Ciproheptadina

Chapter4 Titrimetric, spectrophotometric and chromatographic assay of cyproheptadine
SECTION 4.0
DRUG PROFILE AND LITERATURE SURVEY
4.0.1 DRUG PROFILE
Cyproheptadine hydrochloride (CPH) is chemically known as 4-(5Hdibenzo [a,d]-

cyclohepten-5-ylidene)-1-methylpiperidine hydrochloride [1]. Its molecular formula is
C21H21N.HCl and molecular weight is 323.86 g mol-1. CPH has the following chemical
structure:
NH Cl
CH3
It is white to slightly yellow, crystalline powder, odourless, slightly bitter taste. It

is soluble in water, alcohol and practically insoluble in ether and other organic solvents. It
was synthesized by Engelhrdt et al., [2].
CPH is a sedating antihistamine with antimuscarinic, serotonin-antagonist, and

calcium-channel blocking action in pancreatic islet cells and smooth muscle [3]. It is used
to treat some hormonal disorders and may also be used for treating side effects of taking
antidepressants, other uses of CPH can also seen in clinical and veterinary medicine as an
antiserotonergic and antihistaminic agent with sedative and anticolinergic effects, CPH is
also used to stimulate appetite and weight gain in human and veterinary medicine [4].
The drug is official in Indian Pharmacopeia [5] which describes a UV-

spectrophotometric method for its assay in tablet in 0.1 M HCl at 286 nm. The United
Sates Pharmacoepia [6] describes non-aqueous titration with perchloric acid as titrant
where the end point is located visually using crystal violet as indicator.
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4.0.2 LITERATURE SURVEY OF ANALYTICAL METHODS FOR

CYPROHEPTADINE HYDROCHLORIDE
4.0.2.1 Titrimetric and UV-spectrophotometric methods
There is only one report [7] on the use of titrimetric technique for the
determination of CPH in pharmaceuticals, in which the drug is treated with known excess
of bromate-bromide mixture in HCl medium followed by the determination of unreacted
bromine iodometrically. The method is applicable over a concentration range of 2-15 mg.
Application of derivative UV-spectrophotometry for the assay of CPH in two- component
system has also been reported [8] by Zhou et al.
4.0.2.2 Visible spectrophotometric methods
Eight visible spectrophotometric methods [9-15] were reported for the assay of
CPH in pharmaceuticals. Adamski [9] was, perhaps, the first to report a colorimetric
method for the assay of CPH in tablets. The method involved the extraction of the drug
with chloroform, the chloroform extract with bromocresol green in phosphate buffer of
pH 5.4, re- extraction of the aqueous layer with chloroform, and finally with 0.1 M
sodium hydroxide followed by absorbance measurement at 615 nm. The method is
applicable over a concentration range of 1-10 µg mL-1.In another extractive colorimetric
method [10], the drug was precipitated with reineckate, the ion-pair complex was filtered,
dissolved in acetone and absorbance measured at 525 nm. Beer’s law is obeyed over a
concentration range of 100 -600 µg mL-1.Sane et al. [11] have reported a similar ion-pair
extraction photometric method using three dyes Solochrome Black-T, Solochrome Dark
Blue and Fast Sulphon Black FF, the absorbance of the complex being measured at 520
nm. The drug has also been determined spectrophotometrically based on ion-pair
complex formation with benzyl orange [12] at pH 4.7-4.9 followed by extraction into
dichloromethane and measurement at 404 nm. Basavaiah and Charan [13] have also
reported a spectrophotometric method based on complexation reaction using
bromophenol blue. Basavaiah [7] reported another spectrophotometric method based on
addition of excess of bromine to CPH in acid medium and the residual bromine is
determined by treating with methyl orange and measuring the absorbance of resulting
157
solution at 520 nm. In the same article, kinetic assay of CPH is also described.
Chloranilic acid has been used for the assay of CPH based on charge –transfer reaction
[14], Shingbal and Naik reported another spectrophotometric method based on
complexation [15].
4.0.2.3 Chromatographic methods
A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was
developed by Feás et al., [16] for the assay of CPH syrup formulations. Gas-
chromatographic procedures were also developed for the assay of CPH in tablets [17, 18].
Perhaps the most widely used technique for the assay of CPH in both pharmaceuticals
and body fluids has been the high-performance liquid chromatography (HPLC). Novak et
al., [19] have reported a HPLC method for quantification of CPH in serum or plasma
using micro Bondpak C18 column with a 41:15:44 mixture of methanol, acetonitrile and 5
mM-pentanesulphonic acid solution as a mobile phase, on 0.1 M-phosphate buffer of pH
4.7. Another quantification procedure for CPH in plasma and urine by HPLC has been
reported by Foda et al. [20] utilized a mobile phase, acetonitrile-0.05 M acetate buffer of
pH 3.5(4:1), for HPLC on a column (15 cm × 4 mm) of Ultrasphere octyl (5 µm) with
detection at 254 nm. Another HPLC method for determination of CPH using micro
Bondapak phenyl column in a normal-phase mode with acetonitrile-methanol-0.05 M
NH4H2PO4 of pH 2.5 and UV detection at 280 nm [21]. RP-HPLC method for the
determination of CPH in urine was developed by Kountourellis and Ebete [22].
Burrows and Alliger [23] have reported a method for the determination of CPH in
tablet formulations using Radial-PAK (10 µm) with a mobile phase of acetonitrile-water
(17:3). The calibration graph was rectilinear for 27-80 ng mL-1. Another method [24]
utilizing amino bonded micro Bondapak column with methanol as mobile phase and
detection at 254 nm. The calibration graph was rectilinear over a concentration range of
15-672 ng. Mao and Wang [25] developed a HPLC method for the assay of CPH in
tablets. The method was performed on a Hypersil BDS C18 column (15 cm × 4 mm i.d.),
operated at 25°C, with 0.1M-ammonium hydrogen phosphate/acetonitrile (13:7;
containing 0.08% triethylamine at pH 5.7) as mobile phase at 1 mL min-1 and detection at
240 nm. The calibration graph for CPH was linear from 1-20 mg L-1.
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Basavaiah et al. [26] have developed isocratic high performance liquid chromatographic
method for the determination of CPH in tablets. The method involves elution of the drug
at 30.2°C from a reversed-phase column packed with C18 5 µm, using a mobile phase
acetonitrile/0.1% orthophosphoric acid (80 + 20), pH adjusted to 3.0 at a flow rate of 1
mL min-1, and detection made at 222 nm. The detector response was linear within the
range 5-206 µg mL-1.Stability-indicating HPLC method was developed by Abounassif et
al. [27] for the assay of CPH. Procedure for Simultaneous determination of
cyproheptadine, multi vitamins and sorbic acid was developed by Gindy et al. [28].
Recently, HPLC has been used for the assay of CPH in feed stuff [29].
4.0.2.4 Other techniques
Liu and Lu [30] developed chemiluminescence method for the determination of

CPH, where riboflavin was used as chemiluminescence reagent. Feng and Guo [31]
developed method for the assay of CPH in serum, urine and in pharmaceuticals based on
the measurement of enhancement of resonance light scattering at 364 nm after formation
of ion-association complex with ammonium molybdate. Ion-selective based
potentiometry is another technique which has found application in the analysis of CPH-
containing tablets. The drug has been assayed by potentiometry using CPH-
tetraphenylborate [32], CPH-dinonylnaphthalenesulphonicacid [33], CPH-tetrakis(4-
chlorophenyl)borate [34] as electroactive compounds.
From the literature survey presented in the foregoing paragraphs, it is obvious that
the reported titrimetric method is deficient in one way or the other. The only UV-
spectrophotometric method [8] reported is applicable to two-component system and is not
stability-indicating. The method based on ion-pair formation [9, 11-13] requires strict pH
control, labor-intensive and have disadvantages like poor sensitivity, tedious and time-
consuming liquid-liquid extraction step and use of large amount of organic solvents. A
procedure [10] is cumbersome and involve precipitation and filtration steps and are prone
to loss of analyte there by affecting the accuracy of the methods. The method based on
charge-transfer complexation reaction and involving the use of chloranilic acid is
handicapped by narrow linear dynamic range and poor sensitivity.
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Keeping the above points in view, the author has developed three titrimetric, two
UV and five visible spectrophotometric methods, the latter being based on ion-pair
reaction without extraction and reaction of unreacted N-bromosuccinimide with two
reagents viz, erioglaucine and meta-cresol purple. Also, ultra performance liquid
chromatography (UPLC) has been applied for the first time to the determination of CPH
in pharmaceuticals. The details about the method development and validation of these
methods are presented in this chapter.
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SECTION 4.1
APPLICATION OF ION-ASSOCIATION TITRATION FOR THE ASSAY OF

CYPROHEPTADINE HYDROCHLORIDE IN PHARMACEUTICALS
4.1.1. INTRODUCTION
Titration of analyte in the presence of two immiscible solvents is known as two

phase titration. The titration end point can be detected either visually using an indicator
or potentiometrically. There are two types of ion-pair titrations, namely, titrations using
indicator dyes and titrations using iodide as a lipophilic anion used in pharmacopoeial
assays of many pharmaceutical compounds [35]. Two phase titrations (ion-pair extraction
titrations or ion-association titrations) are widely used in the cosmetics and detergents
industry since it is very useful for estimating surfactants, which often cannot be analyzed
by spectrophotometric methods because they lack chromophores. The characteristic of
the ion-association titration methods consist in the use of a two-phase (water-organic
solvent) system. The end point detection is based on the different stabilities of the ion-
associations formed between the determined substance with the titrant and with the
indicator [36]. Of the two types of ion-association titrations mentioned above, the one
which is of concern in this Section (4.1) is the titrations using indicator dyes. In this type,
the author has used anionic surfactant such as sodium lauryl sulphate (SLS) or
tetraphenylborate (TPB) as titrant, chloroform or 1,2-dichloroethane along with water as
two immiscible solvents, and dimethyl yellow or tetrabromophenolphthalein ethyl ester
as indicator.
There are two different principles in the ion-association titrations based on the
surfactant (titrant) and the indicator used. In the first principle, the titration of the analyte
such as quanternary ammonium compounds and certain tertiary amines with sodium
lauryl sulfate; in the presence of acid, chloroform and dimethyl yellow as indicator; leads
to the formation of colorless ion-association complex extractable to the chloroform layer.
The dimethyl yellow indicator lies in the organic layer in its molecular form making the
organic phase yellow in color. When the equivalence point is reached and one drop

This work has been published in ISRN Analytical Chemistry, 2012, Article ID 816349, 7 pages
161
excess of titrant is added, the color of chloroform layer will be changed from yellow to
pink due to the formation of cationic form of the indicator. In the second principle, the
mixing of the analyte such as certain tertiary amines with tetrabromophenolphthalein
ethyl ester indicator, in the presence of borate-phosphate buffer of pH 6.0 and 1,2-
dichloroethane, will form ion-association complex between the analyte and the indicator
which is extractable to the organic layer making this layer red-violet in color. When the
analyte is titrated with tetraphenylborate under the same conditions, a colorless ion-
association complex between the analyte and titrant will be formed and extracted to the
organic layer. After the equivalence point, the color of the organic phase will be changed
from red-violet to yellow due to the formation of molecular form of the indicator. This
type of ion-association titrations offer two major advantages [37]. Firstly, the color
change at the end-point occurs in the organic phase which overcomes the difficulty in
detecting the color change due to reflection in the case of phase transfer indicator.
Secondly, inherently colored sample in aqueous phase can be easily detected.
The two-phase ion-association titration was applied successfully for the assay of
many pharmaceutical compounds such as cetylpyridinium chloride [38],
chlorpheniramine maleate, chlorhexidine dihydrochloride, diphenhydramine HCl,
ephedrine HCl and dl-methylephedrine HCl [39], local anesthetics (procaine HCl,
dibucaine HCl, tetracaine HCl) [40], cinnarizine and dipyridamole [41], imidazole
derivatives [42], timolol, cilazapril and bromhexine [43], hydroxyzine hydrochloride
[44], bupropion hydrochloride [45].
From the literature survey presented in Section 4.0.2, there is only one report [7]
dealing with titrimetric determination of CPH in pharmaceuticals. In this section, the
author describes two methods for the assay of CPH in bulk drug as well as in tablets. The
methods employ sodium lauryl sulphate or sodium tetraphenylborate as the titrant with
the determinations carried out in the presence of sulphuric acid- chloroform or Walpole
buffer (pH 4.5) and 1,2-dichloroethane with dimethyl yellow or
tetrabromophenolphthalein ethyl ester (TBPE) as the indicator. The details of method
development and validation of the two titrimetric determinations of CPH are presented in
this Section (4.1).
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4.1.2 EXPERIMENTAL
4.1.2.1 Apparatus
Elico LI 610 digital pH meter provided with a combined glass-SCE electrode

system was used to record the pH and Equip-Tronics magnetic stirrer model EQ-770 was
used to carry out the titration successfully.
4.1.2.2 Materials
All chemicals used were of analytical reagent grade and distilled water was used
throughout. A pharmaceutical grade (99.90 %) cyproheptadine hydrochloride (Cipla
India Ltd, Bangalore, India) Practin 4 mg (Wockhardt Ltd., India) and Ciplactin 4 mg
(Cipla India Ltd., Bangalore, India) tablets were purchased from local commercial
sources. Chloroform and 1, 2- dichloroethane (DCE) (both from Merck, Mumbai, India)
and absolute ethanol were used without any purification.
Standard CPH solution
A stock standard solution containing 2.0 mg mL-1 of pharmaceutical grade CPH

solution was prepared in water and used in method A, and the same was diluted to1.0 mg
mL-1 with water for use in method B.
4.1.2.3 Reagents and solutions
Sodium lauryl sulfate (SLS): A 0.01 M solution of this chemical (SLS) (99.0%, LOBA
Chemie Pvt. Ltd., Mumbai, India) was prepared in water and standardized using
benzethonium chloride [46] then diluted to 0.008 M with water.
Dimethyl yellow: A 0.01% (w/v) dimethyl yellow (DMY) (Rolex Laboratory Reagent,
Mumbai, India) was prepared in absolute ethanol.
Sulphuric acid: A 2 M solution of H2SO4 (Merck, Mumbai, India, Sp. gr. 1.84) was
prepared by appropriately diluting concentrated sulfuric acid with water.
Tetraphenylborate: A 0.04 M solution of tetraphenylborate (TPB) was prepared by

dissolving the required amount of sodium tetraphenylboron (99.5%, S. D. Fine Chem.,
Mumbai, India) in water then diluted to 250 mL with 0.001 M sodium hydroxide solution
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and standardized following the recommended procedure in Japanese Pharmacopoeia [47]

and diluted to 0.004 M using water.
A 0.2 % (w/v) potassium salt of tetrabromophenolphthalein ethyl ester (TBPE) (Sigma-

Aldrich, Inc., USA) was prepared in absolute ethanol.
Walpole buffer: Buffer solution of pH 4.5 was prepared by mixing 0.2 M acetic acid
solution (Merck, Mumbai, India) with 0.2 M sodium acetate solution (S. d. fine Chem
Ltd., Mumbai, India) and adjusting the pH with acetic acid.
4.1.2.4 ASSAY PROCEDURES
Method A (using SLS)
Different aliquots (0.5-10 mL) of standard CPH (2 mg mL-1) solution were

transferred into a 100 mL beaker and the volume was adjusted to 20 mL with water. Two
milliliters of 2 M H2SO4, 0.5 mL of 0.01% DMY and 10 mL of chloroform were added
and the mixture was stirred on a magnetic stirrer for 1 min. The mixture was then titrated
with 0.008 M SLS with vigorous stirring until a color change from yellow to pink occurs
in the organic phase at the end-point.
A blank titration was also performed and the necessary volume corrections were
made. The amount of the drug in the measured aliquot was calculated from:
VM w R
Amount(mg) 
n
where V = volume of SLS, mL; Mw = relative molecular mass of the drug; R = molarity of
the SLS and n = number of moles of SLS reacting with each mole of CPH.
Method B (using TPB)
Different aliquots of the standard solution (2.0-9.0 mL, 1 mg mL-1) of pure CPH
were accurately transferred into a 100 mL beaker and the volume was adjusted to 10 mL
with water. Five milliliters of the Walpole buffer of pH 4.5, 2 drops of TBPE indicator
solution and 10 mL of DCE were added and mixed well by magnetic stirring. The
mixture was titrated against 0.004 M TPB solution with vigorous stirring until the color
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of the organic phase changes from red-violet to yellow at the end point. It is not
necessary to make a blank titration because the color of the organic phase is yellow in the
absence of the drug. The amount of drug in the aliquot was calculated from the equation
given under method A.
Procedure for tablets
Sixty tablets containing CPH (practin and ciplactin- 4 mg each) were weighed
accurately and ground into a fine powder. An amount of powder equivalent to 200 mg of
CPH was weighed into a 100 mL calibrated flask containing about 60 mL of water. The
extraction was done by shaking thoroughly for about 20 min; then the volume was made
up to the mark with water, mixed well and filtered using a Whatman No. 42 filter paper.
The first 10 mL portion of the filtrate was discarded in order to avoid small dilution in the
concentration of CPH because of the wetted filter paper. The resulting (2 mg mL-1) CPH
solution was subjected to titration in method A, following the procedures described above.
The solution was diluted with water to get 1 mg mL-1 CPH and used in method B.
Placebo blank analysis and synthetic mixture analysis
A placebo blank of the composition: talc (100 mg), starch (160 mg), calcium
gluconate (90 mg), lactose (120 mg), magnesium stearate (80 mg) and sodium alginate
(60 mg) was made and its solution was prepared in 50 mL calibration flask as described
under “Procedure for tablets”. A convenient aliquot of the placebo blank solution was
subjected to analyses following the recommended procedures.
To 100 mg of the placebo blank of the composition described above, 200 mg of

CPH was added, homogenized, transferred to a 100 mL calibrated flask and the solution
was prepared as described under ‘Procedure for tablets’. Then the analysis of synthetic
mixture solution was performed for suitable aliquots by following the recommended
procedures.
4.1.3 RESULTS AND DISCUSSION
The two-phase ion-association titration was applied to the determination of some

basic pharmaceutical compounds using indicators for visible end-point detection. Earlier,
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the end-point detection in this type of titrations was based on the movement of the
indicator from one phase to other and it was difficult to detect the end point because the
color of the indicator in the aqueous phase or organic phase will be reflected in the other
phase [48].
The use hydrophobic indicator is an alternative approach, which remains in the
organic phase throughout the titration and gives a very sharp color change [49].
Tsubouchi et al. [50] used the potassium salt of the ethyl ester of
tetrabromophenolphthalein (anionic) indicator in their titration whereas dimethyl yellow
(cationic) indicator was used by Eppert and Liebscher [51] for the two-phase titration.
The above two indicators are useful in the detection of the end point as the change in
color depends on the pH [44, 45].
4.1.3.1 Method development
Method A (using SLS)
This is based on the formation of an ion association complex between the CPH
and the titrant, i.e., sodium lauryl sulphate which is used as titrant with dimethyl yellow
as indicator in the presence of chloroform. After treating CPH tertiary amine (R3N) with
H2SO4, the resulting protonated amine (R3NH+) was titrated with sodium lauryl sulphate
using DMY. The effects of the acid and the extracting solvent were optimized and it was
found that 2 mL of 2 M H2SO4 in a total volume of 20 mL of aqueous phase, and
chloroform as solvent (10 mL) gave a good reproducible and stoichiometric results when
compared to 1,2-DCE and dichloromethane (DCM), in the range investigated.
When the mixture of CPH solution, sulphuric acid, chloroform and the DMY
indicator solution was mixed well, the aqueous phase became colorless, because the
indicator itself was not soluble in water, and a yellow color was developed in the
chloroform phase, because the presence of the indicator in a molecular form. When the
drug sample was titrated with SLS solution, the protonated drug (R3NH+) formed
colorless ion association complex (R3NH+·-Titrant‫ )ــ‬which will be extracted into the
organic phase. When the equivalence point was reached and one drop excess of the titrant
was added, the color of organic phase changed from yellow to pink due to the formation
166
of cationic form of DMY indicator, i.e., DMYH+, which is stabilized by the formation of
stable ion-pair complex with the titrant [DMYH+·SLS‫ ]ــ‬in the organic phase.
The chemical reactions which form the basis for this method can be formulated as
follow:
Before the addition of titrant:

chloroform
R3NH+ + DMYaq R3NH+aq + DMYorg
colorless yellow yellow
Before the equivalence point:
R3NH+aq + SLS- aq [R3NH+ ·SLS -]org
colorless
After the equivalence point:
- -
DMYorg + H+ + SLS [DMYH+·SLS ]org
yellow pink
Method B (using TPB)
This is based on the formation of an ion-pair complex between the CPH and TPB
as titrant in a solution buffered at pH 4.5 using TBPE as indicator and 1, 2-dichloroethane
as extracting solvent. The complex formed in this method is highly pH dependent, so the
effect of pH was studied carefully and it was found that 5 mL of Walpole buffer of pH
4.5 in a total volume of 10 mL of aqueous phase and 10 mL DCE solvent gave the best
end-points and most consistent titers than chloroform or DCM.
The chemical reactions for this titration can be explained as:
Before addition the titrant:

DCE
R3NH+ + TBPE [R3 NH+·TBPE-]org
colorless blue red-violet
Before the equivalence point:

- -
R3NH+ + Titrant [R3NH+-Titrant ]org
colorless colorless
After the equivalence point:
- -
Titrant + H + + [R3NH+·TBPE-]org [Titrant ·R3 NH+ ]org + TBPEH
red-violet yellow
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When the mixture of CPH solution, buffer, DCE and TBPE indicator solution was
mixed well, the aqueous phase became colorless, and a red-violet color developed in the
DCE phase, because the indicator forms an organophilic ion-pair complex with the drug.
When the drug sample was titrated with TPB solution, the protonated drug (R3NH+)
formed colorless ion association (R3NH+·Titrant‫ )ـــ‬which will be extracted into the
organic phase. Near the equivalence point of the titration, the organic phase starts to turn
green and when one drop excess of the titrant was added, the color of the organic phase
changed from red-violet to yellow due to the formation of molecular form of the
indicator, i.e., TBPEH, Scheme 4.1.1.
a)
CH3 H CH3
+H+
N N N N N N
CH3 -H+ CH3
DMY(molecular form) DMYH+ (cationic form)

yellow red-pink
b)
COOC2H5 COOC2H5
Br C +H+ Br C
Br Br
-H+
O O O OH
Br Br Br
Br
TBPE - (anionic form) TBPEH (molecular form)

blue yellow
Scheme 4.1.1 Effect of pH on the indicator color

a, Dimethyl yellow; b, Tetrabromophenolphthalein ethyl ester.
4.1.3.2Method validation
The validation of the methods was done according to the present ICH guidelines
[52].
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Range and Stoichiometry
The proposed procedures are applicable over the ranges of 1.0-20 and 2.0-9.0 mg
of CPH for method A and method B, respectively. The reaction stoichiometry was
calculated to be 1:1 for both methods (CPH:SLS) in method A and (CPH:TPB) in method
B, owing to the presence of one basic nitrogen atom in the CPH.
Accuracy and precision
Accuracy was evaluated as percentage relative error between the measured and
taken amounts of CPH (Bias %). The results, compiled in Table 4.1.1, show that the
accuracy is good for both methods. Precision of the methods was calculated in terms of
intermediate precision (intra-day and inter-day). Three different amounts of CPH (within
the working limits) were analyzed in seven replicates during the same day and five
consecutive days. RSD (%) values of the intra-day and inter-day studies showed that the
precision was good for both methods, (Table 4.1.1).
Table 4.1.1 Results of intra-day and inter-day accuracy and precision study
Intra-day accuracy and Inter-day accuracy and
precision precision
PAM
PAM PAM
Method taken,
found, RE,% RSD,% found, RE,% RSD, %
mg
mg mg
A 6.00 6.04 1.08 0.65 6.08 1.36 1.68
12.0 12.20 0.96 1.32 12.24 1.24 1.17
18.0 18.12 1.18 1.24 18.15 1.15 1.33
4.00 4.06 1.29 1.08 4.09 1.55 1.36
B 6.00 6.16 1.92 1.78 6.18 2.18 2.08
8.00 8.14 1.75 1.44 8.16 1.16 1.92
RE-relative error, RSD- relative standard deviation.
Selectivity
To determine the selectivity of the methods, the analytical placebo was prepared
and subjected to analysis by the proposed methods. To identify the interference by
common tablet excipients, synthetic mixture was prepared and subjected to analysis by
the proposed methods after solution preparation using the procedure described earlier.
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The percent recoveries of CPH were 98.46 ± 1.64 (n =5) and 99.41 ± 1.21 (n =5) by
method A and B, respectively, suggesting no interference by the excipients in the assay of
CPH under the described optimum conditions.
Robustness and ruggedness
The robustness of the methods was evaluated by making small incremental

changes in volumes of H2SO4 (2±0.5 mL) and CHCl3 (10±1 mL) in method A, Walpole
Buffer of pH 4.5 (5±0.5 mL) and DCE (10±1 mL) in method B and the effect of the
changes was studied by calculating the RSD values. The changes had negligible influence
on the results as revealed by small intermediate precision values expressed as % RSD.
The values were lying in the range 1.35-2.85 %.Method ruggedness was expressed as the
RSD of the same procedure applied by four different analysts as well as using four
different burettes. The inter-analysts RSD were within 2.65 % whereas the inter-burettes
RSD for the same CPH amount were ranged from 1.54 to 3.35 % suggesting that the
developed method was rugged.
Application to tablets
The proposed methods were successfully applied to the determination of CPH in
two representative tablets Practin and ciplactin. The results obtained are shown in Table
4.1.2 and were compared with those obtained by the reference method [5] by means of
Student’s t- and F-tests [53] at 95 % confidence level. The reference method consisted of
the measurement of the absorbance of CPH tablet extract 0.1 M HCl at 286 nm. In all the
cases, the average results obtained by the proposed methods and reference method were
statistically identical, as the difference between the average values were not significant at
95 % confidence level with respect to accuracy and precision.
Recovery studies
Accuracy of the proposed methods was further confirmed using the standard
addition procedure. Pre-analyzed tablet powder was spiked with pure CPH at three
different levels (50, 100 and 150% of the quantity present in the tablet powder) and the
total was measured by the proposed methods. The determination with each amount was
repeated three times and the results of this study presented in Table 4.1.3.
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Table 4.1.2 Results of analysis of tablets by the proposed methods and statistical comparison with
reference method
Found (% of nominal amount ± SD)*
Tablet brand Nominal Reference method Proposed methods
name amount, mg Method A Method B
98.86 ± 1.04 99.48 ± 1.12
a
Practin 4 100.3±0.76 t = 3.3 t = 2.9
F= 4.5 F= 5.2
98.76 ± 1.87 99.26 ± 2.00
b
Ciplactin 4 100.8±0.85 t = 3.6 t = 3.2
F= 3.5 F= 4.3
*
Mean value of five determinations.
**
Marketed by: aWockhardt Ltd., India, bCipla India Ltd., India.
Tabulated t-value at the 95% confidence level is 2.78; Tabulated F-value at the 95% confidence level is 6.39.
Table 4.1.3 Results of recovery study via standard addition method

Method A Method B
CPH Pure CPH Pure
Total Pure CPH Total Pure CPH
Tablet in CPH in CPH
found, recovered*, found, recovered*,
studied tablet, added, tablet, added,
mg Percent ±SD mg Percent ± SD
mg mg mg mg
7.90 4.0 11.86 99.26±1.11 2.98 1.5 4.49 99.33±1.06
Practin 7.90 8.0 16.15 103.2±1.82 2.98 3.0 6.10 102.3±1.97
7.90 12.0 19.83 99.35±0.98 2.98 4.5 7.55 101.8±1.01
5.92 3.0 8.96 101.7±1.45 2.96 1.5 4.40 99.25±1.26
Ciplactin 5.92 6.0 11.97 100.8±1.68 2.96 3.0 6.16 101.9±1.63
5.92 9.0 15.22 103.1±2.07 2.96 4.5 7.72 102.0±1.12
*Mean value of three determinations.
171
SECTION 4.2
SENSITIVE AND SELECTIVE METHODS FOR THE DETERMINATION OF

CYPROHEPTADINE IN TABLETS USING N-BROMOSUCCINIMIDE AND
TWO DYES
4.2.1INTRODUCTION
N-bromosuccinimide (NBS), chemically known as 1-Bromo-2,5-

pyrrolidenedione, is a brominating and oxidizing agent that is used as source for bromine
in radical reactions i.e. allylic and benzylic bromination and various electrophilic addition
reactions [54]. In oxidimetric reaction, the actual oxidizing agent is either the
hypobromous acid, formed by hydrolysis of NBS or the monovalent positive bromine of
the NBS (the bond between bromine and nitrogen is polarized by the two neighbouring
carbonyl groups). In both cases, bromide is formed during the reaction and the
stoichiometric relations are therefore the same [55]:
O O
N Br + H2O HBrO + N H
O O
BrO + H2O + Br + 2 OH
O O
N Br + 2 H + + N H + HBr
O O
Various methods have been developed and reported for the bromination of many
organic compounds using a variety of brominating agents such as molecular bromine or
Br2 in combination with an acid or an oxidizing agent. But, these reagents are potentially
hazardous, and it is difficult to handle and store elemental halogens [56]. From the ‘green
chemistry’ point of view, the replacement of such harmful reagents with non-toxic,
inexpensive, commercially available, non-polluting, and more selective reagents is an
 This work has been published in Chem. Ind. Chem. Eng. Quart. 2012,18 (3), 449−458.
172
important goal. Among various brominating reagents, NBS is environmentally friendly,

easy to work with and is extensively used for allylic, benzylic, and aromatic nuclear
bromination under mild conditions. A major advantage of the use of NBS is that the by-
product succinimide can be easily recovered and recycled to NBS [57].
The compiled recent research work revealed that NBS is widely used in the
development of analytical methods. In the monograph compiled by Mathur and Narang
[58], a survey of the analytical applications of NBS for the determination of organic
compounds is given. The analytical applications of NBS based on bromination and
oxidation reactions have found extensive applications in the determination of a variety of
organic compounds including those of pharmaceutical interest. For e.g., phenothiazines
[59-61], antiinflamatory drugs [62], ethorvynol [63], clotrimazole [64], pyridoxine
hydrochloride [65], benzodiazopines [66], thioxanthene derivatives [67], acetylenic
hypnotis [68] isomiazid [69], salbutamol sulphate [70] and oxyphen butazone [71] are
some of the pharmaceutical substances which have been assayed using NBS. Likewise,
NBS either alone or in combination with some other reagents has been widely applied in
the direct or indirect spectrophotometric determination of several pharmaceutical
substances which include phenothiazines [59-61], chindamycin [72], sulphonamides [73]
diclofenac sodium [74], propanolol [75], piperezine [76], sulphonamide diuretis [77],
paparerine HCl [78], nifartimox [79], ascorbic acid [80], astemizole [81], amoxycilin and
cafadroxil [82], omeprazole [83], cefotaxime sodium [84], gatifloxacin [85], olanzapine
[86], oxcarbazapine [87].
From the literature survey presented in Section 4.0.2, NBS was not used before
for assaying CPH by any methods. The present author developed one titrimetric and two
spectrophotometric methods for the determination of CPH in bulk drug and tablets
employing NBS as a brominating agent and two dyes, erioglaucine (EG) and meta-cresol
purple (MCP) as auxiliary reagents. In titrimetry, a measured excess of NBS is added to
an acidified solution of CPH and the unreacted NBS is determined iodometrically.
Spectrophotometry involves the addition of a known excess of NBS to CPH in acid
medium followed by estimation of residual NBS by reacting with a fixed amount of
either erioglaucin and measuring the absorbance at 630 nm (method A) or meta-cresol
173
purple and measuring the absorbance at 540 nm (method B). The details about the
method development and validation are presented in this Section (4.2).
4.2.2 EXPERIMENTAL
4.2.2.1 Apparatus
The instrument is the same that was described in Section 2.2.2.1.
4.2.2.2 Materials
All the reagents were of analytical-reagent grade and distilled water was used
throughout the investigation. The pure CPH and its tablets used were the same described
in Section 4.1.
N-bromosuccinimide (NBS): An approximately 0.01 M solution was prepared by

dissolving about 1.8 g of NBS (SRL Research Chemicals, Mumbai, India) in water with
the aid of heat and diluted to one litre with water. The solution was standardized
iodometrically [55] and kept in an amber coloured bottle and stored in a refrigerator; and
used in titrimetry. It was diluted appropriately to get 65 and 120 g mL-1 NBS for use in
spectrophotometric method A and method B, respectively.
Hydrochloric acid: Concentrated hydrochloric acid (Merck, Mumbai, India; sp. gr. 1.18)
was diluted appropriately with water to get 2 M.
Potassium iodide: A 10% solution was prepared by dissolving 10 g of the

chemical(Merck, Mumbai, India) in 100mL of water and used in titrimetric method.
Starch (1%) was prepared as described in Section 3.1.2.3.
Erioglaucine solution, EG, (300 µg mL-1): The solution was prepared by dissolving 30 mg
of dye (Loba Chemie, Mumbai, India) in water and diluting to the mark with water in a
100 mL calibrated flask and used in spectrophotometric method A.
Meta-cresol purple solution, MCP, (80 µg mL-1): A 200 µg mL-1 stock solution was
first prepared by dissolving 20 mg of dye (Loba Chemie, Mumbai, India) in 2 mL of 0.1
M NaOH, and diluted to volume with water in a 100 mL calibrated flask. The solution
174
(200 µg mL-1) was diluted with water to get the working concentration of 80 µg mL-1
MCP for use in spectrophotometric method B.
Stock standard solution of CPH: A stock standard solution equivalent to 1.5 mg mL-1
CPH was prepared by dissolving accurately weighed 150 mg of pure drug in water, and
diluted to the mark in a 100 mL calibrated flask and used in titrimetric work. Another
stock solution equivalent to 200 µg mL-1 of CPH was prepared by dissolving accurately
weighed 20 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 concentrations of 5 and
20 µg mL-1 of CPH for use in spectrophotometric method A and method B, respectively.
Titrimetry
A 10 mL aliquot of pure drug solution containing 1.5-15 mg of CPH was

accurately measured and transferred into a 100 mL titration flask. The solution was
acidified by adding 5 mL of 2 M HCl followed by the addition of 10 mL of 0.01 M NBS
by means of pipette. The content was mixed well and the flask was kept aside for 10 min
with occasional swirling. Then, 5 mL of 10 % potassium iodide was added to the flask
and the liberated iodine was titrated with 0.02 M sodium thiosulphate to a starch end
point. A blank titration was run under the same conditions. The drug content in the
aliquot was calculated.
V  Mol.wt  R
Amountmg  
n
where V = volume of NBS solution reacted with the drug, mL; Mol.wt = relative
molecular mass of drug; R = strength of NBS, mol mL-1 and n = number of moles of
NBS reacting with each mole of drug.
Spectrophotometry using erioglaucine (Method A)
Different aliquots (0.2,0.5,1.0,----4.0 mL) of a standard 5 g mL-1 CPH solution

were transferred into a series of 10 mL calibrated flasks by means of a micro burette and
175
the total volume was adjusted to 4 mL by adding adequate quantity of water. To each
flask were added 1 mL each of 2 M HCl and 1.0 mL of 65 g mL-1NBS solution, the last
being measured accurately. The flasks were stoppered, content mixed and let stand for 10
min with occasional shaking. Finally, 1.0 mL of 300 g mL-1 EG solution (accurately
measured) was added and the volume was made up to 10 mL and mixed. The absorbance
of each solution was measured at 630 nm against a reagent blank after 5 min.
Spectrophotometry using meta-cresol purple (Method B)
Varying aliquots (0.2,0.5,1.0,----6.0 mL) of a standard 20 g mL-1 CPH solution

were transferred into a series of 10 mL calibrated flasks by means of a micro burette and
the total volume was brought to 6 mL by adding water . To each flask were added 1 mL
of 2 M hydrochloric acid and 1.0 mL of NBS solution (120 g mL-1) by means of a micro
burette. The content was mixed well and the flasks were kept aside for 10 min with
intermittent shaking. Finally, 1.0 mL of 80 g mL-1 MCP solution was added to each
flask, the volume was made up to 10 mL, mixed well and absorbance measured against a
reagent blank at 540 nm after 5 min.
In either spectrophotometric method, a standard graph was prepared by plotting

the absorbance versus the concentration of CPH. The concentration of the unknown was
read from the calibration graph or computed from the regression equation derived using
Beer’s law data.
Fifty tablets (Practin and ciplactin) each containing 4 mg of CPH were weighed
accurately and ground into a fine powder. An amount of the powder equivalent to 150 mg
of CPH was accurately weighed into a 100 mL volumetric flask, 60 mL water was added
and content shaken thoroughly for about 20 min. The volume was diluted to the mark
with water, mixed well and filtered using Whatman No.42 filter paper. First 10 mL
portion of the filtrate was rejected and a convenient aliquot of filtrate (containing 1.5 mg
mL-1 CPH) was taken for assay by titrimetric procedure. The tablet extract was diluted
stepwise to get 5 and 20 g mL-1 CPH concentrations for use in spectrophotometric
176
method A and method B, respectively. A suitable aliquot was then subjected to analysis
following the procedures described earlier.
Placebo blank and synthetic mixture analyses
Hundred mg of the placebo blank prepared in Section 4.1.2.4 was taken and its
solution prepared as described under ‘Procedure for tablets’ and then analyzed using the
procedures described above.
To 75 mg of the placebo blank, 100 mg of CPH was added and homogenized,

transferred to 50 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. .
The present work involves the bromination of CPH by NBS followed by

determination of surplus NBS after the bromination reaction is ensured to be complete. In
titrimetry, the unreacted NBS is determined iodometrically. The piperidine ring in CPH,
because of steric hindrance is not vulnerable to bromination, and hence, preferably
bromination takes place in the cycloheptene ring. While in spectrophotometric methods;
it is determined by reacting with a fixed amount of either EG and measuring the
absorbance at 630 nm or MCP and the absorbance at 540 nm. The spectrophotometric
methods make use of the bleaching action of NBS on either of the two dyes, where the
discoloration is caused by oxidative destruction of the dye. The tentative reaction
pathway of titrimetric and spectrophotometric methods is shown in Scheme 4.2.1.
O
O
2 N Br + 2H 2O 2HBrO
+ 2 N H
O
O
NBS
Br Br
H+
+ 2HBrO
+ 2H2O
.HCl
N .HCl
CH3 N
CH3
CPH Brominated CPH
177
H+
CPH + Known excess of NBS Reaction product of the drug + Unreacted NBS
Liberated I2 titrated against thiosulphate
+KI Titrimetry
Spectrophotometry
Unreacted NBS Unbleached color of EG measured at 630 nm(Method A)
+ EG
+MCP Spectrophotometry
Unbleached color of MCP measured at 540 nm(Method B)
Scheme 4.2.1Tentative reaction pathway for titrimetry, spectrophotometric method A and

method B
4.2.3.1Titrimetry
Direct titration of CPH with NBS in acid medium was not successful. However
the back titrimetric assay was found feasible when the reactants were allowed to stand for
some time in acid medium. In this procedure, a known excess of NBS was allowed to
react with CPH in acid medium and the unreacted NBS was subsequently determined
iodometrically. The reaction stoichiometry was found to be 1:2 (CPH:NBS). Reproduci-
ble and stoichiometric results were obtained when HCl medium (0.2 to 0.6 M) was
employed. At optimum acid concentration of 0.4 M (5 mL of 2 M HCl in a total volume
of 25 mL), the reaction goes to completion in 10 min and contact time upto 45 min had
no effect on the stoichiometry or the results.. At lower acid concentration (less than 5 mL
of 2 M HCl) the reaction stoichiometry was slightly less than 2 and higher acid
concentrations exceeding 0.6 M HCl overall resulted in slightly higher ‘n’ values. Under
the optimized reaction condition, there was found to be a definite reaction stoichiometry
of 1:2 between CPH and NBS was found within the range of 1.5-15 mg CPH. A 10 mL
volume of 0.01 M NBS was found adequate for quantitative reaction CPH in the range
178
investigated and contact time of 5 minute was sufficient for the complete liberation of
iodine from the unreacted NBS.
4.2.3.2 Spectrophotometry
Absorption spectra
The proposed methods are based on the determination of unreacted bromine after
the reaction between the drug and NBS is judged to be complete. The greenish color of
the unreacted EG in acid medium absorbed maximally at 630 nm (method A) and the
reddish-pink color of unreacted MCP in acid medium peaked at 540 nm (method B). The
absorption spectra of both the methods are presented in Figure 4.2.1.
Many dyes are irreversibly destroyed to colorless species by oxidizing agents in

acid medium [88] and this observation has been exploited for the indirect
spectrophotometric determination of some pharmaceutical compounds [80-87]. In the
proposed spectrophotometric methods, the ability of NBS to effect bromination of CPH
and irreversibly destroy EG and MCP to colourless products in acid medium has been
capitalized. Both methods are based on the bromination of the drug by measured excess
of NBS and subsequent determination of the latter by reacting with EG or MCP, and
measuring the absorbance at 540 or 630 nm. In either method, the absorbance increased
linearly with increasing concentration of drug. CPH when added in increasing
concentrations to a fixed concentration of NBS consumes the latter and there will be a
concomitant decrease in the concentration of NBS. When a fixed concentration of either
dye is added to decreasing concentration of NBS, a concomitant increase in the
concentration of dye is obtained. This is observed as a proportional increase in the
absorbance at the respective wavelengths of maximum absorption with increasing
concentration of CPH (Figure 4.2.1).
179
0.50
0.8
0.45
i i
0.7
0.40 ii ii
iii iii
0.6
0.35 iv iv
0.30 0.5
A b s o rb a n c e
A b so rb a n ce
0.25
0.4
0.20
0.3
0.15
0.2
0.10
0.05 0.1
0.00 0.0
510 540 570 600 630 660 690 720 450 480 510 540 570 600 630 660 690 720
Wavelength, nm Wavelength, nm
(a) (b)
Figure 4.2.1 Absorption spectra:
a) Method A. i- without CPH; ii- 1 µg mL-1 CPH; iii-0.75 µg mL-1 CPH and
iv-0.5 µg mL-1 CPH.
b) Method B. i-without CPH; ii- 8 µg mL-1 CPH; iii-6 µg mL-1 CPH and iv-
4 µg mL-1 CPH.
Effect of reagent concentration
Preliminary experiments were performed to fix the upper limits of the EG and
MCP that could produce a reasonably high absorbance, and these were found to be 30 µg
mL−1 EG in method A and 8 µg mL−1 MCP in method B. To fix the optimum
concentration of NBS, different concentrations of NBS were reacted with a fixed
concentration of EG or MCP (30 and 8 µg mL−1) in HCl medium and the absorbance was
measured at 630 and 540 nm, respectively. A constant and minimum absorbance resulted
with 6.5 and 12 µg mL-1 NBS for method A and method B, respectively. Different
concentrations of CPH were reacted with 1mL NBS of 65 µg mL−1 in method A and 120
µg mL-1 in method B in HCl medium before determining the residual NBS via the
180
reaction scheme illustrated earlier. This facilitated the optimization of the linear dynamic
range over which each procedure could be applied for the assay of CPH.
Effect of reaction medium
The reaction between CPH and NBS was performed in different acid media viz.,
sulphuric acid,hydrochloric acid and perchloric acid. Hydrochloric acid was found to be
the ideal medium for the bromination of CPH by NBS as well as the latter’s
determination employing either dye. The effect of acid concentration on the reaction
between CPH and NBS was studied by varying the concentration of HCl keeping the
concentrations of NBS and drug fixed. Higher the acid concentrations showed lower
sensitivity, hence 1 mL of 2 M HCl in a total volume of 10 mL was found optimal to
achieve maximum absorbance of sample and minimum absorbance of blank in both the
methods.
Study of reaction time and stability
Under the described experimental conditions, for a quantitative reaction between

CPH and NBS, contact time of 10 min was found necessary in both methods at room
temperature. After addition of dye, the reaction between NBS and dye was instantaneous
and absorbance of the unreacted dye was stable at least 45 min in method A and 60 min
in method B.
4.2.3.4 Method validation
Linearity and sensitivity
The proposed methods were validated for linearity, selectivity, precision,

accuracy, robustness and ruggedness, and recovery. Over the range investigated (1.5-15
mg), a fixed stoichiometry of 1:2 (CPH:NBS) was obtained in titrimetry which served as
the basis for calculations. In spectrophotometry, a linear correlation (Figure 4.2.2) was
found between absorbance at max and concentration of CPH in the ranges given in Table
4.2.1. 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 values obtained from this investigations are presented also presented in the same
table. Sensitivity parameters such as apparent molar absorptivity and Sandell sensitivity
181
values and the limits of detection and quantification are calculated as per the current ICH
guidelines [52] which are compiled in Table 4.2.1 that speaks of the excellent sensitivity
of the proposed method. Limits of detection (LOD) and quantification (LOQ) were also
calculated and present in the same table.
1.0
0.8
0.8
0.6
0.6
Absorbance
Absorbance
0.4
0.4
0.2 0.2
0.0 0.0
0.0 0.5 1.0 1.5 2.0 0 2 4 6 8 10 12
-1 -1
Concentration of CPH, µg mL Concentration of CPH, µg mL
Method A Method B
Figure 4.2.2 Calibration curves
Table 4.2.1 Sensitivity and regression parameters

Parameter Method A Method B
max, nm 630 540
-1
Beer's law limits, µg mL 0.1 – 2.0 0.4 – 12
-1 -1
Molar absorptivity, L mol cm 1.4×105 2.2×104
Sandell sensitivity*, µg cm-2 0.0023 0.0141
-1
Limit of detection, g mL 0.03 0.24
-1
Limit of quantification, g mL 0.09 0.71
Regression equation, Y**
Intercept, (a) 0.0051 0.0095
Slope, (b) 0.4389 0.0658
Standard deviation of intercept (Sa) 0.4765 0.0605
Standard deviation of slope (Sb) 0.3552 0.0166
Regression coefficient (r) 0.9992 0.9997
a
Limit of determination as the weight in µg mL-1 of solution, which corresponds to an
absorbance ofA = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm.b
Y=a+bX, Where Y is the absorbance, X is concentration in µg mL-1
182
In order to study the precision and accuracy of the proposed methods, three
amounts/concentrations of pure CPH within the linearity range were analyzed, each
determination being repeated seven times (intra-day precision) on the same day and one
time each for five days (inter-day precision). The percentage relative standard deviation
(%RSD) was ≤ 2.7 % (intra-day) and ≤ 3.14% (inter-day). In addition, the accuracy of the
proposed method was measured by calculating the percentage relative error (%RE),
which varied between 0.46% and 3%. The results of this study compiled in Table 4.2.2
indicate the high accuracy and precision of the proposed methods.
precision precision
CPH
(n=5) (n=5)
taken
CPH CPH
Method mg/
found found
µg mL-1 %RE %RSD %RE %RSD
mg/ mg/
-1 -1
µg mL µg mL
3.0 2.92 2.22 2.03 2.91 2.83 3.14
Titrimetric
6.0 6.10 2.34 1.46 5.89 1.88 2.62
method
9.0 9.15 2.58 2.59 8.77 2.59 1.93
0.5 0.49 1.81 2.71 0.48 2.91 3.12
Spectrophotometric
1.0 1.02 2.27 2.24 0.98 1.26 2.39
Method A
1.5 1.47 1.89 1.67 1.46 2.29 2.91
4.0 3.92 2.01 1.82 3.87 3.02 2.03
Spectrophotometric
6.0 6.08 1.46 2.06 5.83 2.75 2.72
Method B
8.0 8.13 1.73 1.68 7.78 2.78 3.08
RE- Relative error and RSD- Relative standard deviation
mg in titrimetry and µg mL-1 in spectrophotometry.
Selectivity
Selectivity was evaluated by both placebo blank and synthetic mixture analyses.
The placebo blank, consisting the composition as mentioned under ‘Placebo blank
analysis’ was prepared and analyzed as described under the recommended procedures.
The resulting absorbance readings for the methods were same as the reagent blank,
inferring no interference from the placebo. The selectivity of the methods was further
183
confirmed by carrying out recovery study from synthetic mixture. The percent recoveries
of CPH were 102.1±1.35 for titrimetry 98.7±1.18 and 101.4±1.63 for spectrophotometric
method A and method B, respectively. This confirms the selectivity of the proposed
methods in the presence of the commonly employed tablet excipients.
To evaluate the robustness of the methods, two important experimental variables,

viz., the amount of acid and reaction time, were slightly varied, and the capacity of the
methods was found to remain unaffected by small deliberate variations. The results of this
study are presented in Table 4.2.3 and indicate that the proposed methods are robust.
Method ruggedness is expressed as %RSD of the same procedure applied by three
analysts and using three different spectrophotometers by the same analyst. The inter-
analysts’ and inter-instruments’ RSD values were ≤ 3.04 % indicating ruggedness of the
proposed methods. The results of this study are presented in Table 4.2.3.
Table 4.2.3 Results of robustness and ruggedness expressed as intermediate precision

(% RSD)
Robustness Ruggedness
Nominal Reaction Volume Inter- Inter-
Method amount/ times** of acid$ analysts instruments#
concentration* (n=3) (n=3) (n=3)
3.0 1.58 1.23 0.76 0.62
Titrimetry 6.0 1.26 1.41 0.85 0.72
9.0 1.34 1.38 1.04 0.58
Spectrophotometr 0.5 2.65 2.13 1.04 2.42
ic 1.0 3.14 2.86 1.28 2.16
Method A 1.5 3.38 2.73 0.92 1.85
Spectrophotometr 4.0 1.85 1.05 0.73 2.16
ic 6.0 2.16 1.27 1.12 2.72
Method B 8.0 2.08 1.97 1.08 3.04
* mg in titrimetry and µg mL-1 in spectrophotometry.
** Reaction time was 10±1 min,
$
Volume of HCl, 5±0.5 mL in titrimetry, 1±0.1 mL in spectrophotometry.
#
burettes in titrimetry and spectrophotometers in method A and method B
184
The results presented in Table 4.2.4 showed that there was a close agreement
between the results obtained by the proposed methods and the label claim. The results
were also compared with those of the reference method [6] statistically by a Student's t-
test for accuracy and variance ratio F- test for precision at 95 % confidence level. The
reference method describes non-aqueous titration with perchloric acid as titrant where the
end point was located visually using crystal violet as indicator. The calculated t- and F-
values indicate that there is no significant difference between the proposed methods and
the reference method with respect to accuracy and precision.
Recovery studies
To further ascertain the accuracy of the proposed methods, a standard addition
technique was followed. A fixed amount of drug from pre-analyzed tablet powder was
taken and pure drug at three different levels (50, 100 and 150 % of that in tablet powder)
was added. The total was found by the proposed methods. The determination at each
level was repeated three times and the percent recovery of the added standard was
calculated. Results of this study presented in Table 4.2.5.
185
Table 4.2.4 Results of analysis of tablets by the proposed methods and statistical comparison with the official
method
Founda (Percent of label claim ±SD)
Tablet Label Proposed methods
Reference
Brand name claim* Spectrophotometric Spectrophotometric
method Titrimetric
Method A Method B
97.98± 0.86 98.78± 1.42 98.62± 1.38
b
Ciplactin 4 99.36±1.65 t =2.61 t =2.80 t =2.90
F=3.68 F =1.35 F =1.42
100.8±1.27 100.65 ±1.58 99.16 ± 1.22
c
Practin 4 100.4±1.86 t=2.09 t =2.79 t =2.94
F=2.14 F=1.38 F =2.32
a
Mean value of five determinations,
b
Cipla India Ltd., India. cWockhardt Ltd., India.
The value of t and F (tabulated) at 95 % confidence level and for four degrees of freedom are 2.77 and 6.39, respectively.

Titrimetry Spectrophotometric Method A Spectrophotometric Method B
Tablet CPH in Pure Total Pure CPH CPH in Pure Total Pure CPH CPH in Pure Total Pure CPH
studied tablet, CPH found, recovered* tablet, CPH found, recovered *
tablet, CPH found, recovered*
mg added, mg Percent ± SD µg mL-1 added, µg mL-1 Percent ± SD µg mL-1 added, µg mL-1 Percent ± SD
mg µg mL-1 µg mL-1
4.43 3.0 7.53 103.33±1.45 0.48 0.25 0.73 97.85 ± 1.19 2.96 1.5 4.43 98.55 ± 0.81
Ciplactin 4.43 4.5 9.04 102.42±1.02 0.48 0.50 1.09 101.50 ± 0.79 2.96 3.0 5.93 99.26± 0.58
4.43 6.0 10.58 102.51±0.98 0.48 0.75 1.27 102.50 ± 1.76 2.96 4.5 7.50 100.91 ± 0.84
4.52 3.0 7.55 101.0±0.74 0.53 0.25 0.74 98.56 ± 1.19 3.98 2.0 5.96 98.48 ± 1.62
Practin 4.52 4.5 9.12 102.22±1.24 0.53 0.50 1.02 102.00 ± 0.79 3.98 4.0 8.07 101.79 ± 1.02
4.52 6.0 10.61 101.5±1.16 0.53 0.75 1.28 103.02 ± 1.27 3.98 6.0 10.19 103.82 ± 1.82
*Mean value of three determinations
186
SECTION 4.3
RAPID AND SENSITIVE SPECTROPHOTOMETRIC METHODS FOR THE

DETERMINATION OF CYPROHEPTADINE USING THREE
SULPHONTHALEIN DYES
4.3.1 INTRODUCTION
The chemistry and analytical utility of ion-pair complexation reaction is described

in Section 2.3.1.
In the literature survey presented in Section 4.0.2, six extractive
spectrophotometric methods for the determination of CPH based on ion-pair complex
formation with bromocresol green [9], Solochrome Black-T, Solochrome Dark Blue and
Fast Sulphon Black FF [11], benzyl orange [12] and bromophenol blue [13] have been
reported .These methods require strict pH control, tedious and time consuming extraction
step, and are prone to inaccuracy due to incomplete extraction of the analyte. In this
Section (4.3), three simple extraction-free spectrophotometric methods using three
sulphonthalein dyes are described. The methods are based on formation of yellow ion-
pairs between base form of the drug and three sulphonthalein dyes; bromocresol green
(method A), bromocresol purple (method B), and thymol blue (method C) in
dichloromethane medium followed by absorbance measurement at 430, 415 and 425 nm,
respectively. The method development, validation and its applications are presented in
this Section (4.3).
4.3.2 EXPERIMENTAL
4.3.2.1 Apparatus
The instrument is the same that was described in Section 2.2.2.1.
4.3.2.2 Materials
All the reagents were of analytical-reagent grade and distilled water was used
throughout the investigation. Dichloromethane and acetone were purchased from Merck
(Mumbai, India).The pure CPH and its tablets used were the same described in Section
4.1.
187

Bromocresol green (BCG), bromocresol purple (BCP) and thymol blue (TB): The
solutions (all from Loba Chemie Ltd, Mumbai, India) were prepared daily as 0.1% BCG
and 0.1% BCP in dichloromethane and 0.025% TB in acetone.
Standard drug solution in free base form (CPT)
Into a 125 mL separating funnel, an accurately weighed 22.57 mg of pure CPH

was transferred and dissolved in about 20 mL of water and the solution rendered alkaline
by adding ammonia (concentrated) and the content was shaken for 5 min. The free base
(CPT) formed was extracted with four 20.0 mL portions of dichloromethane, the extract
was passed over anhydrous sodium sulphate and collected in a 100 mL volumetric flask.
The volume was made up to mark with dichloromethane and the resulting solution (200
µg mL-1 CPT) was further diluted with dichloromethane to get a working concentration
of 15 µg mL-1 CPT for method A and 20 µg mL-1 CPT for both method B and method C.

Method A (using BCG)
Varying aliquots (0.25, 0.5, 1.0, 2.0, 3.0, 4.0 mL) of 15 µg mL-1 standard CPT
solution in dichloromethane were measured accurately and transferred into a series of 5
mL volumetric flasks. To each flask was added 1 mL of 0.1 % BCG, the content was
mixed well and diluted to the mark with dichloromethane. After 5 minutes, the
absorbance of each solution was measured at 430 nm against the respective reagent blank
prepared without addition of CPT solution.
Method B (using BCP) and method C (using TB)
Different aliquots (0.25, 0.5, 1.0, 2.0, 3.0, 4.0 mL) of CPT standard solution (20
µg mL-1) were measured accurately using microburette and transferred into a series of 5
mL volumetric flask. To each flask, 1 mL of 0.1 % BCP solution in method B or 0.025%
TB in method C, was added, diluted to the mark with dichloromethane and mixed well.
The absorbance of the resulting yellow colored chromogen was measured after 5 min at
415 nm in method B and 425 nm in method C against respective reagent blank.
188
In all the methods, a standard graph was prepared by plotting the increasing
absorbance values vs concentration of CPT. 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.
Twenty tablets (Practin and Ciplactin 4 mg) were weighed and pulverized. The
amount of tablet powder equivalent to 22.57 mg of CPH was transferred into a 100 mL
volumetric flask containing 60 mL of water. The content was shaken well for 20 min and
diluted to the mark with water. The resulting solution was filtered through Whatmann
No. 42 filter paper and the filtrate was collected in to a 125 mL separating funnel. The
salt (CPH) was converted to free base as described earlier, CPT solutions of
concentrations 15 for method A and 20 µg mL-1 for method B and method C, were
prepared as described under the general procedure for pure drug and a suitable aliquot
was used for assay by applying procedures described earlier.
Thirty mg of the placebo blank prepared in Section 2.1.2.4 was taken and its

by the procedures described above.
4.3.3.1 Absorption spectra

The absorption spectra of the ion-pair complexes, formed between CPT and each
of BCG, BCP and TB recorded at 340-500 nm against the respective blank solution are
shown in Figure 4.3.1. The yellow ion-pair complexes showed maximum absorbance at
189
430, 415 and 425 nm for CPT-BCG, CPT-BCP and CPT-TB, respectively. The
measurements were thus made at these wavelengths.
0.6 0.7
0.6 c
0.5 d
a
b 0.5
0.4
Absorbance
Absorbance
0.4
0.3
0.3
0.2 0.2
0.1
0.1
0.0
0.0 340 360 380 400 420 440 460 480 500
340 360 380 400 420 440 460 480 500
Wavelength, nm
Wavelength, nm
0.6
0.5 e
f
0.4
Absorbance
0.3
0.2
0.1
0.0
340 360 380 400 420 440 460 480 500 520
Wavelength, nm
Figure 4.3.1 Absorption spectra of:

a. CPT-BCG ion-pair complex (6.0 µg mL-1 CPT), b. blank (Method A),
c. CPT-BCP ion-pair complex (8.0 µg mL-1 CPT), d. blank (Method B),
e. CPT-TB ion-pair complex (8.0 µg mL-1 CPT) and f. blank (Method C).
4.3.3.2 Reaction pathway
Extraction-free spectrophotometry based on ion-pair complexation reaction has

received considerable attention in recent years for the quantitative determination of many
pharmaceuticals [89-93]. Cyproheptadine in its base form (CPT) was found to react with
the cited dyes in dichloromethane medium to produce intense colored and a highly stable
ion-pair complex. Chemically, the structure of CPT possesses a tertiary amino group and
190
features its basic nature. This structure suggests the possibility of utilizing an anionic dye
as chromogenic reagent. The solution of CPT in dichloromethane does not absorb in the
visible region. The solutions of the dye employed too have insignificant absorbance (Fig.
2). However, the reaction of CPT with cited dyes results in the formation of intense
yellow colored product with an absorption maximum at 430, 415 or 425 nm and this is
due to an opening of lactoid ring and subsequent formation of quinoid group [94, 95]. It
is supposed that the two tautomers are present in equilibrium but due to strong acidic
nature of the sulphonic acid group, the quinoid body predominates. Finally, protonated
CPT forms ion-pair with the dye. The possible reaction scheme is shown in Scheme
4.3.1.
Br Br Br Br Br Br
HO OH
HO O HO O
Br + H+
Br O Br O O Br Br O
S O Br
O S S
O OH- O-
BCG BCG
(lactoid ring) (quinoid ring)
Br Br Br Br
HO O
HO O
+ + H+
Br Br Br
O O Br
S N+ SO3-
N
O- H CH
CH3 3
CPT 1:1 complex CPT:BCG
191
OH O
H 3C Br H3C Br O
CH3 CH3
H3C Br
CH3
C OH
OH + H+
C OH
Br C
SO3 Br
SO3H Br
SO 3-
BCP
(lactoid ring) (quinoid ring)
O O
H3C Br H3C Br
CH3 CH3
+ + H+
OH C OH
C
N N+ - Br
Br
CH3 SO3- H CH SO3
3
CPT 1:1 complex CPT:BCP
OH
O OH HO OH
+ H+
HO O O O O O O
S S
S OH O-
O
TB
(quinoid ring)
(lactoid ring)
HO OH HO OH
+
O + H+ O O
O S
N S +
O- O-
CH3 H N
CH3
CPT 1:1 complex CPT:TB
Scheme 4.3.1 The possible reaction pathway for the formation of CPT-BCG/BCP/TB
ion-pair complex
192
Effect of solvent
Several organic solvents such as chloroform, dichloromethane, 1,2-dichloroethane

were tried for the extraction of base form of the cyproheptadine. Only dichloromethane
favored the extraction of the drug to its base form. Few organic solvents such as
dichloromethane, chloroform, acetone, methanol and 1,4-dioxan were examined to
dissolve cited dyes. Among these solvents, dichloromethane was preferred as the most
suitable solvent to carry out the experiments in method A (BCG) and method B (BCP),
and acetone in method C(TB); because in this medium, the reagent blank gave negligible
blank absorbance and the ion-pair complex formed was found to exhibit higher sensitivity
and stability. In other solvents, the reagent blank yielded high absorbance value.
Effect of volume of dye, and reaction time, and stability of the ion-pair complex
In order to find out the optimum amount of dye required to obtain maximum
absorbance, experiments were performed separately by measuring the absorbance of the
final solution resulting from the reaction mixture containing a fixed concentration of CPT
and various amounts of the dye. It was found that 1 mL of dye solution (0.1% BCG in
method A, 0.1% BCP in method B and 0.025% TB in method C) was sufficient to
produce maximum and reproducible absorbance (Figure 4.3.2). A 5 min standing time
was sufficient for the complete formation of ion-pair complex. The absorbance of the
resulting ion-pair complex was found to be stable for at least 1 h in method A, 3h in
method B and 2h in method C at room temperature (28±2 °C).
Investigation of composition of ion-pair complex and its conditional stability

constant
The composition and conditional stability constant of the CPT-BCG , CPT-BCP

or CPT-TB complex formed were evaluated by applying Job’s method of continuous
variations [96] using equimolar concentrations of CPT (prepared by following the general
procedure) and the dye. In method A, CPT and dye concentration used were 6.79 ×10-5M,
4.20×10-5 M each in method B, where as 5.61 × 10-5 M each in method C. The
193
experiments were performed by mixing equimolar solutions of the drug and dye by
maintaining the total volume at 5.0 mL. In all the cases, the plot reached a maximum
value at a mole fraction of 0.5 which indicated the formation of 1:1 (CPT:dye) complex
(Figure 4.3.3), and the results revealed that the formation of ion -pair complex between
drug and reagent followed a 1:1 reaction stoichiometry.
0.65
0.60
0.55
0.50
0.45
a
0.40 b
Absorbance
c
0.35 d
e
0.30
f
0.25
0.20
0.15
0.10
0.05
0.00
0.5 1.0 1.5 2.0 2.5 3.0
Volume of reagent, mL
Figure 4.3.2 Effect of reagent concentration on the formation of ion pair complex
a-blank, b- CPT-BCG ion-pair complex (Method A, 6 µg mL-1 CPT)),
c-blank, d- CPT-BCP ion-pair complex (Method B, 8 µg mL-1 CPT)
e-blank and f- CPT-TB ion-pair complex (Method C, 8 µg mL-1 CPT )
0.6
0.6
0.5
0.5
0.4
0.4
Absorbance
Absorbance
0.3
0.3
0.2
0.2
0.1
0.1
0.0 0.0
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0
Mole ratio Mole ratio

VCPT/(VCPT+VBCG) VCPT /(VCPT+VBCP)
(a) (b)
194
0.5
0.4
0.3
Absorbance
0.2
0.1
0.0
0.0 0.2 0.4 0.6 0.8 1.0
Mole ratio
VCPT/(VCPT+VTB)
(c)
Figure 4.3.3 Job’s plots obtained for: (a) CPT-BCG, (b) CPT-BCP
and c) CPT-TB ion-pair complex.
The conditional stability constant (Kf) of the ion-association complex was

calculated [97]. The log Kf values were found to 5.432, 7.562 and 6.225 for method A,
method B, and method C, respectively.
A linear correlation was found between absorbance at max and concentration of

CPT (Figure 4.3.4) in the ranges given in Table 4.3.1. 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 data obtained from this
investigation are presented in Table 4.3.1. Sensitivity parameters such as apparent molar
absorptivity and sandell sensitivity values, the limits of detection and quantification are
calculated as per the current ICH guidelines [52] are compiled in the same table, speak of
the excellent sensitivity of the proposed method. The high values of ε and low values of
Sandell’s sensitivity and LOD indicate the high sensitivity of the proposed methods.
195
1.0 1.2
1.0
0.8
0.8
Absorbance
Absorbance
0.6
0.6
0.4
0.4
0.2
0.2
0.0 0.0
0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 16 18
-1 -1
Concentration of CPT, µg mL Concentration of CPT, µg mL
Method A Method B
1.2
1.0
0.8
Absorbance
0.6
0.4
0.2
0.0
0 2 4 6 8 10 12 14 16 18
-1
Concentration of CPT, µg mL
Method C
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) of the methods. These assays were performed for three levels of analyte.
The results of this study are summarized in Table 4.3.2. The percentage relative standard
deviation (%RSD) values were ≤ 1.68% (intra-day) and ≤ 1.91% (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 CPT, and
it was ≤ 2.24% demonstrate the high accuracy of the proposed methods.
196

Parameter Method A Method B Method C
max, nm 430 415 425
-1
Linear range, µg mL 0.75-12 1.0-16 1.0-16
Color stability, hrs 1 3 2
-1 -1
Molar absorptivity(ε), L mol cm 2.6 × 104 2.2 × 104 1.9 × 104
a -2
Sandell sensitivity , µg cm 0.0123 0.0144 0.0162
-1
Limit of detection (LOD), g mL 0.20 0.22 0.31
-1
Limit of quantification (LOQ), g mL 0.60 0.67 0.92
Regression equation, Yb
Intercept (a) 0.0018 0.0010 0.0223
Slope (b) 0.0822 0.0705 0.0706
Standard deviation of a (Sa) 0.0426 0.0877 0.0923
Standard deviation of b (Sb) 0.0015 0.0048 0.0052
Regression coefficient (r) 0.9999 0.9999 0.9991
a
Limit of determination as the weight in µg mL-1 of solution, which corresponds to an absorbance ofA =
0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm.b Y=a+bX, Where Y is the
absorbance, X is concentration in µg mL-1
precision precision
CPT
(n=5) (n=5)
taken
CPT CPT
Method µg mL-1
found %RE %RSD found %RE %RSD
µg mL-1 µg mL-1
3.0 3.05 1.78 1.25 3.03 1.28 1.42
A 6.0 6.02 0.36 0.96 6.11 1.86 1.57
9.0 9.11 1.31 1.14 9.15 1.72 1.30
4.0 3.98 1.82 1.66 4.02 0.58 1.24
B 8.0 8.04 1.64 0.58 8.05 0.72 1.36
12.0 12.12 1.38 1.54 12.07 0.64 1.12
4.0 3.94 1.37 1.07 4.09 2.24 1.32
C 8.0 7.89 1.33 1.28 8.12 1.52 1.56
12.0 11.80 1.47 1.68 12.16 1.36 1.91
RE- Relative error and 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
197
interference. To assess the role of the inactive ingredients on the assay of CPT, the
general procedure was followed by taking the synthetic mixture extract at three different
concentrations of CPT. The percentage recovery values obtained were in the range
97.87– 102.12% with RSD < 1.6% with clear indication of non-interference by the
inactive ingredients in the assay of CPT.
The robustness of the methods was evaluated by making small incremental

changes in the volume of dye (1±0.2 mL) and contact time (5±1 min), and the effect of
the changes were studied on the absorbance of the ion-pair complex. At three
concentrations of CPT studied, the changes had negligible influence on the results as
revealed by small intermediate precision values expressed as % RSD (≤ 1.82%). Method
ruggedness was demonstrated by having the analysis done by four analysts, and also by a
single analyst performing analysis on four different instruments in the same laboratory.
Intermediate precision values (%RSD) in both instances were in the range 0.76-3.65%
indicating acceptable ruggedness. These results are presented in Table 4.3.3.
Table 4.3.3 Results of robustness and ruggedness expressed as intermediate precision

(%RSD)
Method robustness
Parameters altered Method ruggedness
CPT b
Time, min Inter- Inter-
Method taken, Dye, mLa
µg mL -1 RSD,% analysists’ instruments’
RSD, %
(n=3) RSD, % RSD, %
(n = 3)
(n = 4) (n = 4)
3.0 1.13 1.28 1.46 2.86
A 6.0 1.19 1.54 1.72 3.27
9.0 1.26 1.31 1.34 1.64
4.0 1.34 1.82 0.76 2.66
B 8.0 1.28 1.12 1.26 3.65
12.0 1.23 1.64 1.01 3.42
4.0 1.26 1.42 1.56 2.83
C 8.0 1.21 1.17 0.84 2.46
12.0 1.17 1.29 1.72 3.14
a
Dye (BCG, BCP and TB) volumes used were 0.8, 1.0 and 1.2 mL.
b
Reaction time were 4.0, 5.0 and 6.0 min
198
The proposed methods were applied to the quantification of CPH in commercial

tablets. The same batch tablets were assayed by the reference method [6]. The results
obtained by the proposed methods agreed 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-values. The results of assay are given in Table 4.3.4.
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 CPT at
three concentration levels (50, 100 and 150 % of that in tablet powder) and the total was
found by the proposed methods. In all the cases, the added CPT recovery percentage
values ranged from 99.80–102.81% with standard deviation of 0.76-2.62 (Table 4.3.5)
indicating that the recovery was satisfactory, and that the co formulated substance did not
interfere in the determination.
199
Table 4.3.4 Results of analysis of tablets by the proposed methods and comparison with the reference method
Founda (Percent of label claim ±SD)
Tablet Label claim
Reference Proposed methods
Brand name (mg)
method A B C
98.28± 1.22 98.98± 1.13 98.92± 1.09
Ciplactinb
4 99.36±1.65 t =1.24 t =0.50 t =0.43
F=1.92 F =2.37 F =2.17
100.2±1.37 100.65 ±1.28 99.16 ± 1.31
c
Practin 4 100.4±0.86 t=2.39 t =2.59 t =1.76
F=2.53 F=2.21 F =2.32
a
Mean value of five determinations,
b
Cipla India Ltd., India.cWockhardt Ltd., India.
The value of t and F (tabulated) at 95 % confidence level and for four degrees of freedom are 2.77 and 6.39, respectively.

Method B Method B Method C
Tablet CPT in Pure Total Pure CPT CPT in Pure Total Pure CPT CPT in Pure Total Pure CPT
studied tablet, CPT found, recovered* tablet, CPT found, recovered *
tablet, CPT found, recovered*
µg mL-1 added, µg mL-1 Percent ± SD µg mL-1 added, µg mL-1 Percent ± SD µg mL added, µg mL-1
-1
Percent ± SD
µg mL-1 µg mL-1 µg mL-1
2.97 1.5 4.50 102.4± 2.18 3.96 2.0 6.00 102.8 ± 2.19 3.93 2.0 5.98 102.6 ± 2.13
Ciplactin 2.97 3.0 6.01 101.6± 1.81 3.96 4.0 8.01 101.4 ± 1.51 3.93 4.0 8.72 102.8 ± 2.03
2.97 4.5 7.52 101.2± 2.05 3.96 6.0 9.95 99.97 ± 1.33 3.93 6.0 9.95 101.6 ± 2.62
3.02 1.5 4.47 101.2 ± 1.25 4.04 2.0 6.07 101.9 ± 1.28 3.96 2.0 5.99 101.7 ± 2.62
Practin 3.02 3.0 6.00 101.5± 1.49 4.04 4.0 8.12 102.3 ±1.17 3.96 4.0 7.99 100.8 ± 1.72
3.02 4.5 7.57 102.2± 1.03 4.04 6.0 10.03 99.80 ± 1.06 3.96 6.0 10.03 101.3 ± 0.76
200
SECTION 4.4
A STABILITY INDICATING UPLC METHOD FOR THE DETERMINATION

OF CYPROHEPTADINE HYDROCHLORIDE: APPLICATION TO
PHARMACEUTICAL ANALYSIS
4.4.1 INTRODUCTION
Ultra performance liquid chromatography (UPLC) is a relatively new technique

giving new possibilities in liquid chromatography, especially concerning decrease of time
and solvent consumption [98]. UPLC system is designed in a special way to withstand
high system back-pressure. Special analytical columns UPLC Acquity UPLC BEH C18
packed with 1.7 µm particles are used in the system. The UPLC system allows shortening
analysis time upto nine times compared to the conventional HPLC system, but separation
efficiency remains the same or is even improved. As efficiency and speed of analysis are
of great importance in many applications of liquid chromatography, especially in
pharmaceutical , toxicological and chemical analyses, where it is important to increase
throughput and reduce analysis costs, UPLC could play a significant role in the future of
liquid chromatography. In recent years, there has been an increasing tendency towards
development of stability- indicating assays [99-102], using the approach to stress testing
enshrined in International Conference on Harmonisation (ICH) guideline Q2A(R2) [103].
This approach is being extended to pharmaceuticals to enable accurate and precise
quantification of drugs in the presence of their degradation products.
In the literature survey presented in Section 4.0.1, many HPLC methods are
presented for the determination of CPH but some of them fail to show their adoption to
the assay in pharmaceuticals, and they are not stability-indicating. To the best of author’s
knowledge, no UPLC method has ever been reported for the determination of CPH in
bulk drug as well as in tablets. To fill this gap, the author has developed a rapid, precise,
accurate and validated stability-indicating UPLC method for the determination of CPH in
bulk drug and in tablets. 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 via acidic,
201
basic, hydrolytic, oxidative, thermal and photolytic stress conditions. The optimization
parameters and the validation results in detail are presented in this Section (4.4).
4.4.2 EXPERIMENTAL
4.4.2.1Materials
All the reagents used were of analytical grade. Doubly distilled water was used
throughout the investigation. Pure CPH used was the same as described in Section 4.1.
HPLC grade acetonitrile was purchased from Merck India .Pvt. Ltd, Mumbai, India.,
Doubly distilled water was used throughout the investigation.
1M hydrochloric acid, 1M sodium hydroxide, 5 % hydrogen peroxide were prepared

as described in Section 3.4.2.3.
The pure CPH and its tablets used were the same described in Section 4.1.
Preparation of stock solution
Accurately weighed 100 mg of pure CPH was dissolved in and diluted to mark in
a 100 mL standard flask with the mobile phase.
Mobile phase preparation
Dissolved 0.2 g sodium hydrogenorthophosphate, 0.8 g potassium dihydrogen

orthophosphate and 0.5 g of 1-pentane sulphonic acid in 1000 mL of water and the pH
was adjusted to 3.5 using orthophosphoric acid. A 600 mL portion of this resulting buffer
was mixed with 400 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.
Chromatographic conditions and equipments
Chromatography was performed on a Waters Acquity UPLC™ system (Waters,

Manchester, UK) using an Acquity BEH column (C-18 100 mm, 2.1 mm and 1.7 µm;
202
Waters, Manchester, UK) equipped with binary solvent delivery pump, auto sampler and
tunable UV (TUV) detector. The column oven temperature was maintained at 30 °C and
the autosampler temperature maintained at ambient. Isocratic mobile phase flow was
carried out throughout the run. Total cycle time was 5 min with a flow rate of 0.25 mL-1
min and an injection volume of 2 µL using partial loop mode. The output signal was
monitored and processed using Empower-2 software.
Instrumental parameters
The isocratic flow rate of mobile phase was maintained at 0.25 mL min-1. The column
temperature was adjusted to 30 °C. The injection volume was 2.0 µL. The sample run
was monitored at 254 nm and the run time was 5.0 min. The retention time of the sample
was observed at about 2.2 min.
Stress study
All stress decomposition studies were performed at an initial drug concentration

of 80 µg mL-1 in mobile phase. Acid hydrolysis was performed in 1 M HCl at 80 °C for 2
h. The study in alkaline condition was carried out in 1 M NaOH at 80 °C for 2 h. For
study in neutral condition, drug dissolved in water was heated at 80 °C for 2 h. Oxidative
studies were carried out at 80 °C in 5% hydrogen peroxide for 2 h. For photolytic
degradation studies, pure drug in solid state was exposed to 1.2 million lux hours in a
photo stability chamber. Additionally, the drug powder was exposed to dry heat at 105 °C
for 3h. Samples were withdrawn at appropriate time, cooled and neutralized by adding
base or acid and subjected to UPLC analysis after suitable dilution.
Procedure for calibration curve
Working standard solutions containing 3-120 µg mL-1 CPH were prepared by

serial dilutions of aliquots of the stock solution. Aliquots of 2 µL were injected (six
injections) and eluted with the mobile phase under the reported chromatographic
conditions. The average peak area versus the concentration of CPH in µg mL-1 was
plotted. Alternatively, the regression equation was derived using mean peak area-
203
concentration data and the concentration of the unknown was computed from the
regression equation.
Twenty tablets (both practin-4 mg and ciplactin- 4 mg) were accurately weighed
and ground into a fine powder. Powder equivalent to 20 mg CPH was transferred into a
100 mL volumetric flask and 60 mL of the mobile phase was added. The mixture was
sonicated for 20 min to achieve complete dissolution of CPH, and the content was then
diluted to volume with the mobile phase to yield a concentration of 200 µg mL-1 CPH,
and filtered through a 0.22 µm nylon membrane filter. The tablet extract was injected to
the UPLC column after diluting to 80 µg mL-1.
4.4.2.4 Procedure for method validation
Six injections, of three different concentrations (60, 80 and 100 µg mL-1), were
given on the same day and the values of relative standard deviation (RSD) were
calculated to determine intra-day precision. These studies were also repeated on different
days to determine inter-day precision.
Limits of detection (LOD) and quantification (LOQ)
Signal to noise (S/N) ratio method was adopted to obtain the limit of quantification
(LOQ) and limit of detection (LOD). Series of dilutions of the CPH stock solution was
made to attain LOQ and LOD in acceptable values. LOQ solution was injected six times
(n=6) and calculated the % RSD values for the obtained CPH peak area and retention
time.
Linearity
Seven-point calibration curves were obtained in a concentration range from 3 to 120

µg mL−1 for CPH; three independent determinations were performed at each
concentration.
204
To determine the robustness of the method the experimental conditions were

deliberately changed. The flow rate of the mobile phase (0.25±0.05 mL min-1), column
oven temperature (30±5 ºC), mobile phase composition (65:35, 60:40, 55:45; buffer:
solvent mixture v/v) and detection wavelength (254±1 nm) were the varied parameters. In
each case, the %RSD values were calculated for the obtained peak area and retention
time. The number of theoretical plates and tailing factors were compared with those
obtained under the optimized conditions. Three different columns of same dimensions
were used for the analyses. The study was performed on the same day and three different
days by three different analysts for three different concentrations of CPH (triplicate
injections). The area obtained from each concentration was compared with that obtained
under optimized conditions. The relative standard deviation values were evaluated for
each concentration.
Solution stability and mobile phase stability
Stability of CPH solution was studied by injecting the sample into the
chromatographic system at different time intervals. The peak area was recorded in the
time intervals of 0, 12 and 24 hrs and the RSD values were calculated. Freshly prepared
solution was injected at the same time intervals for mobile phase stability (0, 12 and 24
hours) and RSD values of the peak areas were calculated.
4.4.3.1Method 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.
205
Alternative combinations of gradient and isocratic methods were also performed to obtain
a suitable peak. Finally isocratic method was found suitable for the assay.
When CPH 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 CPH 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 Acquity
BEH C18, (100 × 2.1) mm, 1.7 µm column. Acetonitrile was the next solvent option.
Better results were obtained when CPH eluted in acetonitrile and
sodiumhydrogenorthophosphate, potassium dihydrogenorthophosphate and of 1-pentane
sulphonic acid buffer. CPH eluted late with higher peak shape and theoretical plates. The
peak symmetry was optimized with varying ratios of buffer and acetonitrile. Best peak
was obtained with buffer-acetonitrile ration (60:40 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. Summary of solvents used are summarized as in Table 4.4.1.
Table 4.4.1 Summary of solvent optimization

Solvent A Solvent B Observations/ Remarks
5mM Ammonium acetate Methanol Very broad peak with peak splitting
5mM Ammonium acetate Acetonitrile Peak eluted too early
Di basic potassium phosphate (pH Very broad peak with peak
Methanol
3.0 with 10% H3PO4) splitting.
Di basic potassium phosphate (pH Less theoretical plates and peak
Acetonitrile
3.0 with 10% H3PO4) fronting
Mono basic potassium phosphate (pH Broad peak and less theoretical
Methanol
3.0 with 10% H3PO4) plates.
Sodium hydrogen ortho phosphate,
potassium dihydrogen Acetonitrile Good peak shape with theoretical
orthophosphate and 1-pentane at 40% ratio plates above 2000.
sulphonic acid (pH 3.5) at 60% ratio
In order to achieve symmetrical peak of CPH, various stationary phases like C8

(different dimension), C18 (different brand), length (50 mm and 100 mm) and phenyl
columns were studied. Summary of stationary phases are represented as in Table 4.4.2.
From the summary, it is concluded that acquity BEH C18, (100 × 2.1) mm, 1.7 µm
206
column have the ideal stationary phase for the determination. The column oven
temperature was studied at higher (35°C) and room (25°C) temperatures and then found
that 30°C is the optimum. Shimadzu Pharmaspec 1700 UV/Visible spectrophotometer
was used for absorbance measurements. A 80 µg mL-1 of CPH solution in acetonitrile
was scanned from 400 to 200 nm against acetonitrile as blank and wavelength of the
method was optimized to 254 nm. Overlay chromatograms of Blank and CPH solutions
are as shown in Figure 4.4.1.
Table 4.4.2 Summary of stationary phase optimization

Stationary phase Dimension Observations/ Remarks
Acquity BEH Phenyl (100 x 2.1) mm, 2 µm Peak eluted very early with higher tailing
Acquity BEH C8 (50 x 2.1) mm, 1.7 µm No retention of CPH, eluted very early
Eclipse Plus C18,
(50 x 2.1) mm, 1.8 µm Peak fronting observed
RRHD
Acquity BEH C8 (100 x 2.1) mm, 1.7 µm Peak eluted before 1 min
Acquity BEH C18 (100 x 2.1) mm, 1.7 µm Satisfactory peak shape observed
(a)
(b)
Figure 4.4.1 Typical chromatograms obtained under optimized conditions for: (a) 80 µg
mL-1 CPH and (b) blank.
207
4.4.3.2 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. CPH 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 5 %. The chromatograms obtained for CPH
after subjecting to degradation are presented in Figure 4.4.2. Assay study was carried out
by the comparison with the peak area of CPH sample without degradation.
(a)
(b)
208
(c)
(d)
(e)
(f)
Figure 4.4.2 Chromatograms obtained for CPH (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.
209
The described method for the assay of CPH was validated as per the current ICH
Guidelines. Parameters such as system suitability, specificity, precision, robustness,
ruggedness, linearity, accuracy, LOQ, LOD, solution stability, filter compatibility were
studied for the suitability of the method.
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. As seen from the data the
theoretical plates are 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
0.6.
Analytical parameters
A linear correlation was obtained between the peak area and the concentration in
the range of 3 – 120 µg mL-1 CPH from which the linear regression equation was
computed and found to be:
y = 35,970x + 53,024 r² = 0.9999
where y is the mean peak area, x is the concentration of CPH 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 4.4.3. These results confirm the
linear relation between concentration of CPH and the peak areas as well as the sensitivity
of the method.
210
Table 4.4.3 Linearity and regression parameters

Parameter Value
Linear range, µg mL-1 3-120
Limits of quantification, (LOQ), µg mL-1 2.85
Limits of detection, (LOD), µg mL-1 0.94
Regression equation
Slope (b) 35970.1
Intercept (a) 53024.0
Correlation coefficient (r) 0.9999
The percent relative error which is an indicator of accuracy is ≤ 0.89 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 < 0.40%. The results obtained for the evaluation of accuracy and precision of
the method are compiled in Tables 4.4.4 and 4.4.5.
Table 4.4.4 Results of accuracy study (n=5)

Concentration Intra-day Inter-day
of CPH Concentration RE,% Concentration RE,%
injected, of CPH of CPH
-1
µg mL found, found,
µg mL-1 µg mL-1
60.0 60.36 0.61 60.53 0.89
80.0 80.24 0.29 80.47 0.58
100.0 99.63 0.37 99.39 0.62
Table 4.4.5 Results of precision study (n=5)

Con Inter-day
Intra-day precision
injected, precision
-1
µg mL Mean area±SD %RSDa %RSDb Mean area %RSDa %RSDb
± SD
60.0 2225795±5825 0.26 0.24 2230424±7868 0.35 0.29
80.0 2924419±10910 0.37 0.17 2912796±11769 0.40 0.21
100.0 3650935±2741 0.08 0.13 3649373±5084 0.13 0.17
a
Relative standard deviation based on peak area;
b
Relative standard deviation based on retention time.
211
The robustness of an analytical procedure is a measure of its capacity to remain

unaffected by small, but deliberate variations in method parameters, and provides an
indication of its reliability during normal usage. At the deliberate varied chromatographic
conditions (flow rate, temperature, wavelength of detection and mobile phase
composition), the analyte peak % RSD, tailing factor and theoretical plates remained
closer to the actual values. The RSD values ranged from 0.10 to 2.20 % resumes the
robustness of the proposed method. In method ruggedness, different columns (same lot),
at different day by different analysts were performed. The results are summarized in
Table 4.4.6 and 4.4.7.
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 (Figure 4.4.3).
(a)
(b)
Figure 4.4.3 Chromatograms obtained for (a) tablet extract (80 µg mL-1) and (b) for
placebo blank.
212
Stability of the solution
At the specified time interval, % RSD for the peak area obtained from drug
solution stability and mobile phase stability were within 1%. This shows no significant
change in the elution of the peak and its system suitability criteria (%RSD, tailing factor,
theoretical plates). The results also confirmed that the standard solution of drug and
mobile phase were stable at least for 24 hours during the assay performance.
Stability of the solution and mobile phase stability
At the specified time interval, % assay of CPH obtained from drug solution
stability and mobile phase stability were within 1%. For solution stability same sample
solution was injected at 0, 6, 12 and 24 hours and for mobile phase stability, separately
prepared CPH sample injected at the same time interval as above. The results confirmed
that the CPH solution of drug and mobile phase were stable at least for 24 hours during
the assay performance, which are represented in Table 4.4.8.
213
Table 4.4.6 Results of method robustness study expressed as intermediate precision (%RSD
Mean Mean
Mean peak
Condition Modification RSD,% Mean Rt± SD* RSD,% theoretical RSD,% tailing RSD,%
area± SD*
plates ±SD* factor ± SD*
25ºC 2878765±4637 0.16 2.188±0.008 0.37 2702±59.65 2.20 1.32±0.02 1.51
Temperature 30ºC 2932419±6736 0.22 2.195±0.006 0.28 2767±39.36 1.42 1.30±0.01 0.76
35ºC 2947846±5823 0.19 2.188±0.008 0.37 2786±47.56 1.70 1.28±0.02 1.56
65:35 2943821±5638 0.19 2.187±0.012 0.53 2689±53.36 1.98 1.23±0.02 1.62
Mobile phase
60:40 2875296±4728 0.16 2.186±0.003 0.15 2772±32.56 1.17 1.27±0.02 1.57
composition
55:45 2985915±6281 0.21 2.187±0.013 0.57 2865±55.97 1.95 1.35±0.01 0.74
0.20 2915998±5246 0.18 2.185±0.007 0.34 2805±48.16 1.71 1.28±0.02 1.96
Flow rate,
0.25 2908856±2857 0.10 2.209±0.004 0.17 2698±42.84 1.58 1.32±0.02 1.62
min
0.30 2906684±4313 0.15 1.989±0.005 0.25 2791±53.16 1.90 1.22±0.02 1.63
253 2906684±4479 0.15 2.188±0.008 0.37 2695±35.16 1.30 1.34±0.02 1.49
Wavelength,
254 2883363±3005 0.10 2.195±0.006 0.28 2747±42.3 1.53 1.25±0.01 0.80
nm
255 2944213±8138 0.28 2.184±0.007 0.34 2698±38.58 1.42 1.29±0.02 1.55
*Mean value of three determination
Table 4.4.7 Results of method ruggedness study expressed as intermediate precision (%RSD)
Variable Mean RSD,% Mean Rt ±SD* RSD, % Mean RSD, % Mean RSD, %
peak area ±SD* theoretical tailing
plates± SD* factor± SD*
Analyte 2937022±5845 0.20 2.195±0.006 0.28 2783±54.34 1.95 1.26±0.03 2.38
(n=3)
Column 2944213±8138 0.28 2.209±0.004 0.18 2804±59.78 2.13 1.24±0.02 1.61
(n=3)
214
Table 4.4.8 Solution stability results

Initial 6 hours 12 hours 18 hours 24 hours
% Assay for solution 99.8 100.1 99.6 99.6 99.3
stability
% Assay for mobile 99.7 100.0 100.1 99.8 99.7
phase stability
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 4.4.9 illustrates the results obtained from this study.
Table 4.4.9 Results of determination of CPH in tablet and statistical comparison with the
reference method
Formulation Nominal % CPH found* ± SD t- F- value
brand name amount, mg Reference Proposed value
method method
a
Practin 4.0 101.6±1.64 100.1±0.65 2.31 6.36
Ciplactinb 4.0 101.2±1.36 99.93±0.81 2.73 2.81

a
Wockhardt Ltd., India.
b
Cipla India Ltd., India.
*
Mean value of five determinations. Tabulated t-value at 95% confidence level is 2.78; Tabulated F-value
at 95% confidence level is 6.39
Recovery studies
A standard addition procedure was followed to further evaluate the accuracy of

the method. The solutions were prepared by spiking pre-analyzed tablet with pure drug at
three different levels and injected to chromatographic column. The recovery of the
known amount of added analyte was computed. The percentage recovery of CPH from
215
pharmaceutical dosage forms ranged from 99.49 to 100.8%. The results presented in
Table 4.4.10 reveal good accuracy of the proposed method.

CPH in Pure CPH Total Pure CPH
Tablet
tablet, added, found, recovered*
studied
µg mL-1 µg mL-1 µg mL-1 (%CPH±SD)
40.05 20.0 60.53 100.8±1.25
Practin 40.05 40.0 80.37 100.4±0.98
40.05 60.0 99.73 99.69±0.86
39.97 20.0 60.26 100.5±0.52
Ciplactin 39.97 40.0 79.56 99.49±0.73
39.97 60.0 99.55 99.58±0.47
*Mean value of three determination
216
SECTION 4.5
DEVELOPMENT AND VALIDATION OF UV-

SPECTROPHOTOMETRIC METHODS FOR THE DETERMINATION
OF CYPROHEPTADINE IN PHARMACEUTICALS, AND ITS
DEGRADATION STUDIES
4.5.1 INTRODUCTION
A smart profile and utilization of UV-spectrometry in different assay have been
presented in Section 2.5. From the literature survey presented Section 4.0 it is evident
that, application of derivative UV-spectrophotometry for the assay of CPH in two-
component system has also been reported [8]. Another official (USP) [6] UV-
spectrophotometric method was developed for the assay of CPH in tablets, which
describes measurement of absorbance in 0.1 M HCl at 286 nm.
In the literature, no stability-indicating UV-spectrophotometric methods have ever
been reported for the assay of CPH. In the present Section (4.5), two simple, inexpensive,
accurate and reproducible UV- spectrophotometric methods for the assay of CPH are
described. The methods are based on the measurement of absorbance of CPH solution
either in 0.1 M H2SO4 at 223 nm (method A), or methanol at 222 nm (method B).
Besides, method A was used to study the degradation of the drug under different stress
conditions as per the ICH guidelines [103].
4.5.2 EXPERIMENTAL
4.5.2.1 Apparatus
The instrument used for absorbance measurement is the same as described in

Section 2.5.2.1.
4.5.2.2 Materials
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 4.1.2.
217
Sulphuric acid (0.1 M): Prepared by diluting concentrated acid (Merck, Mumbai, India;
Sp.gr. 1.84) with water.
Methanol (Merck, Mumbai, India), was used as such as a solvent in method B.
5 M hydrochloric acid, 5% hydrogen peroxide, 5 M sodium hydroxide were prepared

as described under Section 3.4.2.3.
Standard drug solution
Standard drug solutions of 20 µg mL-1 CPH prepared separately in 0.1 M H2SO4

and in methanol and used for assay in method A and method B, respectively.
Method A (using 0.1 M H2SO4)
Different aliquots (0.00, 0.25, 0.5, 1.0, -----5.0 mL) of a standard CPH (20 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 M H2SO4. The absorbance of each solution
was measured at 223 nm against 0.1 M H2SO4.
Method B (using methanol)
Aliquots (0.00, 0.25, 0.5, 1.0,-----5.0 mL) of a standard CPH (20 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 methanol. The absorbance of each solution was
measured at 222 nm against methanol.
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.
218
Twenty tablets (Practin and Ciplactin 4 mg) were weighed and pulverized. The
amount of tablet powder equivalent to 10.0 mg of CPH was transferred into two separate
100 mL volumetric flasks. Sixty mL of 0.1 M H2SO4 was added to one and methanol 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 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 CPH) was diluted to get 20 µg mL-1 CPH and analysed for CPH
following the procedures described above.
Thirty mg of the placebo blank prepared in Section 2.1.2.4 was taken and its

by the procedures described above.
Forced degradation study
Five mL of 50 µg mL-1 CPH 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 HCl 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 CPH solution in 0.1 M
H2SO4 was prepared and absorbance measured. For UV degradation study, the stock
solutions of the drug (100 µg mL-1) were exposed to UV radiation of wavelength 254 nm
219
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.
4.5.3 RESULTS AND DISCUSSIONS
4.5.3.1 Spectral characteristics
In the present work, two UV-spectrophotometric methods for the determination of

CPH in bulk drug and in tablets have been described. In method A, 0.1 M H2SO4 was
used as the solvent and the absorbance was measured at 223 nm (Figure 4.5.1a) whereas
in method B, methanol was used as a solvent with the measurement being made at 222
nm (Figure 4.5.1b).
(a)
(b)
Figure 4.5.1 Absorption spectra: a) Method A (0.1 M H2SO4), b) Method B (methanol).
220
4.5.3.2 Forced degradation studies
In method A, the absorption spectra of the CPH 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 CPH
samples” with that of the “standard CPH solution”. The absorption spectrum of CPH
solution treated with 5 M hydrochloric acid (Figure 4.5.2a) showed the same spectrum of
the standard solution (Figure 4.5.1a) which indicated that CPH does not undergo
degradation under acidic condition. Also, CPH solution in 0.1 M H2SO4 was treated with
5 M sodium hydroxide and the absorption spectrum was run and it showed minor
degradation under basic conditions as the absorbance value was reduced slightly (Figure
4.5.2b). From Figure 4.5.2c and 4.5.2d it is clear that CPH is quite stable under dry heat
and UV-light exposure stress conditions. Contrary to the above discussions, the
absorption spectrum of CPH solution subjected to oxidative stress conditions showed that
CPH undergoes significant degradation due to its susceptibility to oxidation (Figure
4.5.2e). The results are given in Table 4.5.1.
(a)
(b)
221
(c)
(d)
(e)
Figure 4.5.2 Absorption spectra of 10 µg mL-1 CPH a) acid hydrolysis, b) base

hydrolysis, C) thermal hydrolysis, d) photolytic degradation and e) peroxide
degradation.
222
Table 4.5.1 Results of degradation study

Degradation condition % Degradation
No degradation ( control) Zero
Acid hydrolysis (5 M HCl , 80°C, 2 hours) Zero
Base hydrolysis (5 M NaOH , 80°C, 2 hours) 74.8
Oxidation (5% H2O2 , 80°C, 2 hours) undetectable
Thermal (100°C, 24 hours) Zero
Photolytic (1.2 K flux, 48 hours) Zero

A linear correlation was found between absorbance at max and concentration of

CPH (Figure 4.5.3). The slope (b), intercept (a) and correlation coefficient (r) for each
system were evaluated by using the method of least squares. Optical characteristics such
as Beer’s law limits, molar absorptivity and Sandell sensitivity values of the methods
were calculated. The limits of detection (LOD) and quantitation (LOQ) were also
calculated according to ICH guidelines [52], and all these data are presented in Table
4.5.2. The high values of ε and low values of Sandell sensitivity and LOD indicate the
high sensitivity of the proposed methods.
1.4
1.2
1.2
1.0
1.0
0.8
Absorbance
Absorbance
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0 0.0
0 2 4 6 8 10 0 2 4 6 8 10
-1 -1
Concentration of CPH, µg mL Concentration of CPH, µg mL
Method A Method B
223

Parameter Method A Method B
max, nm 223 222
-1
Linear range, µg mL 0.5 – 10.0 0.5 – 10.0
Molar absorptivity(ε), L mol-1cm-1 3.5 × 104 3.9 × 104
Sandell sensitivity*, µg cm-2 0.0091 0.0083
Limit of detection (LOD), µg mL-1 0.15 0.13
-1
Limit of quantification (LOQ), µg mL 0.45 0.39
Regression equation, Y**
Intercept (a) 0.0027 0.0033
Slope (b) 0.1105 0.1220
Standard deviation of a (Sa) 0.0568 0.0585
Standard deviation of b (Sb) 0.0114 0.0122
Regression coefficient (r) 0.9996 0.9998
*
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, a is
intercept, b is slope.
Accuracy was evaluated as percentage relative error between the measured

concentrations and the concentrations taken for CPH (Bias %). The results obtained are
compiled in Table 4.5.3 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 CPH 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.
precision precision
CPH
(n=7) (n=5)
Method taken,
CPH CPH
µg mL-1
found, %RE %RSD found %RE %RSD
-1 -1
µg mL µg mL
2.0 2.02 1.27 1.82 2.04 1.60 1.74
A 4.0 4.03 0.98 0.78 4.05 1.56 1.65
6.0 6.03 0.65 1.08 6.06 1.43 1.79
2.0 1.97 1.23 1.42 2.02 1.25 1.67
B 4.0 4.02 0.68 1.67 4.04 1.63 1.52
6.0 6.06 1.04 1.68 6.08 1.82 1.46
RE: Relative error and RSD: Relative standard deviation
224
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 CPH in its tablets. A separate
experiment was performed with the synthetic mixture. The analysis of synthetic mixture
solution yielded percent recoveries of 98.92±0.98 for method A and 100.4±1.08 for
method B, indicating the non-interference by inactive ingredients.
Method robustness and ruggedness were demonstrated by determination of CPH

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 4.5.4.
Table 4.5.4 Results of robustness and ruggedness expressed as intermediate

precision(%RSD)
Ruggedness
CPH Inter-
Robustness#
Method taken, Inter-analysts cuvettes
(%RSD)
µg mL-1 (%RSD), (n=4) (%RSD),
(n=4)
2.0 0.94 0.35 1.26
A 4.0 0.86 0.48 1.78
6.0 0.78 0.26 1.04
2.0 0.72 0.76 1.85
B 4.0 0.68 0.48 1.26
6.0 0.86 0.54 1.46
#
Wavelengths used were 222,223and 224 in method A and 221, 222 and 223 in method B.
In order to evaluate the analytical applicability of the proposed method to the

quantification of CPH in commercial the tablets, results obtained by the proposed
225
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 4.5.5) 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 4.5.5 Results obtained by the analysis of tablets by the proposed method and
statistical comparison of results with the reference method
Nominal Found* (Percent of label claim ± SD)
Tablet brand
amount Reference
name Method A Method B
(mg/tablet) method
101.42±1.17 101.86±1.46
Practin* 4 102.6±0.68 t=2.01 t=1.10
F= 2.96 F= 4.60
97.48±0.89 98.68±0.89
Ciplactin# 4 97.08±0.72 t= 1.45 t= 1.70
F= 1.52 F= 2.77
*
Mean value of 5 determinations.
*
Wockhardt Ltd., India.
#
Cipla India Ltd., India.
Tabulated t-value at the 95 % confidence level and for four degrees of freedom is 2.77.Tabulated F-
value at the 95 % confidence level and for four degrees of freedom is 6.39.
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 CPH at
three concentration levels (50, 100 and 150 % of that in tablet powder) and the total was
found by the proposed methods. The added CPH recovery percentage values ranged from
98.75–102.90 % with standard deviation of 0.96-2.06 (Table 4.5.6) indicating that the
recovery was good, and that the co-formulated substance did not interfere in the
determination.
226

Method A Method B
Pure Pure
Tablets CPH in Total Pure CPH CPH in Total Pure CPH
CPH CPH
studied tablet, found, recovered tablet, found, recovered
-1 added, -1 * -1 added, -1
µg mL -1 µg mL (Percent±SD ) µg mL -1 µg mL (Percent±SD*)
µg mL µg mL
3.04 1.5 4.57 102.01±1.28 3.05 1.5 4.54 99.80±1.31
Practin 3.04 3.0 6.56 100.86±1.28 3.05 3.0 6.13 102.90±1.15
3.04 4.5 7.60 101.37±1.53 3.05 4.5 7.67 102.72±1.37
2.92 1.5 4.42 100.08±1.68 2.96 1.5 4.44 99.32±1.74
2.92 3.0 6.36 98.75±2.06 2.96 3.0 5.92 98.96±0.96
Ciplactin
2.92 4.5 7.39 98.91±0.87 2.96 4.5 7.40 98.75±0.63
*Mean value of three determinations.
227
SECTION 4.6
SUMMARY AND CONCLUSIONS –Assessment of methods
In all, three titrimetric, two UV-spectrophotometric and five visible

spectrophotometric, and one UPLC methods were developed and validated for the
determination of cyproheptadine hydrochloride in bulk drug and in tablet form. In
addition, effect of several stress conditions on the stability of CPH was also investigated
with the help of a UV-spectrophotometry and UPLC.
From the literature survey presented in Section 4.0.2, other than official method
[5] there is only one titrimetric method for the determination of CPH in pharmaceuticals.
Titrimetry may serve as useful alternative to many of the sophisticated techniques
because of its cost-effectiveness, ease of operation, remarkable accuracy and precision
and wide applicability. Due to the scarcity of titrimetric procedure, the author developed
three simple, selective and rapid titrimetric procedures for the assay of CPH in bulk drug
as well as in pharmaceutical formulations. The first two titrimetric reactions are specific
for the tertiary amino group present in CPH using ion-association titration technique.
Another titrimetric method is based on bromination of CPH with NBS in acid medium.
The proposed titrimetric methods are simple, precise, accurate and applicable over either
semi micro scale (1.5-15 mg CPH using NBS and 1-20 mg CPH using SLS) or micro
scale (2-9.0 mg CPH using TPB) thus offering an additional advantage.
Spectrophotometry, particularly in the visible region of the electromagnetic

spectrum, is one of the most widely used methods of analysis in clinical, environmental
and pharmaceutical analysis labs because many substances can be selectively converted
to a colored derivative. In addition, the instrumentation is readily available and generally
fairly easy to operate. Considering these advantages and based on various reaction
chemistries, eight reports employing visible spectrophotometry are found in the literature
for the assay of CPH in pharmaceuticals. However, most of the reported methods suffer
from one or other disadvantage such as poor sensitivity, poor selectivity, tedious time
consuming liquid-liquid extraction step, strict pH control and narrow linear range, etc., as
indicated in Table 4.6.1. In this Chapter, the author has made an attempt to develop and
228
validate five visible spectrophotometric methods for the determination of CPH using
different reagents and based on different reaction schemes.
Table 4.6.1 Comparison of performance characteristics of the proposed methods with the
existing methods
I Titrimetry
Sl. Range,
Reagent Titration conditions Remarks Ref.
No. mg
Non aqueous titration
in glacial acetic acid Use of non-
1. Perchloric acid medium, end point NA aqueous 5
detected visually using medium
crystal violet indicator
Liberated iodine
titrated against 0.15 M
2. KBr03-KBr Na2S203 in HCl 1-15 - 7
medium, end point
detected iodometrically
Liberated iodine
titrated against 0.02 Rapid,
M Na2S203 in HCl applicable to Present
3. NBS 1.5-15
medium, end point semimicro work
detected scale samples
iodometrically
Sodium lauryl Two phase titration

Present
4. sulfate & H2SO4 using dimethyl yellow 1.0-20
Applicable to work
as indicator
micro and
semimicro
Two phase titration
sodium size samples,
using
tetraphenylborate & sensitive and Present
5. tetrabromophenolpht 2.0-9
Walpole buffer (pH selective work
hale-in ethyl ester as
4.5)
indicator
229
II Spectrophotometry
Linear range
Sl. max
Reagent/s used Methodology (µg mL-1) Remarks Ref
No. (nm)
(ε = L/mol/cm)
1 Bromocresol Green Chloroform extractable ion-pair Required close pH control and involved
complex measure 615 1-10 extraction steps 23
2 a)Solochrome Black T
b)Solochrome Dark Blue Fast Extractable ion-pair 520 4-18 Require extraction 25
c) Sulphon Black FF Complex measured
3
Dichloromethane extractable ion- 404 10-60 Required close pH control and involved
Benzyl orange 26
pair complex measured extraction step
4 Reineckate Ion-pair complex measured after 525 100-600 Less sensitive, involves precipitation,
filtering Precipitate formed filtration and dissolution steps 24
5. Yellow colored chloroform 420 2-12
extractable 1:1ion-pair complex Required close pH control and involved 27
Bromophenol Blue was measured extraction steps
Chloroform extractable turbid 650 10-70
suspension was measured
Residual bromine treated with 0.5-4 Less sensitive
6 Bromate-Bromide
Methyl Orange and measured 520 (5.25 x 104) 22
7 Chloranilic acid Charge-transfer complex was 520 25-125 Less sensitive, narrow linear ranges
measured (1.48 x 103) 28
8 a)Erioglaucin In both the methods unbleached 630 0.1-2.0 Highly sensitive and selective, no
dye color measured (1.4×105) extraction step, no pH-adjustment, Present
employ mild acid conditions, work
b)Meta-cresol purple 540 0.4-12.0 inexpensive instrumental setup.
(2.2×104)
9 a)Bromocresol Green 430 0.75-12 Highly sensitive with wide linear

In all three methods, resulting (2.6 × 104) dynamic ranges, no pH-adjustment, Present
b)Bromocresol purple yellow colored ion-pair 415 1.0-16 single step reaction and inexpensive work
complexes were measured. (2.2 × 104) instrumental setup.
230
c)Thymol Blue 425 1.0-16

(1.9 × 104)
10 a)0.1 M H2SO4 Absorbance measured in 0.1 M 223 0.5-10 Simple, sensitive, no optimization of Present
H2SO4 (3.5 × 104) experimental conditions, stability- work
b) Methanol Absorbance measured in 0.5-10 indicating
methanol 222 (3.9 × 104)
The outstanding performance characteristics of the proposed methods are simplicity and sensitivity; and selectivity in
the case of ion-pair complexation reaction methods. Among the five proposed methods, two methods employing NBS are the
most sensitive one as can be seen from the molar absorptivity values which range from 1.4  105 to 2.2  104 L mol-1 cm-1.
Also, among the proposed methods, three methods based on extraction-free ion-pair complexation reaction are the very simple
ones since they are based on one step reaction i.e., CPH in dichloromethane mixed with the BCG/BCP/TB dye
dichloromethane or acetone and the formed yellow colored ion-pair complex was directly measured at the respective
wavelength. The UV- spectrophotometric methods are simple and cost-effective in terms of the media employed (H2SO4 and
methanol). Wide linear dynamic ranges (0.5-10 µg mL-1), high sensitivity (=3×104) and low LOQ values (< 0.5 µg mL-1) are
the features of the proposed UV-spectrophotometric methods.
Though a few chromatographic techniques are available for the assay of CPH in body fluids [16-22] and in
pharmaceuticals [23-29], only one method was stability-indicating [27]. UPLC, which is becoming popular because of its
231
speed and cost-effectiveness, was not earlier used for the assay of CPH 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 1.9
min enables rapid determination 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 revealed CPH was found to be more stable under all subjected stress
conditions rather than oxidative stress conditions.
232
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Chapter4 Titrimetric, spectrophotometric and chromatographic assay of cyproheptadine

DRUG PROFILE AND LITERATURE SURVEY

4.0.1 DRUG PROFILE

Cyproheptadine hydrochloride (CPH) is chemically known as 4-(5Hdibenzo [a,d]-

It is white to slightly yellow, crystalline powder, odourless, slightly bitter taste. It

CPH is a sedating antihistamine with antimuscarinic, serotonin-antagonist, and

The drug is official in Indian Pharmacopeia [5] which describes a UV-

4.0.2 LITERATURE SURVEY OF ANALYTICAL METHODS FOR

4.0.2.1 Titrimetric and UV-spectrophotometric methods

4.0.2.2 Visible spectrophotometric methods

4.0.2.4 Other techniques

Liu and Lu [30] developed chemiluminescence method for the determination of

APPLICATION OF ION-ASSOCIATION TITRATION FOR THE ASSAY OF

Titration of analyte in the presence of two immiscible solvents is known as two

Elico LI 610 digital pH meter provided with a combined glass-SCE electrode

Standard CPH solution

A stock standard solution containing 2.0 mg mL-1 of pharmaceutical grade CPH

4.1.2.3 Reagents and solutions

Tetraphenylborate: A 0.04 M solution of tetraphenylborate (TPB) was prepared by

and standardized following the recommended procedure in Japanese Pharmacopoeia [47]

A 0.2 % (w/v) potassium salt of tetrabromophenolphthalein ethyl ester (TBPE) (Sigma-

4.1.2.4 ASSAY PROCEDURES

Method A (using SLS)

Different aliquots (0.5-10 mL) of standard CPH (2 mg mL-1) solution were

Method B (using TPB)

Procedure for tablets

Placebo blank analysis and synthetic mixture analysis

To 100 mg of the placebo blank of the composition described above, 200 mg of

4.1.3 RESULTS AND DISCUSSION

The two-phase ion-association titration was applied to the determination of some

4.1.3.1 Method development

Method A (using SLS)

Before the addition of titrant:

Method B (using TPB)

The chemical reactions for this titration can be explained as:

Before addition the titrant:

Before the equivalence point:

DMY(molecular form) DMYH+ (cationic form)

TBPE - (anionic form) TBPEH (molecular form)

Scheme 4.1.1 Effect of pH on the indicator color

Range and Stoichiometry

Accuracy and precision

Robustness and ruggedness

The robustness of the methods was evaluated by making small incremental

Table 4.1.3 Results of recovery study via standard addition method

SENSITIVE AND SELECTIVE METHODS FOR THE DETERMINATION OF

N-bromosuccinimide (NBS), chemically known as 1-Bromo-2,5-

important goal. Among various brominating reagents, NBS is environmentally friendly,

The instrument is the same that was described in Section 2.2.2.1.

4.2.2.3 Reagents and solutions

N-bromosuccinimide (NBS): An approximately 0.01 M solution was prepared by

Potassium iodide: A 10% solution was prepared by dissolving 10 g of the

4.2.2.4 ASSAY PROCEDURES

A 10 mL aliquot of pure drug solution containing 1.5-15 mg of CPH was

Spectrophotometry using erioglaucine (Method A)

Different aliquots (0.2,0.5,1.0,----4.0 mL) of a standard 5 g mL-1 CPH solution

Spectrophotometry using meta-cresol purple (Method B)

Varying aliquots (0.2,0.5,1.0,----6.0 mL) of a standard 20 g mL-1 CPH solution