section 7.0 drug profile and literature survey...

Chapter 7 Titrimetric, spectrophotometric and Chromatographic assay of Ketotifen

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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.


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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


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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


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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).


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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.


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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


375

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


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


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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


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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


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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)


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



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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


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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


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


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.


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.


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


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.


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.


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


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


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)



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


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.


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


0 5 10 15 20 250.0

0.2

0.4

0.6

0.8

1.0

Abs

orba

nce



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.


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.


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


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).


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.


393


Tablets analysed

Label claim,

mg/tablet



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.



µg mL-1

Pure KTF added

µg mL-1

Total found

µg mL-1


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


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.


395


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.


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.


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


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.


7.3.3.1 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


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)


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.


399



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


0 5 10 15 20 250.0

0.2

0.4

0.6

0.8

1.0

Abs

orba

nce



400


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.


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


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.


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.


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


Tablets analysed

Label claim,

mg/tablet



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


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.



µg mL-1

Pure KTF added

µg mL-1

Total found

µg mL-1


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


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.


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.


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.


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


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.


7.4.3.1Method 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.


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)


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)


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.


409


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


410


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.


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


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


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


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


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.


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


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.


417

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section 7.0 drug profile and literature survey...

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