresponding quinoimine derivative [31], measured at 460 nm and in method C, the
surplus oxidant was treated with o-dianisidine in acid medium to form the orange red
colour product and measured at 470 nm (Scheme 7.1.2 and 7.1.3).
In methods B and C, the increasing concentrations of drug added to a fixed
concentration of Ce(IV) yield decreasing concentrations of Ce(IV), this concomitant
decrease in concentration results in decreasing absorbance values of its reaction product
with p-dimethylaminobenzaldehyde in method B and with o-dianisidine in method C.
7.1.4.3 Method development
Selection of reaction medium
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)-p-DMAB reaction product at 460 nm. This may be attributed to the highest
340 360 380 400 420 440 460 480 500 520 540 5600.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Abso
rban
ce
Wavelength, nm
a b c d
360 400 440 480 520 560 600
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
e f g h
Abso
rban
ce
Wavelength, nm
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
378
oxidation potential of Ce(IV) in HClO4 (Eo = 1.75 V) as compared to that of Ce(IV) in
H2SO4 (Eo = 1.44 V), HNO3 (Eo = 1.61 V) or HCl (Eo = 1.28 V) [32]. Therefore, all the
solutions [ALB, Ce(IV) and p-DMAB] were prepared in 4 M perchloric acid through out
the investigation and the same was maintained as reaction medium in method B.
p-DMAB
O
N
H2O
N
OHHO
+Ce4+
-e-
N+
O
OH
H
+Ce4+
-e-
O
N
O
+H2OHCOOH
OH
N
2Ce4+
-2e-
O
N+
(measured at 460 nm)
+4Ce3+
Scheme 7.1.2 The possible reaction pathway for the oxidation of p-DMAB by Ce(IV) in
the presence of HClO4 .
H3CO
H2N NH2
OCH3 H3CO
HN NH
OCH3H+
+Unreacted Ce(IV)
o-dianisidine orange red colour Maximum absorption at 470 nm
Scheme 7.1.3 The possible reaction pathway for the oxidation of O-dianisidine by
Ce(IV).
Optimization of Ce(IV) for methods B and C
In method B, to fix the optimum concentration of Ce(IV), different concentrations
of oxidant were reacted with a fixed concentration of p-DMAB in HClO4 medium and the
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
379
absorbance measured at 460 nm. A constant and maximum absorbance resulted with 30
µg mL-1 Ce(IV), and hence, different concentrations of KTF were reacted with 1 mL of
300 µg mL-1 Ce(IV), in HClO4 medium before determining the residual Ce(IV), via the
reaction scheme illustrated earlier. This facilitated the optimization of the linear dynamic
range over which procedure could be applied for the assay of KTF. In method C, 10 µg
mL-1 Ce(IV) gave a constant and maximum absorbance with o-dianisidine at 470 nm,
hence, 1 mL of 100 µg mL-1 Ce(IV), was fixed as optimum.
Study of reaction time and stability of the coloured species
Under the described experimental conditions, the reaction between KTF and
Ce(IV), was complete within 10 min at room temperature (28 ± 2 °C). After the addition
of p-DMAB, a standing time of 5 min was necessary for the formation of coloured
product, and thereafter, the absorbance of the coloured product was stable for more than
an hour in method B. In method C, with fixed concentrations of KTF, Ce(IV), ODS and
acid, the absorbance was measured by different intervals of time, showed that reaction
time is five min, and the coloured product is stable for 15 min.
Effect of diluent
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,
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, o-dianisidine prepared in
ethanol and drug in water, water as diluent gave maximum absorbance.
7.1.4.5 Method validation
Linearity and sensitivity
The present methods were validated for linearity, selectivity, precision, accuracy,
robustness and ruggedness, and recovery. Over the range investigated (2-18 mg), a fixed
stoichiometry of 1:2 [KTF:Ce(IV)] was obtained in titrimetry which served as the basis
for calculations. In spectrophotometry, the calibration graphs (Figure 7.1.2) were found
to be linear from 0.4–8.0 µg mL-1 and 0.4-10.0 µg mL-1 KTF in method B and method C,
respectively, in the inverse manner. Sensitivity parameters such as molar absorptivity and
Sandell sensitivity values and the limits of detection (LOD) and quantification (LOQ)
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
380
were calculated according to the ICH guidelines [33]. These are summarized in Table
7.1.1.
Method B Method C
Figure 7.1.2 Calibration curves
Table 7.1.1 Sensitivity and regression parameters Parameter Method B Method C max, nm 460 470 Colour stability, min. 60 15 Linear range, µg mL-1 0.4–8.0 0.4–10 Molar absorptivity(ε), L mol-1 cm-1 4.0 x 104 3.7 x 104
Sandell sensitivity*, µg cm-2 0.0105 0.0136 Limit of detection (LOD), g mL-1 0.39 0.43 Limit of quantification (LOQ), g mL-1 1.19 1.32 Regression equation, Y**
Intercept (a) 0.8744 0.8082 Slope (b) -0.0914 -0.0815 Standard deviation of a (Sa) 2.2 x 10-2 6.6 x 10-2 Standard deviation of b (Sb) 4.9 x 10-3 7.3 x 10-3 Regression coefficient (r) -0.9998 -0.9990 *Limit of determination as the weight in µg per mL of solution, which corresponds to an absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. **Y=a+bX, Where Y is the absorbance, X is concentration in µg mL-1, a is intercept and b is slope.
Accuracy and precision
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
0 2 4 6 80.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Abs
orba
nce
Concentration of KTF, µg mL-1
0 2 4 6 8 100.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Abso
rban
ce
Concentration of KTF, µg mL-1
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
381
percentage relative error between the measured and taken amounts/concentrations. The
results of this study are compiled in Table 7.1.2 and speak of the excellent intermediate
precision (%RSD ≤ 2.90) and accuracy (%RE ≤ 2.42) of the results.
Table 7.1.2 Results of intra-day and inter-day accuracy and precision study
Method*
KTF Taken mg/
µg mL-1
Intra-day accuracy and precision (n=7)
Inter-day accuracy and precision (n=7)
KTF found mg/
µg mL-1 RE% RSD%
KTF found
mg/ µg mL-1
RE % RSD %
A 5.00 10.0 15.0
5.06 10.12 14.80
1.20 1.62 1.34
1.80 1.40 0.94
5.12 10.15 14.72
2.42 1.51 1.58
2.90 2.43 1.92
B 2.0 4.0 6.0
2.02 3.96 6.08
1.23 1.62 1.33
1.72 0.87 1.46
2.03 3.94 6.17
1.62 1.26 1.90
1.18 1.58 0.88
C 4.0 6.0 8.0
3.94 6.06 7.90
0.73 1.06 1.21
1.43 1.09 1.18
3.95 6.11 7.88
1.14 1.84 1.47
2.12 1.79 1.09
In method A, KTF taken/found, are in mg and they are µg mL-1 in method B and C. RE-Relative error (%); RSD-.Relative standard deviation (%).
Robustness and ruggedness
To evaluate the robustness of the methods, two important experimental variables,
viz., 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 7.1.3 and indicate that the proposed methods are robust.
Method ruggedness was demonstrated having the analysis done by four analysts, and also
by a single analyst performing analysis on four different apparatus or instruments in the
same laboratory. Intermediate precision values (%RSD) in both instances were in the
range 0.85–2.96% indicating acceptable ruggedness. The results are presented in Table
7.1.3.
Selectivity
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
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
382
recorded in method B and method C, suggesting the non-interference 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 (n = 5), and yielded a % recovery of 102.3 ± 0.62 KTF. In
spectrophotometry, 2 mL of 20 µg mL-1 KTF in method B and 3 mL of 20 µg mL-1 KTF
in method C when subjected to analysis (n = 5) yielded % recoveries of 98.24 and
101.8% KTF with standard deviations of 1.74 and 2.32, respectively. These results
complement the findings of the placebo blank analysis with respect to selectivity.
Table 7.1.3 Results robustness and ruggedness expressed as intermediate precision (%RSD)
Method
KTF taken mg/
µg mL-1
Robustness Ruggedness
Iner-analysis (% RSD),
n=4
Inter-cuvettes/ burettes
(% RSD), n=4
H2SO4 volume,
mLa (%RSD),
n=3
Reaction Time, b
min (%RSD),
n=3
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
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
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
Application to tablets
Commercial KTF tablets were analyzed using the developed methods and also a
reference method [5]. The reference method involves potentiometric titration of KTF in
anhydrous acetic anhydride - acetic acid medium with acetous perchloric acid. The
results obtained were compared statistically by the Student’s t-test and the variance-ratio
F-test. The calculated t- and F- values did not exceed the tabulated values of 2.78 and
6.39 at the 95 % confidence level and for four degrees of freedom, indicating close
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
383
similarity between the proposed methods and the reference method with respect to
accuracy and precision. These results are summarized in Table 7.1.4.
Table 7.1.4 Results of analysis of tablets by the proposed methods and comparison with the official method
Tablets analysed
Label claim,
mg/tablet
Found* (Percent of label claim ± SD) Reference
method Proposed methods
Method A Method B Method C
aAsthafen 1 100.8 ± 0.91 101.4 ± 0.89 102.8 ± 1.91 102.1 ± 1.75
t= 1.05 t= 2.11 t= 2.16 F= 1.05 F= 4.41 F= 3.7
bKetasma 1 102.3 ± 1.02 100.6 ± 0.98 102.8 ± 1.36 101.3 ± 0.88
t= 2.69 t= 0.66 t= 1.66 F= 1.08 F= 1.78 F= 1.34
*Mean value of five determinations; aTorrent pharmaceuticals, Sikkim, India,b Sun pharmaceuticals, Sikkim, India. Tabulated t-value at the 95% confidence level is 2.78; Tabulated F-value at the 95% confidence level is 6.39.
Recovery studies
To further ascertain the accuracy and reliability of the methods, recovery
experiments were performed via standard-addition procedure. Pre-analysed 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 7.1.5) was within the permissible limits indicating the absence of
inactive ingredients in the assay.
Table 7.1.5 Results of recovery study via standard addition method Method Tablet
studied KTF in tablet
mg/ µg mL-1
Pure KTF added mg/
µg mL-1
Total found mg/
µg mL-1
Pure KTF * Percent ± SD
A
6.03 3.0 9.24 102.3 ± 1.98 Ketasma-1 6.03 6.0 12.22 101.6 ±1.25
6.03 9.0 15.29 101.3 ± 2.08
B
3.03 1.5 4.54 101.1 ± 1.26 Ketasma-1 3.03 3.0 6.09 102.1 ± 1.98
3.03 4.5 7.59 101.4 ± 1.34
C
2.05 1.0 3.06 101. 1 ± 1.63 Ketasma-1 2.05 2.0 4.03 99.36 ± 1.28
2.05 3.0 5.12 102.4± 1.53 * mg in method A; µg mL-1 in methods B and C, * Mean value of three determinations
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
384
SECTION 7.2
SPECTROPHOTOMETRIC DETERMINATION OF KETOTIFEN FUMARATE
IN PHARMACEUTICALS USING IRON(III)CHLORIDE AND TWO
CHELATING AGENTS
7.2.1 INTRODUCTION
Iron(III) chloride, also called ferric chloride, is an industrial scale commodity
chemical compound, with the formula FeCl3. Ferric chloride is a mild oxidising agent.
The standard reduction potential for the reduction of Fe3+ (aq) to Fe2+ (aq) under standard
acidic condition is 0.771 V.
Many pharmaceutical compounds like raloxifene hydrochloride [34], antibacterial
drugs such as norfloxacin, ciprofloxacin, cinoxacin and nalidixic acid [35], piperazine
derivatives, namely ketoconazole, trimetazidine hydrochloride and piribedil [36],
nitrofurazone [37], nelfinavir mesylate [38], cefuroxime sodium [39], oxcarbazepine
[40], cephalexin [41], etamsylate [42, 43], furosemide [44], pentaprazole [45] etc. have
been determined using ferric chloride as oxidizing agent.
A scan of the literature survey presented in Section 7.0.2.0 and the above
paragraph reveals that ferric chloride has not been applied to the determination for KTF.
Utilizing the oxidizing property of ferric chloride, the author has developed two visible
spectrophotometric methods, which are convenient for the determination of KTF in pure
form and in dosage forms. The procedures involved oxidation of KTF with
iron(III)chloride and determining the resuting iron (II) by complexing with either 1, 10-
phenanthroline (method A) or 2, 2΄-bipyridyl (method B). The complex formation of Fe2+
with 1, 10-phenanthroline [46] and 2, 2΄-bipyridyl [47-49] has long been recognized. The
proposed methods are characterized by simplicity, sensitivity, wide linear dynamic
ranges, mild experimental conditions and above all cost-effectiveness.
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
385
7.2.2 EXPERIMENTAL
7.2.2.1 Materials All the reagents used were of analytical-reagent grade and distilled water was
used throughout the investigation. The pure KTF and its tablets and injection used were
the same described in Section 7.1.
7.2.2.2 Reagents and solutions
Ferric chloride (hydrated, 3mM): The aqueous solution of 0.05 M ferric chloride (S.D.
Fine Chem., Mumbai, India) was prepared by dissolving 1.35 g of the chemical in 100
mL of distilled water and stored in a dark bottle. The stock solution was then diluted
appropriately with distilled water to get 3mM working concentration for both methods.
The solution was prepared afresh just before the experiment.
1,10-phenanthroline (phen) (0.01 M):The solution was prepared by dissolving 198 mg
of the chemical (Qualigens Fine Chemicals, Mumbai,India, assay 100%) in distilled
water and diluted to 100 mL with distilled water.
2,2’-Bipyridyl (bipy) (0.01 M):The solution was prepared by dissolving 156 mg of the
chemical (Qualigens Fine Chemicals, Mumbai,India, assay 100%) in distilled water and
diluted to volume in a100 mL calibrated flask.
Orthophosphoric acid (0.02 M): Concentrated acid (Merck, Mumbai, India, sp. gr.
1.75) was appropriately diluted with distilled water to get the required concentration.
Stock standard solution: A stock solution equivalent to 100 µg mL-1 of KTF was
prepared by dissolving accurately weighed 10 mg of pure drug in water and diluting to
the mark in a 100 mL calibrated flask. This was diluted appropriately with water to get
working concentration 20 and 50 µg mL-1 KTF for use in method and method B,
respectively.
7.2.2.3 ASSAY PROCEDURES
Method A (Using 1, 10-Phenanthroline)
Different aliquots (0.2, 0.5, 1.0, 2.0, 3.0 and 4.0 mL) of the standard 20 µg mL-1
KTF solution were accurately measured and transferred into a series of 10 mL calibrated
flasks and the total volume was adjusted to 4 mL by adding water. To each flask, 2 mL of
a ferric chloride (3 mM) and 2 mL of 0.01 M 1,10-phenanthroline were added, followed
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
386
by 1 mL of orthophosphoric acid (0.02 M), and the volume was brought to 10 mL with
distilled water. The flasks were stoppered, the content mixed well and the flasks were let
stand for 10 min with occasional shaking. Then, the absorbance of each solution was
measured at 510 nm against the reagent blank.
Method B (Using 2, 2΄-Bipyridyl)
Varying aliquots (0.2, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mL) of the standard KTF
solution (50 µg mL-1) were accurately measured into a series of 10 mL calibrated flasks
by means of a micro-burette and the total volume was brought to 5 mL by adding water.
To each flask 2 mL of 3 mM ferric chloride and 2 mL of 0.01 M 2,2’-bipyridyl were
added followed by 1 mL of orthophosphoric acid (0.02 M). The content was mixed well
and diluted to the mark with distilled water. The absorbance of each solution was
measured at 520 nm against reagent blank after 10 min.
A standard graph was prepared by plotting the increasing absorbance values
versus concentration of KTF (µg mL-1). The concentration of the unknown was read from
the standard graph or computed from the respective regression equation derived using the
Beer’s law data.
Procedure for tablets
An amount of finely ground tablet powder equivalent to 10 mg of KTF was
accurately weighed and transferred into a 100 mL calibrated flask, 60 mL of water was
added and the content shaken thoroughly for 15-20 min to extract the drug into the liquid
phase; the volume was finally diluted to the mark with water, mixed well and filtered
using a Whatman No. 42 filter paper. First 10 mL of the filtrate was discarded and a
suitable aliquot of the filtrate (100 µg mL-1KTF) was diluted stepwise with water to get
20 and 50 µg mL-1concentrations for method A and method B, respectively.
Placebo blank and synthetic mixture analyses
Fifty mg of the placebo blank prepared in Section 7.1.2.4 was taken and its
solution prepared as described under ‘Procedure for tablets’ and then analyzed using the
procedures described above.
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
387
To 20 mg of the placebo blank, 10 mg of KTF was added and homogenized,
transferred to 100 mL volumetric flask and the solution was prepared as described under
“Procedure for tablets”. A convenient aliquot was diluted and then subjected to analysis
by the procedures described above.
7.2.3 RESULT AND DISCUSSION
KTF contains a sulphur group, which could be oxidized to sulfoxide group and
the drug was found to undergo oxidation with FeCl3 in neutral medium. The complex
formation of Fe2+ with 1, 10- phenanthroline and 2, 2΄-bipyridyl has long been
recognized. The proposed methods involved two steps:
The first step is the oxidation of KTF with excess FeCl3 in neutral medium, and
the second step is the determination of the resulting Fe2+ by subsequent chelation with
either phen or bipy and measuring the absorbance at the respective wavelength (Figure
7.2.1). The probable reaction mechanism is shown in Scheme 7.2.1.The amount of Fe2+
formed was found to be proportional to the amount of KTF serving as basis for its
quantification. KTF, when added in increasing concentrations, consumes Fe3+, and
consequently there will be concomitant increase in Fe2+ concentration. This is observed as
a proportional increase in the absorbance of the coloured species with increasing
concentration of KTF and fixed concentration of reagent. The increasing absorbance
values at 510 nm in method A and at 520 nm in method B were plotted against the
concentration of KTF to obtain the calibration graph.
Figure 7.2.1 Absorption spectra of:
a-Fe(II)-phen complex, b-blank (Method A) c-Fe(II)-bipy complex and d-blank (Method B)
400 450 500 550 6000.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
Abso
rban
ce
Wavelength, nm
a b c d
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
388
Fe2+
+
N
N
N
N
Fe2+
Ferroin (orange-red colour)measured at 510 nm.
N
N
+N
N
Fe2+
Method A
Method B
Fe(II)-2,2'-bipyridyl chelate (red colour)measured at 520 nm.2,2'-Bipyridyl
1,10-Phenanthroline
3
3
3
3
Excess of Fe3++
O
S
NCH3
+
O
S
NCH3
HO OH
O
O
HO OH
O
O
O
Fe2+
KTF Oxidation product of KTF
Scheme 7.2.1 Possible reaction scheme for the proposed methods
7.2.3.1 Optimization of the reaction conditions
Investigations were carried out to achieve maximum color development in the
quantitative determination of KTF. When the Fe3+ concentration was increased, the
absorbance value of reagent blank was found to increase. Hence, by considering the
sensitivity of the reaction with a minimum blank absorbance, 2 mL of 3 mM ferric
chloride in a total volume of 10 mL were found optimum in both methods. Even though,
oxidation of KTF by Fe3+ and subsequent chelation of Fe2+ with either phen or bipy was
found to occur in neutral medium, the presence of o-phosphoric acid was necessary to
increase the stability of the developed red color chelate by maintaining the desired pH. A
1 mL of 0.02 M O-phosphoric acid in a total volume of 10 mL was found adequate in
both the methods. Several experiments were carried out to study the effect of phen and
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
389
bipy concentrations on the color development. In order to determine the optimum
concentration of Phen, different volumes (0.5-2.5 mL) of 0.01 M Phen solution were used
with a fixed concentration of KTF (6 µg mL-1) in a total volume of 10 mL and 1.5-2.5
mL of Phen solution was found to give constant absorbance readings. Hence, 2 mL of
phen solution in a total volume of 10 mL is fixed (Figure 7.2.2). In method B, 2 mL of
0.01 M bipy solution in a total volume of 10 mL was found to give the maximum
absorbance value (Figure 4), hence, the same volume was used. The standing times for
full color development were found to be 10 min for both methods; and the color was
stable for 30 min thereafter in both methods.
Figure 7.2.2 Effect of 1, 10-phenanthroline (4 µg mL-1 KTF) and 2, 2΄-bipyridyl (15 µg mL-1 KTF)
7.2.3.2 Method validation
Linearity and sensitivity
A linear correlation was found between absorbance at max and concentration of
KTF (Figure 7.2.3) in the ranges given in Table 7.2.1. Regression analysis of the Beer’s
law data using the method of least squares was made to evaluate the slope (b), intercept
(a) and correlation coefficient (r) for each system and the analytical results obtained from
this investigations are presented in Table 7.2.1. The optical characteristics such as Beer’s
law limits, molar absorptivity and Sandell sensitivity values of both the methods are also
given in Table 7.2.1. The high values of ε and low values of Sandell sensitivity and LOD
indicate the high sensitivity of the proposed methods.
0.5 1.0 1.5 2.0 2.5 3.00.30
0.35
0.40
0.45
0.50
0.55
0.60
Abso
rban
ce
Volume of reagent, mL
phen bipy
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
390
Method A Method B
Figure 7.2.3 Calibration curves
Table 7.2.1 Sensitivity and regression parameters Parameter Method A Method B max, nm 510 520 Linear range, µg mL-1 0.4-8.0 1.0-25.0 Molar absorptivity(ε), L mol-1 cm-1 5.40 × 104 1.6 × 104
Sandell sensitivity*, µg cm-2 0.0079 0.0254 Limit of detection (LOD), g mL-1 0.15 0.27 Limit of quantification (LOQ), g mL-1 0.46 0.80 Regression equation, Y**
Intercept (a) 0.0061 0.0024 Slope (b) 0.1227 0.0392 Standard deviation of a (Sa) 0.0674 0.0130 Standard deviation of b (Sb) 0.0150 0.0010 Regression coefficient (r) 0.9997 0.999 *Limit of determination as the weight in µg per mL of solution, which corresponds to an absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. **Y=a+bX, Where Y is the absorbance, X is concentration in µg mL-1, a is intercept, b is slope.
Accuracy and precision
The precision and accuracy of the proposed methods were studied by repeating
the experiment seven times within the day to determine the repeatability (intra-day
precision) and five times on different days to determine the intermediate precision (inter-
day precision). The assay was performed for three levels of analyte in each method. The
results of this study are summarized in Table 7.2.2. The percentage relative standard
0 2 4 6 80.0
0.2
0.4
0.6
0.8
1.0
Abs
orba
nce
Concentration of KTF, µg mL-1
0 5 10 15 20 250.0
0.2
0.4
0.6
0.8
1.0
Abs
orba
nce
Concentration of KTF, µg mL-1
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
391
deviation (%RSD) values were ≤ 1.92 % (intra-day) and ≤ 1.94 % (inter-day) indicating
good precision of the methods. Accuracy was evaluated as percentage relative error
(%RE) between the measured mean concentrations and taken concentrations of KTF, and
it was ≤ 2.36 % demonstrating the high accuracy of the proposed methods.
Table 7.2.2 Results of intra-day and inter-day accuracy and precision study
Methods* KTF taken
µg mL-1
Intra-day accuracy and precision (n = 7)
Inter-day accuracy and precision (n = 5)
KTF found µg mL-1 %RE %RSD KTF found
µg mL-1 %RE %RSD
A
2.00 2.03 1.60 1.92 2.04 2.36 1.94 4.00 3.95 1.02 0.90 3.94 1.35 0.82 6.00 6.04 0.75 1.29 6.06 1.06 1.32
B
10.0 9.92 0.74 0.62 9.92 0.74 0.66 15.0 15.15 1.10 1.02 15.20 1.40 1.04 20.0 20.29 1.50 0.45 20.35 1.80 0.42
RE. Relative error; RSD. Relative standard deviation.
Selectivity
A systematic study was performed to determine the effect of matrix on the
absorbance by analyzing the placebo blank. In the analysis of placebo blank solution the
absorbance in each case was equal to the absorbance of blank which revealed no
interference. To assess the role of the inactive ingredients on the assay of KTF, the
general procedure was applied on the synthetic mixture extract by taking three different
concentrations of KTF (2, 4 and 6 µg mL-1 in method A and 10, 15 and 10 µg mL-1 in
method B). The percentage recovery values were in the range 97.38 – 102.3% with RSD
< 3% indicating clearly the non-interference from the inactive ingredients in the assay of
KTF.
Robustness and ruggedness
The method robustness was tested by making small incremental changes in FeCl3
and phen/bipy concentrations (n = 3) and performing the experiments using 2, 4 and 6 µg
mL-1 (method A) and 10, 15 and 10 µg mL-1 KTF (method B). The RSD with the altered
FeCl3 and Phen/Bipy concentrations were < 1.5%. In order to demonstrate the ruggedness
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
392
of the methods, a drug solution at different concentration levels were analyzed by four
different analysts, and also with three different cuvettes by a single analyst. The inter-
analysts RSD were < 1.07 %, whereas the inter-instrumental variation expressed as RSD
were < 1.5 %. These low values of intermediate precision demonstrate the robustness and
ruggedness of the proposed methods (Table 7.2.3).
Application to tablets
The proposed methods were applied to the quantification of KTF in commercial
tablets. The tablets were assayed by the reference method [5]. The results obtained by the
proposed methods agree well with the claim and also are in agreement with those by the
reference method. Statistical analysis of the results did not detect any significant
difference between the performance of the proposed methods and reference method with
respect to accuracy and precision as revealed by the Student’s t-value and variance ratio
F-value. The results of assay are given in Table 7.2.4.
Table 7.2.3 Results of robustness and ruggedness expressed as intermediate precision (%RSD)
Method
KTF taken
(µg mL-1)
Method robustness Method ruggedness Parameter altered Ferric
chloride* mL
%RSD (n = 3)
Reaction time**
%RSD (n=3)
Inter-analysts’ %RSD (n = 4)
Inter-instruments’
%RSD (n = 3)
A
2.00 0.94 1.02 0.71 1.23 4.00 1.05 0.98 0.94 1.49 6.00 0.89 1.09 0.87 1.37
B
10.0 1.12 1.05 1.07 1.21 15.0 1.01 1.16 0.95 1.37 20.0 1.17 1.19 1.01 1.29
*Ferric chloride volumes used were 1.8, 2.0 and 2.2 mL **reaction time of 8, 10 and 12 min in method A.
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
393
Table 7.2.4 Results of analysis of tablets by the proposed methods and comparison with the official method
Tablets analysed
Label claim,
mg/tablet
Found* (Percent of label claim ± SD) Reference
method Proposed methods
Method A Method B
aAsthafen 1 100.8 ± 0.91 100.6 ±0.86 101.2 ± 0.75
t= 1.22 t= 1.55 F= 1.11 F= 1.47
bKetasma 1 102.3 ± 1.02 101.3 ± 1.06 101.5 ± 0.88
t= 2.26 t= 0.97 F= 1.07 F= 1.34
*Mean value of five determinations; aTorrent pharmaceuticals, Sikkim, India,b Sun pharmaceuticals, Sikkim, India. Tabulated t-value at the 95% confidence level is 2.78; Tabulated F-value at the 95% confidence level is 6.39.
Recovery studies
To further assess the accuracy of the methods, recovery experiments were performed
by applying the standard-addition technique. The recovery was assessed by determining the
agreement between the measured standard concentration and added known concentration to
the sample. The test was done by spiking the pre-analyzed tablet KTF with pure KTF at
three different levels (50, 100 and 150 % of the content present in the preparation) and the
total was found by the proposed methods. Each test was repeated three times. In both the
cases, the recovery percentage values ranged between 100.5 and 102.1% with standard
deviation in the range 1.05-1.98%. Closeness of the results to 100% showed the fairly good
accuracy of the methods. The results are shown in Table 7.2.5.
Table 7.2.5 Results of recovery study via standard addition method Method Tablet
studied KTF in tablet
µg mL-1
Pure KTF added
µg mL-1
Total found
µg mL-1
Pure KTF * Percent ± SD
A
3.04 1.5 4.54 100.5 ± 1.98 Ketasma-1 3.04 3.0 6.07 101.3 ±1.05
3.04 4.5 7.63 102.0 ± 1.71
B
7.60 3.75 11.42 100.5 ± 1.26 Ketasma-1 7.60 7.50 15.39 102.1 ± 1.98
7.60 11.25 19.15 101.6 ± 1.34 * Mean value of three determinations
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
394
SECTION 7.3
VALIDATED UV-SPECTROPHOTOMETRIC DETERMINATION
OF KETOTIFEN IN PHARMACEUTICALS AND
ITS STABILITY STUDIES
7.3.1 INTRODUCTION
A smart profile and utilization of UV-spectrometry in different assay have been
presented in Section 2.5. From the literature survey presented Section7.0 it is evident
that, application a UV-spectrophotometric method for the determination KTF in bulk
drug and capsules. The method was based on the measurement of absorbance in acetate
buffer (pH 5) at 300 nm [12]. Beer’s law was obeyed over a concentration range 2-300
µg mL-1. Another method involves the measurement of absorbance of drug solution in 2
M HCl at 298 nm [13].
In the literature, no stability-indicating UV-spectrophotometric methods have ever
been reported for the assay of KTF. In the present Section (7.3), two simple, inexpensive,
accurate and reproducible UV- spectrophotometric methods for the assay of KTF are
described. The methods are based on the measurement of absorbance of KTF solution
either in 0.1 M NaOH at 308 nm in method A, or in 0.1 M Acetic acid at 300 nm in
method B. Besides, method A was used to study the degradation of the drug under
different stress conditions as per the ICH guidelines.
7.3.2 EXPERIMENTAL
7.3.2.1 Apparatus
The instrument used for absorbance measurement is the same as described in
Section 2.5.2.1.
7.3.2.2Materials
All chemicals used were of analytical reagent grade. Doubly-distilled water was
used to prepare solutions wherever required. Pure drug and tablets used were the same as
described in Section 7.1.2.
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
395
7.3.2.3 Reagents and solutions
Hydrochloric acid (2 M), sodium hydroxide (2 M), hydrogen peroxide (5 %)
were prepared as described under Section 3.4.2.3.
Acetic acid (0.1 M): Prepared by diluting concentrated acid (Merck, Mumbai, India, Sp.
gr. 1.18) with water.
Standard drug solution
Standard drug solutions of 50 µg mL-1 KTF prepared separately in 0.1 M NaOH
and in 0.1 M acetic acid and used for assay in method A and method B, respectively.
7.3.2.4 ASSAY PROCEDURES
Method A (using 0.1 M NaOH)
Different aliquots (0.00, 0.2, 0.5, 1.0, -----5.0 mL) of a standard KTF (50 µg mL-
1) solution were accurately transferred into a series of 10 mL volumetric flasks and the
volume was made up to the mark with 0.1 NaOH. The absorbance of each solution was
measured at 308 nm against 0.1 M NaOH.
Method B (using 0.1 M acetic acid)
Aliquots (0.00, 0.2, 0.40, 1.0,-----5.0 mL) of a standard KTF (50 µg mL-1)
solution were accurately transferred into a series of 10 mL volumetric flasks and the
volume was made up to 10 mL with 0.1 M acetic acid. The absorbance of each solution
was measured at 300 nm against 0.1 M acetic acid.
In both the cases, calibration curves were plotted and the concentration of the
unknown was read from the calibration graph or computed from the regression equation
derived using Beer’s law data.
Procedure for tablets
Twenty tablets were weighed accurately and ground into a fine powder. An
accurately weighed amount of the powdered tablet equivalent to 10 mg of KTF was
transferred into two 100 mL calibrated flasks. Sixty mL of 0.1 M NaOH was added to
one and 0.1 M Acetic acid to other flask and the content was shaken thoroughly for 15-20
min to extract the drug into the liquid phase; the volume was finally diluted to the mark
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
396
with the respective solvent, mixed well and filtered using a Whatman No. 42 filter paper.
An aliquot of the filtrate (100 µg mL-1 in KTF) was diluted to get 50 µg mL-1 KTF and
analysed for KTF following the procedures described above.
Placebo blank and synthetic mixture analysis
Fifty mg of the placebo blank prepared in Section 7.1.2.4 was taken and its
solution prepared as described under ‘Procedure for tablets’ and then analyzed using the
procedures described above.
To 20 mg of the placebo blank, 10 mg of KTF was added and homogenized,
transferred to 100 mL volumetric flask and the solution was prepared as described under
“Procedure for tablets”. A convenient aliquot was diluted and then subjected to analysis
by the procedures described above.
Forced degradation study
Five ml of 50 µg ml-1 KTF was taken (in triplicate) in a 25 ml volumetric flask
and mixed with 5 ml of 5 M HCl (acid hydrolysis) or 5 M NaOH (alkaline hydrolysis) or
5% H2O2 (oxidative degradation) and boiled for 2 h at 80 °C in a hot water bath. The
solution was cooled to room temperature and diluted to the mark with 0.1 M NaOH after
neutralization with 5.0 ml of 5 M NaOH (for acid hydrolysis) and 5 ml of 5 M HCl (for
alkaline hydrolysis). In thermal degradation, solid drug was kept in Petri dish in oven at
100 °C for 24 h. After cooling to room temperature, 10 µg ml-1 KTF solution in 0.1 M
NaOH was prepared and absorbance measured. For UV degradation study, the stock
solutions of the drug (50 µg mL-1) were exposed to UV radiation of wavelength 254 nm
and of 1.2K flux intensity for 48 h in a UV chamber. The solutions after dilution with 0.1
M H2SO4 was assayed as described above.
7.3.3 RESULTS AND DISCUSSION
7.3.3.1 Spectral characteristics
The KTF solution in 0.1 M NaOH (method A) and in 0.1 M Acetic acid (method
B) showed absorption maximum at 308 and 300 nm, respectively. At these wavelengths
0.1 M NaOH or 0.1 M Acetic acid had insignificant absorbance. Therefore, further
investigation for the analysis of KTF was carried out at 308 nm (method A) and 300 nm
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
397
(method B). Figure 7.3.1 represents the absorption spectra of KTF in 0.1 M NaOH and
0.1 M Acetic acid along with their respective blanks.
(a) (b)
Figure 7.3.1 Absorption spectra of: a-10 µg mL-1 KTF in 0.1MNaOH (Method A)
b-10 µg mL-1 KTF in 0.1 M Acetic acid(Method B)
7.3.3.2 Stability studies
The absorption spectra of the KTF (Method A) solutions treated with acid, base,
hydrogen peroxide, dry heat and UV radiation were run in the range of (200-400 nm).
The degradation study was based on the comparison of the UV spectra of “stressed KTF
samples” with that of the “standard KTF solution”. The absorption spectrum of KTF
solution treated with 2 M hydrochloric acid (Figure 7.3.2a), 2 M NaOH (Figure 7.3.2b)
and 5% H2O2 (Figure 7.3.2c) showed degradation as the absorbance value was reduced
slightly. There was no degradation when KTF solution in 0.1 M NaOH was subjected to
dry heat (Figure 7.3.2d) and Uv-light (Figure 7.3.2e) exposure stress conditions.
(a)
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
398
(b)
(c)
(d)
(e)
Figure 7.3.2 Absorption spectra of KTF (10 µg mL-1) after subjecting to a) acid
hydrolysis, b) base hydrolysis, c) peroxide degradation, d) thermal hydrolysis and e) photolytic hydrolysis.
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
399
7.3.3.3 Method validation
Linearity and sensitivity
A linear correlation was found between absorbance at max and concentration of
KTF (Figure 7.3.3). The slope (b), intercept (a) and correlation coefficient (r) for each
system were evaluated by using the method of least squares. Sensitivity parameters such
as apparent molar absorptivity and Sandell sensitivity values, the limits of detection and
quantification calculated as per the current ICH guidelines [33] are compiled in Table
7.3.1.
Method A Method B Figure 7.3.3 Calibration curves
Table 7.3.1 Sensitivity and regression parameters Parameter Method A Method B max, nm 308 300 Linear range, µg mL-1 1.0 – 25.0 1.0 – 25.0
Molar absorptivity(ε), L mol-1cm-1 1.78 × 104 1.77 × 104
Sandell sensitivity*, µg cm-2 0.0215 0.0239 Limit of detection (LOD), µg mL-1 0.37 0.42 Limit of quantification (LOQ), µg mL-1 1.12 1.26 Regression equation, Y**
Intercept (a) 0.0102 0.0037 Slope (b) 0.0401 0.0399 Standard deviation of a (Sa) 0.0382 0.0028 Standard deviation of b (Sb) 0.0416 0.0031 Regression coefficient (r) 0.9997 0.9997 *Limit of determination as the weight in µg per mL of solution, which corresponds to an absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1.0 cm2 and l = 1.0 cm.
bXaY ** , where Y is the absorbance and X concentration in µg mL-1.
0 5 10 15 20 250.0
0.2
0.4
0.6
0.8
1.0
1.2
Abso
rban
ce
Concentration of KTF, µg mL-1
0 5 10 15 20 250.0
0.2
0.4
0.6
0.8
1.0
Abs
orba
nce
Concentration of KTF, µg mL-1
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
400
Accuracy and precision
Accuracy was evaluated as percentage relative error between the measured
concentrations and the concentrations taken for KTF (Bias %). The results obtained are
compiled in Table 7.3.2 and show that the accuracy is good. Precision of the method was
calculated in terms of intermediate precision (intra-day and inter-day). Three different
concentrations of KTF were analyzed in seven replicates during the same day (intra-day
precision) and for five consecutive days (inter-day precision). RSD (%) values of the
intra-day and inter-day studies showed that the precision was satisfactory.
Robustness and ruggedness
Method robustness and ruggedness were demonstrated by determination of KTF
at three different concentrations. Method robustness was tested by measuring the
absorbance at different wavelengths whereas the method ruggedness was performed by
four different analysts, and also with three different cuvettes by a single analyst. The
intermediate precision, expressed as percent RSD, which is a measure of robustness and
ruggedness was within the acceptable limits as shown in the Table 7.3.3.
Selectivity
The proposed methods were tested for selectivity by placebo blank and synthetic
mixture analyses. The placebo blank solution was subjected to analysis according to the
recommended procedures and found that there was no interference from the inactive
ingredients, indicating a high selectivity for determining KTF in its tablets.A separate
Table 7.3.2 Results of intra-day and inter-day accuracy and precision study
Method KTF
taken, µg mL-1
Intra-day accuracy and precision
(n=7)
Inter-day accuracy and precision
(n=5) KTF
found, µg mL-1
%RE %RSD KTF found
µg mL-1 %RE %RSD
A 10.0 15.0 20.0
10.06 15.22 20.13
0.65 1.46 0.65
1.30 1.22 1.08
10.16 15.25 20.22
1.60 1.66 1.12
1.64 1.55 1.87
B 10.0 15.0 20.0
10.10 15.15 20.34
0.88 1.10 1.73
1.08 0.99 1.76
10.11 15.26 20.38
1.12 1.73 1.92
1.68 1.70 1.66
%RE. Percent relative error, %RSD. Relative standard deviation.
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
401
experiment was performed with the synthetic mixture. The analysis of synthetic mixture
solution yielded percent recoveries of 98.56±0.98 for method A and 100.4±1.08 for
method B, indicating the non-interference by inactive ingredients.
Table 7.3.3 Results robustness and ruggedness expressed as intermediate precision(%RSD)
Method KTF
taken, µg mL-1
Robustness# (%RSD)
Ruggedness
Inter-analysts (%RSD), (n=4)
Inter-cuvettes (%RSD),
(n=4)
A 10.0 15.0 20.0
0.64 0.86 0.38
0.35 0.48 0.26
1.26 1.78 1.04
B 10.0 15.0 20.0
0.42 0.58 0.36
0.76 0.48 0.54
1.85 1.26 1.46
#Wavelengths used were 307,308and 309 in method A and 299, 300 and 301 in method B.
Application to tablets
In order to evaluate the analytical applicability of the proposed method to the
quantification of KTF in commercial the tablets, results obtained by the proposed
methods were compared to those of the reference method [5] by applying Student’s t-test
for accuracy and F-test for precision. The results (Table 7.3.4) showed that the Student’s
t- and F-values at 95 % confidence level did not exceed the tabulated values, which
confirmed that there is a good agreement between the results obtained by the proposed
methods and the reference method with respect to accuracy and precision
Table 7.3.4 Results of analysis of tablets by the proposed methods and comparison with the official method
Tablets analysed
Label claim,
mg/tablet
Found* (Percent of label claim ± SD) Reference
method Proposed methods
Method A Method B
aAsthafen 1 100.8 ± 0.91 101.26 ± 0.85 101.4 ± 1.04
t= 1.85 t= 2.55 F= 1.14 F= 1.30
bKetasma 1 102.3 ± 1.02 101.3 ± 0.59 100.6 ± 0.87
t= 2.52 t= 2.75 F= 2.98 F= 1.37
*Mean value of five determinations; Tabulated t-value at the 95% confidence level is 2.78; Tabulated F-value at the 95% confidence level is 6.39
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
402
Recovery studies
The accuracy and validity of the proposed methods were further ascertained by
performing recovery studies. Pre-analysed tablet powder was spiked with pure KTF at
three concentration levels (50, 100 and 150 % of that in tablet powder) and the total was
found by the proposed methods. The added KTF recovery percentage values ranged from
99.24–102.0 % with standard deviation of 0.68-1.42 (Table 7.3.5) indicating that the
recovery was good, and that the co-formulated substance did not interfere in the
determination.
Table 7.3.5 Results of recovery study via standard addition method Method Tablet
studied KTF in tablet
µg mL-1
Pure KTF added
µg mL-1
Total found
µg mL-1
Pure KTF * Percent ± SD
A
7.58 3.50 11.37 101.2 ± 1.28 Ketasma-1 7.58 7.50 15.02 99.24 ±1.42
7.58 11.25 18.91 100.7 ±0.71
B
7.54 3.50 11.34 101.5 ± 1.10 Ketasma-1 7.54 7.50 15.18 102.0 ± 0.68
7.54 11.25 18.87 100.8 ± 0.84 * Mean value of three determinations
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
403
SECTION 7.4
STABILITY-INDICATING UPLC METHOD FOR THE DETERMINATION OF
KETOTIFEN IN TABLETS
7.4.1 INTRODUCTION
The utility and importance of UPLC was discussed in Section 4.4.1. The reported
chromatographic methods [16-22] are not stability-indicating.
To the best of the author’s knowledge, no UPLC method has been ever reported
for the determination of KTF in pharmaceuticals, Thus, a need for a rapid, precise,
accurate and validated stability-indicating UPLC method for the determination of KTF in
bulk and tablets was felt. This was accomplished with a Waters Acquity UPLC system
and Acquity BEH column (C18, 100mm, 2.1mm and 1.7 µm). The stability indicating
power of the method was established by comparing the chromatograms obtained under
optimized conditions before forced degradation with those after degradation. The
optimization parameters and the validation results in detail are presented in this Section
(7.4).
7.4.2 EXPERIMENTAL
7.4.2.1Materials
All the reagents used were of analytical grade. Doubly distilled water was used
throughout the investigation. Pure KTF used was the same as described in Section 7.1.
HPLC grade acetonitrile orthophosphoric acid were purchased from Merck India. Pvt
Ltd. Mumbai, India.
Mobile phase preparation
About 1 g of orthophosphoric acid was dissolved in 1000 mL of water. A 500 mL
portion of this resulting solution was mixed with 500 mL of acetonitrile, shaken well and
filtered using 0.22 µm Nylon membrane filter. This solution was also used as diluent in
all subsequent preparations of the sample.
Hydrochloric acid (1 M), Sodium hydroxide (NaOH, 1M), Hydrogen peroxide
(H2O2, 5% v/v) were prepared in usual manner as described in previous Section.
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
404
Preparation of stock solution
A stock standard solution of (1 mg mL-1) was prepared by dissolving an
accurately weighed 100 mg of pure drug in 100 mL volumetric flask using the mobile
phase.
Chromatographic conditions and equipments
Analyses were carried out on a Waters Aquity UPLC with Tunable UV (TUV)
detector. The output signal was monitored and processed using Empower software. The
Chromatographic column used was Agilent Zorbax (50 × 2.1) mm and 5 µm particle size.
Isocratic elution process was adopted throughout the analysis.
Instrumental parameters
The isocratic flow rate of mobile phase was maintained at 0.25 mL min-1. The
column temperature was adjusted to 40 °C. The injection volume was 4 µL. Eluted
sample was monitored at 300 nm and the run time was 4.0 min. The retention time of the
sample was about 1.8 min.
7.4.2.3 ASSAY PROCEDURES
Procedure for preparation of calibration curve
Working solutions containing 1-120 µg mL-1 of KTF were prepared by serial
dilutions of aliquots of the stock solution. Aliquots of 4.0 µL were injected in triplicate
(six injections) and eluted with the mobile phase under the reported chromatographic
conditions. The average peak area versus the concentration of KTF in µg mL-1 was
plotted. Alternatively, the corresponding regression equation was derived using mean
peak area-concentration data and the concentration of the unknown was computed from
the regression equation.
Procedure for tablets
Fifty tablets (Each tablet contained 1.0 mg) were weighed and transferred in to a
clean, dry mortar and powdered. Tablet powder equivalent to 8 mg of KTF was
transferred in to a 100 mL volumetric flask and 60 mL of the mobile phase was added.
The solution was sonicated for 20 min to achieve complete dissolution of KTF, made up
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
405
to the mark with mobile phase and then filtered through a 0.22 µm nylon membrane
filter. The solution (80 µg mL-1 of KTF) obtained was analyzed by UPLC.
Forced degradation study (Stress study)
All stress decomposition studies were performed at an initial drug concentration
of 80 µg mL-1 in mobile phase and added 5 mL of 1 M HCl, 1 M NaOH or 5% H2O2
separately, and the flasks were heated for 2 h on a water bath maintained at 80 C. Then
the solutions were cooled and neutralized by adding base or acid, the volume in each
flask was brought to the mark with mobile phase, and the appropriate volume (4.0 µl)
was injected for analysis. Solid state thermal degradation was carried out by exposing
pure drug to dry heat at 105 C for 3 h. For photolytic degradation studies, pure drug in
solid state was exposed to 1.2 million lux hours in a photo stability chamber. The sample
after exposure to heat and light was used to prepare 80 µg mL-1 solutions in mobile phase
and the chromatographic procedure was followed.
7.4.2.4 Procedure for method validation
The validation parameters viz; accuracy and precision, LOD, LOQ, robustness
and ruggedness, solution stability and mobile phase stability were carried by following
the same procedure as described under Section 5.4.2.4.
7.4.3 RESULTS AND DISCUSSION
7.4.3.1Method development
Different chromatographic conditions were experimented to achieve better
efficiency of the chromatographic system. Parameters such as mobile phase composition,
wavelength of detection, column, column temperature, pH of mobile phase and diluents
were optimized. Several proportions of buffer, and solvents were evaluated in-order to
obtain suitable composition of the mobile phase. Choice of retention time, tailing,
theoretical plates and run time were the major tasks while developing the method.
Alternative combinations of gradient and isocratic methods were also performed to obtain
a suitable peak. Finally, isocratic method was found suitable for the assay.
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
406
When KTF was injected with methanol and potassium phosphate buffer mobile
phases, the resultant peak showed either tailing or much shortened retention time (less
than 1min). As the buffer ratio increased, the retention time of the KTF augmented but
peak eluted with abnormal shape. The concentration of buffer and methanol varied in
different ratios and found incompatible. The separation was carried out with Agilent
Zorbax, (50 × 2.1) mm, 5 µm column. Acetonitrile was the next solvent option. Better
results were obtained when KTF was eluted in acetonitrile, and orthophosphoric acid.
KTF eluted late with higher peak shape and theoretical plates. The peak symmetry was
optimized with varying concentrations of buffer and acetonitrile. Best peak was obtained
with buffer-acetonitrile ratio (50:50 v/v). Different buffers like ammonium acetate and
dibasic potassium phosphate were the other salts used for development. Flow rate of 0.25
mL/min was selected with regard to the back pressure and analysis time as well. In order
to achieve symmetrical peak of KTF, various stationary phases like C8 (different
dimension), C18 (different brand), length (100 mm and 50 mm) and phenyl columns
were studied. It is concluded that Agilent Zorbax, (50 × 2.1) mm, 5 µm column have the
ideal stationary phase for the determination. The column oven temperature was studied at
higher (45°C) and room (35°C) temperatures and then found that 40°C is the optimum.
Shimadzu Pharmaspec 1700 UV/Visible spectrophotometer was used for absorbance
measurements. A 80 µg mL-1 of KTF solution in acetonitrile was scanned from 400 to
200 nm against acetonitrile as blank and wavelength of the method was optimized to 300
nm. Overlay chromatograms of blank and KTF solutions are as shown in Figure 7.4.1.
(a)
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
407
(b)
Figure 7.4.1 Chromatograms of: a) blank, b) pure KTF (80 µg mL-1).
7.4.3.2 Stability studies
All forced degradation studies were analyzed at 80 µg mL-1 concentration level.
The observation was made based on the peak area of the respective sample. KTF was
found to be more stable under acid and alkaline, photolytic (1.2 million lux hours),
thermal (105 0C for 3 hours) in solid state, and hydrolytic (aqueous, 80 0C for 2 hours)
stress conditions. Less degradation occurred under oxidative stress conditions with
percent decomposition being only around 10 %. The chromatograms obtained for KTF
after subjecting to degradation are presented in Figure 7.4.2. Assay study was carried out
by the comparison with the peak area of KTF sample without degradation.
(a)
(b)
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
408
(c)
(d)
(e)
(f)
Figure 7.4.2 Chromatograms obtained for KTF (80 µg mL-1) after subjecting to stress
studies by: (a) acid degradation, (b) base degradation, (c) hydrolytic degradation, (d) thermal degradation (e) photo degradation and (f) oxidative degradation.
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
409
7.4.3.3 Method validation
System suitability
System suitability is the measurement of performance verification of system,
method and column performance. Theoretical plates (should be more than 2000), tailing
factor (should be less than 1.5) and percentage relative standard deviation (should be less
than 2) for the area and retention time of replicate injections were verified on precision,
ruggedness (variation in column, analyst and day) and robustness (variation in
temperature, mobile phase and wavelength) of the validation. The theoretical plates found
more than 2000, tailing factor is less than 1.4 and the percentage relative standard
deviation (%RSD) for area and retention time were less than 0.5.
Analytical parameters
A calibration curve was obtained for KTF from LOQ to 150% of its stock
solution. A linear correlation was obtained between the mean peak area and the
concentration in the range of 1 – 120 µg mL-1 KTF from which the linear regression
equation was computed and found to be:
y = 33902x + 18204 r² = 0.9999
where y is the mean peak area, x is the concentration of KTF in µg mL-1 and r is the
correlation coefficient. The LOD and LOQ values, slope (m), y-intercept (a) and their
standard deviations are evaluated and presented in Table 7.4.3. These results confirm the
linear relation between the mean peak area and concentration as well as the sensitivity of
the method.
Table 7.4.1 Linearity and regression parameters Parameter Value Linear range, µg mL-1 1-120 Limits of quantification, (LOQ), µg mL-1 1.0 Limits of detection, (LOD), µg mL-1 0.3 Regression equation Slope (b) 33902.7 Intercept (a) 18204.0 Correlation coefficient (r) 0.9999
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
410
Accuracy and precision
The percent relative error which is an index of accuracy is ≤ 0.64 and is indicative
of high accuracy. The calculated percent relative standard deviation (%RSD) can be
considered to be satisfactory. The peak area based and retention time based RSD values
were < 1. The results obtained for the evaluation of precision and accuracy of the method
are compiled in Tables 7.4.2 and 7.4.3.
Robustness and ruggedness
At the deliberate varied chromatographic conditions (flow rate, temperature, and
mobile phase composition), the analyte peak %RSD, tailing factor and theoretical plates
remained near to the actual values. The RSD values ranged from 0.1 to 2.0% resumes the
robustness of the proposed method. In method ruggedness, different columns (same lot),
on different days by different analysts were performed. The results were summarized in
Table 7.4.4 and 7.4.5.
Table 7.4.2 Results of accuracy study (n=5) Concentration of KTF injected, µg mL-1
Intra-day Inter-day Concentration of KTF found, µg mL-1
RE,%
Concentration of KTF found, µg mL-1
RE,%
60.00 60.30 0.43 60.38 0.64 80.00 80.34 0.46 80.39 0.51 100.0 99.48 0.52 99.40 0.60
Table 7.4.3 Results of precision study (n=5) Con
Injected,µg mL1
Intra-day precision Inter-day precision Mean area
± SD %RSDa %RSDb Mean area
± SD %RSDa %RSDb
60.00 2078490±5277 0.25 0.41 2130424±6948 0.32 0.38 80.00 2777526±7909 0.31 0.29 2852796±8769 0.29 0.34 100.0 3376954±7228 0.21 0.18 3418865±6987 0.22 0.21
a Relative standard deviation based on peak area; b Relative standard deviation based on retention time
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
411
Table 7.4.4 Results of method robustness study expressed as intermediate precision (%RSD
Condition Modification Mean peak area± SD* RSD,% Mean Rt±
SD* RSD,% Mean theoretical plates ±SD*
RSD, %
Mean tailing factor ± SD* RSD,%
Temperature 35ºC 2745625±5368 0.20 1.749±0.005 0.28 2423±39.65 1.63 1.15±0.01 0.86 40ºC 2782549±6376 0.22 1.745±0.003 0.17 2572±49.16 1.91 1.13±0.02 1.76 45ºC 2813486±6491 0.24 1.750±0.004 0.23 2686±44.28 1.64 1.14±0.02 1.75
Mobile phase composition
55:45 2843821±4982 0.17 1.742±0.005 0.28 2689±50.31 1.87 1.12±0.02 1.78 50:50 2791630±5748 0.21 1.738±0.006 0.34 2772±41.27 1.48 1.17±0.02 1.70 45:55 2985915±7246 0.24 1.749±0.004 0.22 2565±35.93 1.40 1.15±0.01 0.86
Flow rate, min
0.20 2615998±6345 0.23 1.746±0.005 0.21 2705±29.13 1.07 1.19±0.02 1.68 0.25 2708856±7023 0.25 1.738±0.004 0.23 2398±50.80 2.16 1.20±0.01 0.83
0.30 2891589±5949 0.20 1.751±0.003 0.17 2491±43.63 1.75 1.17±0.02 1.69
Wavelength, nm
299 2716684±5481 0.19 1.748±0.008 0.45 2505±41.19 1.64 1.12±0.02 1.49 300 2802479±6805 0.24 1.735±0.006 0.34 2647±35.56 1.34 1.15±0.02 1.73 301 2944213±7238 0.25 1.752±0.004 0.22 2498±47.33 1.89 1.18±0.02 1.69
*Mean value of three determination
Table 7.4.5 Results of method ruggedness study expressed as intermediate precision (%RSD) Variable Mean peak area
±SD* RSD, %
Mean Rt ±SD*
RSD, % Mean theoretical plates± SD*
RSD, % Mean tailing factor± SD*
RSD, %
Analyte (n=3)
2676471±7058 0.26 1.742±0.005 0.28 2457±48.83 1.98 1.14±0.02
1.75
Column (n=3)
2681692±6649 0.24 1.757±0.006 0.34 2492±41.19 1.65 1.16±0.02 1.72
*Mean value of three determinations
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
412
Selectivity
Selectivity of the method was evaluated by injecting the mobile phase, placebo
blank, pure drug solution and tablet extract. No peaks were observed for mobile phase
and placebo blank and no extra peaks were observed for tablet extracts.
Stability of the solution and mobile phase stability
At the specified time interval, % assay of KTF obtained from drug solution
stability and mobile phase stability were within 1%. For solution stability same sample
solution was injected at 0, 5, 10, 20 and 24 hours and for mobile phase stability,
separately prepared KTF sample injected at the same time interval as above. The results
confirmed that the KTF solution of drug and mobile phase were stable at least for 24
hours during the assay performance.
Application to tablet
A 80 µg mL-1 solution of tablets was prepared as per ‘Procedure for tablets’ and
injected in triplicate to the UPLC system. The mean peak area of the tablet extract for this
concentration was found to be equivalent to that of pure drug solution of the same
concentration and the results were compared with those of a reference method [5]. The
accuracy and precision of the proposed method was further evaluated by applying
Student’s t- test and variance ratio F- test, respectively. The t- and F- values at 95%
confidence level did not exceed the tabulated values and this further confirms that there is
no significant difference between the reference and proposed methods with respect to
accuracy and precision. Table 7.4.6 illustrates the results obtained from this study.
Table 7.4.6 Results of determination of KTF in tablet and statistical comparison with the reference method
Formulation brand name
Nominal amount, mg
% KTF found* ± SD t-value
F- value Reference
method Proposed method
Asthafen 1.0 100.8±0.91 100.5±0.56 1.04 2.64
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
413
Recovery studies
A standard addition procedure was followed to evaluate the accuracy of the method.
The solutions were prepared by spiking pre-analyzed tablet powder with pure KTF at
three different levels and injected to chromatographic column after sample preparation as
described before. The recovery of the known amount of added analyte was computed.
The percentage recovery of KTF from pharmaceutical dosage forms ranged from 99.72
% to 100.3%. Detailed results presented in Table 7.4.7 reveal good accuracy of the
proposed method.
Table 7.4.7 Results of recovery study via standard addition method
Tablet studied
KTF in tablet, µg mL-1
Pure KTF added, µg mL-1
Total found, µg mL-1
Pure KTF recovered*
(%KTF±SD)
Asthafen 40.20 40.20 40.20
20.0 40.0 60.0
60.34 80.48 99.92
100.2±0.92 100.3±1.07 99.72±0.66
*Mean value of three determination
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
414
SECTION 7.5
SUMMARY AND CONCLUSIONS –Assessment of methods
For ketotifen, one titrimetric, four visible, two UV-spectrophotometric and one
UPLC methods were developed and validated. One UV-spectrophotometric and UPLC
methods are stability-indicating. The titrimetric/spectrophotometric methods are based on
well established and characterized redox/redox complexation reactions. The proposed
titrimetric and spectrophotometric methods rely on the use of inexpensive and eco-
friendly chemicals, and simple instrumentation.
For the first time visual titrimetric procedure was developed for the determination
of KTF in pharmaceuticals. The method utilizes a stable oxidizing agent Ce(IV) and
ferroin indicator. The method is simple, rapid and easy to perform.
Most of the reported visible spectrophotometric methods suffer from
disadvantages like use of organic solvent/expensive chemical, less sensitivity, heating,
applicable over narrow linear dynamic ranges etc as can be seen from Table 7.5.1. In
contrast, the present methods are simple and sensitive except the method using Ce(IV).
The methods have been demonstrated to be free from rigid experimental conditions like
heating or extraction except one using Ce(IV) and use stable, cheaper and readily
available chemicals. Among the proposed methods, the method employing Fe(III)- 1,10-
phenanthroline and is the most sensitive method with ε value of 5.4 × 104 L mol-1 cm-1 .
Two UV-spectrophotometric methods were developed and found to be superior
compare to the reported methods in terms of simplicity and sensitivity and application.
Among the two developed methods method using 0.1 M NaOH was subjected to forced
studies under different stress conditions. For the first time, author developed a stability-
indicating UV-spectrophotometric method, which can be considered as the simplest
method to evaluate the stability of the drug under various stress conditions.
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
415
Table 7.5.1 Comparison of performance characteristics of the proposed spectrophotometric methods with the existing methods
Sl. No. Reagent/s used Methodology max
(nm)
Linear range (µg/mL)
(ε = L/mol/cm)
Remarks Ref
1 a)Molybdenum-thiocyanate b)DDQ
Methylene chloride extractable ion-pair complex measured
469.5
588
5-37.5
10-80
Employs unstable oxidant, narrow linear range, use of organic solvent
25
2 a)F-C reagent b) NBS-celestine blue c)Sulphanilamide d) Azocarmine G
Measurement of the absorbance of blue colored chromogen Unreacted NBS measured using celestine blue in acid medium Measurement of pink ccolored coupled product of drug with diazotized sulphanilamide with drug Chloroform extractable 1:1ion-pair complex was measured
720
540
520
540
4-28
2-12
1-10
2.5-25
Multi step, unstable oxidant, strict pH control, tedious extraction procedure
26
3 a) bromophenol blue b) bromothymol green c)bromothymol blue d)bromocresol purple
Chloroform extractable 1:1ion-pair complex was measured
- 0 Required close pH control, narrow linear range and involved tedious time consuming extraction steps
27
4 Bromocresol green Chloroform extractable 1:1ion-pair complex was measured
423 5.15-61.91 Required close pH control, use of organic solvent narrow linear range and involved tedious time consuming extraction steps
28
5
Ce(IV)-p-DMAB
Ce(IV)-0-dianisidine
Oxidation form of p-DMAB measured Oxidation form of 0-dianisidine measured
460
470
0.4-8.0 (4.0 x 104)
0.4-10 (3.7 x 104)
Simple, highly sensitive, extraction free, use of stable cerium(IV) solution
Present work
6
Iron(III)-1,10-phenanthroline Iron(III)-2, 2΄-bipyridyl
Red colored chromogen (ferroin) formed is measured Red colored chromogen [Fe(II)-Bipy complex]formed is measured
510
520
0.4-8.0 (5.4 x 104)
1-25 (1.6 x 104)
Simple, sensitive, aqueous medium and extraction free
Present work
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
416
Though a few chromatographic techniques [16-24] are available for the assay of
KTF in pharmaceuticals, none of them is stability-indicating. UPLC, which is becoming
popular because of its speed and cost-effectiveness, was not earlier used for the assay of
KTF. Using this technique, the author has developed a rapid, isocratic RP-UPLC method
for the drug in its pure form and tablet. The results obtained are highly satisfactory with
respect to accuracy, precision, selectivity, robustness and ruggedness. A short retention
time of 1.8 min enables rapid determination of the drug which is of paramount
importance in routine analysis. The method was demonstrated to be stability-indicating
with the results of the degradation study revealing that the drug is stable to all other
conditions with the exception of oxidative degradation with slight degradation.
Selectivity of all the proposed methods was examined by both placebo blank and
synthetic mixture analyses. The methods are free from common excipients found in drug
formulations. Though methods based on the oxidation of KTF by Ce(IV) and
iron(III)chloride require quite longer reaction time of 10 min, they offer the advantages
in terms of sensitivity.
Statistical comparison of the proposed methods with those of the reference
method was performed by applying the Student’s t-test for accuracy and F-test for
precision. All calculated values were found to be less than the tabulated t- and F- values.
The results show that there is no significant difference between the results of the
proposed methods and reference method. The accuracy and validity of the methods were
further ascertained by recovery studies via spike method. The methods have been
demonstrated to be fairly accurate and precise in addition to being sensitive and simple.
Hence, the proposed methods can usefully be employed in quality control laboratories.
Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen
417
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