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Indian Journal of Chemical Technology
Vol. 13, July 2006, pp. 360-366
Application of bromate-bromide mixture and methyl orange in the titrimetric,
spectrophotometric and kinetic assay methods for cyproheptadine in
pharmaceuticals
K Basavaiah*
Department of Chemistry, University of Mysore,
Manasagangotri, Mysore 570 006, India
Email: [email protected]
Received 4 October 2005; revised received 12 April 2006; accepted 28 April 2006
Three new methods have been developed for the assay of cyproheptadine in bulk drug and in tablet formulation based
on the bromination of cyproheptadine hydrochloride (CPH) by bromine generated in situ by the action of acid on bromatebromide mixture. In titrimetry, the drug is treated with a known excess of bromate-bromide mixture in hydrochloric acid
medium followed by the determination of unreacted bromine iodometrically. Spectrophotometry involves the addition of a
measured excess of bromate-bromide reagent in acid medium to CPH, and after the reaction is ensured to be complete, the
residual bromine is determined by reacting with a fixed amount of methyl orange, and measuring the absorbance at 520 nm.
In kinetic method, a mixture containing CPH and methyl orange dye in 1 M sulphuric acid is mixed with bromate-bromide
reagent at 4-5°C and time required for bleaching of methyl orange colour is measured. In titrimetry and spectrophotometry,
the amount of bromine reacted corresponds to the amount of CPH, and in the former method, quantitation is based on a 1:1
reaction stoichiometry (CPH:KBrO3) and the method is applicable over 2-15 mg range. The spectrophotometric method
permits the determination of CPH in 0.5-4.0 μg mL-1 range with an apparent molar absorptivity of 5.25× 104 L mol-1 cm-1
and a Sandell sensitivity of 0.0062 μg cm-2. The limits of detection (LOD) and quantification (LOQ) are calculated to be
0.04 and 0.12 μg mL-1, respectively. Quantitation in kinetic method is based on the linear relationship that exists between the
CPH concentration and the time for bromination as indicated by the bleaching of methyl orange acid color. The method is
applicable over 10-50 μg mL-1 range with LOD and LOQ values being 1.42 and 4.74 μg mL-1 , respectively. The methods
were statistically evaluated by calculating the relative error for accuracy and intra-day and inter-day coefficients of variation
for precision, and were applied successfully to the determination of CPH in tablets and syrup with mean recoveries in the
range of 97.58-100.9%. The accuracy and reliability of the methods were further ascertained by performing recovery tests
via standard-addition technique. There was no interference from tablet excipients and additives.
Keywords:Bromate-bromide mixture, Methyl orange, Cyproheptadine, Pharmaceuticals
IPC Code: G01J3/00
Cyproheptadine
hydrochloride
(CPH)
is
4-(5H-dibenzo [a, d] cyclohepten-5-ylidene)-1-methyl
piperidine hydrochloride. CPH is a serotonin and a
histamine antagonist with anticholinergic and sedative
effects. In addition, it exhibits antimuscarine and
calcium channel blocking activity. CPH is used to
treat some hormonal disorders and may be used for
treating side-effects of taking antidepressants. It has
also been shown to stimulate appetite and weight gain
in children and adults. CPH is some times used as an
adjunct in therapy in children who are taking human
growth harmone1.
Various techniques have been developed for the
assay of CPH in body fluids and pharmaceuticals.
High performance liquid chromatography (HPLC) has
been applied for the determination of the drug in
human milk and other biological fluids2-4. The drug
metabolites5
have
been
assayed
by
ultracentrifugation-mass spectrometry.
The drug is official in the Indian Pharmacopeia6
which describes a UV- spectrophotometric method for
its assay in tablets. Literature survey revealed the
availability of few methods for the assay of CPH in
pharmaceutical formulations. Liquid chromatography
(LC)7, GLC8, and HPLC9 have been used to assay
CPH. 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-tetraphenyl borate10, and
CPH-dinonylnaphthalene
sulphonic
acid11 as
electroactive compounds. Application of derivative
UV-spectrophotometry for the assay of CPH in two-
BASAVAIAH: ASSAY METHODS FOR CYPROHEPTADINE IN PHARMACEUTICALS
component12 and multi- component13 mixtures is also
reported.
There is no report on the titrimetric assay of CPH,
and all the visible spectrophotometric methods
reported are based on the extraction of the ion-pair
complex into an organic solvent before measuring the
absorbance. Adamski14 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, reextraction of the aqueous layer with chloroform, and
finally with 0.1 N sodium hydroxide followed by
absorbance measurement at 615 nm. In another
extractive colorimetric method15, the drug was
precipitated with reineckate, the ion-pair complex was
filtered, dissolved in acetone and absorbance
measured at 520 nm. Sane et al.16, 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 orange17 at
pH
4.7-4.9
followed
by
extraction
into
dichloromethane and measurement at 404 nm.
Basavaiah and Charan18 have also reported a
spectrophotometric method based on complexation
reaction using Bromophenol Blue.
Thus, the reported chromatographic techniques,
although sensitive, require expensive instrumental
set-up. The visible spectrophotometric methods
currently available involve multi-step extraction, rigid
pH control and use of organic solvents. Additionally,
their sensitivity is poor. This communication reports
three methods based on bromination of CPH by in situ
generated bromine, the reaction being followed by
titrimetry, spectrophotometry or reaction rate
measurement, and use of bromate-bromide mixture
and methyl orange as reagents. The methods are
simple, rapid and sensitive, and can be used
conveniently in laboratories which lack modern
expensive instruments such as those required for LC,
HPLC or GLC.
Experimental Procedure
Apparatus
A Systronics Model 106 digital spectrophotometer
provided with matched 1 cm quartz cells was used for
all absorbance measurements.
361
Reagents and materials
All chemicals used were of analytical reagent grade
and distilled water was used to prepare the solutions.
Bromate-bromide mixture equivalent to 0.02 M
KBrO3-0.2 M KBr was prepared by dissolving
accurately weighed 3.34 g of KBrO3 (Sarabhai M.
Chemicals, Baroda, India) and 23.8 g of KBr (S. d.
Fine Chem. Ltd., Mumbai, India) in water and
diluting to 1 litre in a calibrated flask. The solution
was used in titrimetric work and for kinetic work the
same was diluted accurately in 1:1 ratio with water.
Sodium thiosulphate solution (0.15 M), hydrochloric
acid (5 M), 1% starch indicator and potassium iodide
(10%) were prepared in the usual manner. For
spectrophotometric work, a stock standard solution
equivalent to 1000 μg mL-1 KBrO3 containing a large
excess of bromide was prepared by dissolving 100 mg
of KBrO3 and 1 g of KBr in water and diluting to the
mark with water in a 100 mL calibrated flask (KBrO3
to be weighed exactly). The stock solution was diluted
stepwise with water to get a working concentration of
10 μg mL-1 KBrO3. A stock standard solution
equivalent to 500 μg mL-1 methyl orange solution was
prepared by dissolving accurately weighed 55.5 mg of
the dye (S. d. Fine Chem. Ltd., Mumbai, India; dye
content 90%) in water and diluting to the mark in a
100 mL calibrated flask, mixed, and filtered using
glass-wool. The filtrate was diluted 10-fold to obtain a
working concentration of 50 μg mL-1 and used in
spectrophotometric work. For kinetic investigations, a
400 μg mL-1 methyl orange was similarly prepared in
exactly 1 M H2SO4 and diluted 10-fold with same
acid to obtain a working concentration of 40 μg mL-1
dye. Pharmaceutical grade CPH was received as gift
from Cipla India Ltd., Mumbai, India, and was used
as received. A stock standard solution equivalent to 2
mg mL-1 CPH was prepared by dissolving accurately
weighed 500 mg of pure drug in water and diluting to
volume in a 250 mL calibrated flask, and used in
titrimetric studies. The stock solution (2000 μg mL-1
CPH) was diluted appropriately with water to get
working concentrations of 20 and 200 μg mL-1 for
spectrophotometric method and kinetic method,
respectively.
Assay methods
Titrimetry
A 10 mL aliquot of pure drug solution containing
2-15 mg of CPH was accurately measured and
transferred into a 100 mL titration flask. The solution
was acidified by adding 5 mL of 5 M HCl. Then,
362
INDIAN J. CHEM. TECHNOL., JULY 2006
10 mL of KBrO3–KBr mixture (0.02M in KbrO3)was
added, the contents were mixed well and left to stand
for 10 min with occasional swirling. Finally, 5 mL of
10% KI solution was added and the liberated iodine
was estimated. A reagent blank experiment was
performed without CPH. The amount of CPH in the
measured aliquot was calculated based on the amount
of bromate reacted.
orange solution without CPH, and the time required to
bleach the color was noted (Tb). The corrected time
was computed using Tc=Ta-Tb. A calibration graph
was prepared by plotting the corrected time, Tb versus
concentration of CPH, or a regression equation was
derived using Tb and concentration data. The
concentration of the unknown was read from the
calibration graph or computed from the regression
equation.
Spectrophotometry
Different aliquots (0.25, 0.50---------2.0 mL) of 20
μg mL-1 standard CPH solution were accurately
transferred into a series of 10 mL calibrated flasks by
means of micro burette, and the total volume was
adjusted to 2 mL by adding requisite volume of water.
To each flask was added 2 mL of 5 M HCl followed
by 1 mL of bromate-bromide mixture (10 μg mL-1
with respect to KBrO3). The contents were mixed well
and let stand for 10 min with periodic shaking.
Finally, 1 mL of 50 μg mL-1 methyl orange solution
was added to each flask, the volume was diluted to the
mark with water, mixed and absorbance of each
solution was measured at 520 nm against a reagent
blank after 5 min. A standard graph was prepared by
plotting the measured absorbance as a function of
CPH concentration. The concentration of the
unknown was read from the calibration graph or
computed from the regression equation derived using
the Beer’s law data.
Kinetic method
Different aliquots ( 5, 10,-----------25 mL) of 200
μg mL-1 standard CPH solution were accurately
measured and transferred into separate 50 mL
calibrated flasks each containing 25 mL of methyl
orange solution (40 μg mL-1 in 1 M H2SO4) and
diluted to the mark with water. Into separate test tubes
of similar dimensions, pipetted out 5 mL of the above
drug-dye solution and 5 mL of bromate-bromide
mixture (0.01 M with respect to KBrO3). Both the
tubes were immersed in an ice bath until they reached
4-5°C. A stop-clock (accurate to 1 s) was started and
the two solutions were mixed thoroughly noting the
time of mixing (initial time Ti). The solution was
stirred gently with the thermometer used for
temperature measurements. The time at which methyl
orange acid colour was discharged was noted (final
time, Tf). The actual time required for bleaching of the
dye color is given by Ta= Tf –Ti. A blank experiment
was carried out simultaneously by mixing equal
volumes of bromate-bromide mixture and methyl
Method for tablets
Twenty tablets containing CPH were accurately
weighed and ground into a fine powder. A portion of
the powder equivalent to 250 mg of CPH was
accurately weighed into a 250 mL calibrated flask,
150 mL of water was added to the flask and shaken
for 15-20 min, the volume was diluted to the mark
with water, mixed well and the mixture was filtered
using a Whatman No 42 filter paper. The first 10 mL
portion of the filtrate was discarded, and a convenient
aliquot of the subsequent portion was assayed by the
titrimetric procedure. The filtrate equivalent to 1000
μg mL-1 CPH was diluted with water so as to get 20
and 200 μg mL-1 w. r. t. CPH for assay by
spectrophotometric method and kinetic method,
respectively.
Method for syrup
The contents of five bottles of ciplactin syrup
containing CPH were pooled and mixed. A 250 mL
portion of the syrup equivalent to 100 mg of CPH was
measured accurately and transferred into a separator.
The contents were rendered alkaline to litmus paper
with 6 M ammonia and then one mL added in excess.
The contents were then exctracted with four 25 mL
portions of chloroform, the extracts were pooled and
evaporated to dryness on a water bath. The residue
was dissolved in 0.1 M HCl and diluted to 100 mL
with water in a calibrated flask. A suitable aliquot was
then assayed by titrimetry. The syrup solution (1000
μg mL-1 CPH) was diluted appropriately as described
under tablets and analysed by spectrophotometric and
kinetic methods.
Results and Discussion
The bromate-bromide mixture and methyl orange
combination has successfully been employed for the
assay of several bioactive substances19,20. From
preliminary experiments, CPH was found to undergo
bromination by bromine, generated in situ by the
action of acid on bromate-bromide mixture. Based on
BASAVAIAH: ASSAY METHODS FOR CYPROHEPTADINE IN PHARMACEUTICALS
363
this reaction, three new methods were developed for
CPH. In titrimetry, the reaction was followed by back
titration of the unreacted bromine iodometrically, and
in spectrophotometry, it was followed by measuring
the increase in absorbance; the change being caused
by bleaching action of bromine. The same bleaching
action was used to follow the bromination reaction
kinetically.
Method development
Optimisation of reaction conditions
Titrimetry
The quantitative nature of reaction between CPH
and in situ generated bromine was tested by treating
2-15 mg of drug with a known excess of bromatebromide mixture in acid medium, and backtitrating
the residual bromine iodometrically after ensuring the
completion of reaction. The reaction stoichiometry
was calculated to be 1:1(CPH:KBrO3) in conformity
with the probable reaction scheme given in Fig. 1.
Hydrochloric acid medium was found to be better
suited and the reaction stoichiometry was unaffected
when 3-7 mL of 5 M HCl was used in a total volume
of 25 mL. Hence, 5 mL of 5 M HCl was used in the
assay procedure. At the laboratory temperature
(30±2°C), the time required for complete bromination
was found to be 5 min and contact time up to 15 min
did not affect the reaction stoichiometry and the
results. For the range studied (2-15 mg), 10 mL of
0.02 M-KBrO3-0.2 M KBr solution was found
adequate for a quantitative reaction.
Spectrophotometry
In the proposed spectrophotometric method for
CPH, varying amounts of drug were reacted with a
fixed and excess amount of bromate-bromide in HCl
medium, and after a predetermined time, the
unreacted bromine was determined by treating with a
fixed amount of methyl orange and measuring the
absorbance at 520 nm. A linear relation was found
between absorbance and concentration of CPH which
formed the basis for quantification of the drug.
A preliminary study showed that 1 mL of 50 μg mL-1
methyl orange acid form in a total volume of 10 mL
produced a convenient maximum absorbance at 520 nm.
This color was completely and irreversibly bleached by
1 mL of 10 μg mL-1 of KBrO3 in the presence of a large
excess of bromide in HCl medium. Hence, different
amounts of CPH were reacted with 1 mL of 10 μg mL-1
of KBrO3 (plus excess of KBr) and after
the bromination was judged complete, the surplus
Fig. 1⎯ Possible reaction scheme
bromine (in situ generated) was determined by
treating with 1 mL of 50 μg mL-1 methyl orange, and
measuring the absorbance at 520 nm. This step
enabled to determine the concentration range of CPH
which could be quantitated.
CPH, when added in increasing amounts to a fixed
amount of bromate-bromide mixture in acid medium,
consumes bromine proportionately, and there will be
a concomitant fall in the bromine concentration.
When a fixed amount of methyl orange was added to
decreasing concentrations of bromine, a proportional
increase in the dye concentration occurred as
indicated by the proportional increase in the
absorbance at 520 nm as a function of CPH
concentration. Hydrochloric acid medium was ideally
suited for both bromination and bleaching steps. Two
mL of 5 M HCl in a total volume of ~ 5 mL was
found adequate for the bromination step which was
complete in 5 min, and the same quantity of acid was
maintained in the final volume for bleaching of the
dye color. A contact time of 5 min was necessary for
the bleaching of methyl orange acid color. The
measured color was stable for several days in the
presence of the reaction product.
Kinetic method
The bleaching of methyl orange acid color by in
situ generated bromine has previously been applied
for the kinetic determination of various drugs21.
Applying the same concept of reaction rates, micro
amounts of CPH were determined. In the proposed
method for CPH, different amounts of drug were
mixed with a fixed amount of methyl orange and
made 0.5 M with respect to sulphuric acid. Equal
volumes of drug-dye solution and 0.01 M KBrO3 -0.1
M KBr mixture, each maintained at 4-5°C were mixed
and the time required for the red color of methyl
orange to be discharged was measured and corrected
for blank experiment. When increasing amounts of
CPH were reacted with a fixed amount of bromate-
INDIAN J. CHEM. TECHNOL., JULY 2006
364
bromide, the concentration of the latter decreased
concomitantly after the bromination reaction. This
resulted in an increase in time required for bleaching
the color of a fixed amount of methyl orange. This is
observed as proportional increase in corrected time, Tc
with increasing concentration of CPH. A linear
relationship was found between the corrected time, Tc
and concentration of CPH which served as the basis
for the quantitation of the drug.
Method validation
Quantitative data
as a function of CPH concentration, and the linearity
was found in the range of 10-50 μg mL-1 and can be
described by the equation:
Y= -0.36+ 0.258 X (n = 5, r = 0.9992)
where Y is the corrected time, Tc, in seconds and X
concentration in μg mL-1. The LOD and LOQ were
calculated to be 1.42 and 4.74 μg mL-1, respectively.
Precision and accuracy
Titrimetry was found applicable over 2-15 mg
range. Outside these limits deviant results were
obtained. The relationship between the titration end
point and the drug amount was evaluated by
calculating the correlation coefficient, r, via linear
least squares treatment, and the r value was found to
be -0.9917 suggesting that the reaction between CPH
and KBrO3 proceeds stoichiometrically in the ratio
1:1 in the investigated range.
In spectrophotometry, the Beer’s law was obeyed
over the concentration range of 0.5-4.0 μg mL-1. The
apparent molar absorptivity and Sandell sensitivity
were 5.25× 104 L mol-1 cm-1 and 0.0062 μg cm-2,
respectively. The linear plot gave the regression
equation
A= 0.005 + 0.1573 C (r ═ 0.9886, n═ 8)
Intra-day precision was assessed from the results of
seven replicate analyses on pure drug solution. The
mean values and the relative standard deviation
(RSD) values for replicate determinations at three
different
levels
(concentration/amount)
were
calculated. To evaluate the inter-day precision,
analysis was performed over a period of five days
preparing all solutions afresh each day. The accuracy
of the methods was established by calculating the
percentage deviation observed in the analysis of pure
drug solution and expressed as the percent relative
error (RE). Table 1 summarizes the intra-day
precision and accuracy data for the determination of
CPH in pure drug solution by the proposed methods,
which were within 2% (except kinetic method which
was less precise). The inter-day precision was less
than 3%.
Application to assay in dosage forms
where A is the absorbance and C, concentration in μg
mL-1. The limit of detection, LOD (K=3) and the limit
of quantification, LOQ (K=10) were established
according to ICH guidelines 22 and were calculated to
be 0.04 and 0.12 μg mL-1, respectively.
The calibration graph for kinetic method was
obtained by plotting the corrected time (Tc) in seconds
To demonstrate the applicability of the proposed
methods to the assay of CPH in tablets, commercially
available proprietary drugs containing CPH were
subjected to analysis. The results of the assay
compiled in Table 2 reveal good agreement with the
declared content and percent found. The results were
also compared with those obtained by the official
Table 1⎯ Evaluation of intra-day precision and accuracy
Method*
CPH taken
CPH found
Range
RE %
SD
SDM
RSD %
ROE** %
Titrimetry
5.0
9.0
13.0
1.0
2.0
4.0
10.0
30.0
50.0
5.04
9.13
13.29
0.99
2.03
4.05
10.17
30.55
50.24
0.15
0.28
0.33
0.04
0.05
0.12
0.28
0.61
1.81
0.80
1.44
2.23
1.00
1.50
1.25
1.70
1.83
0.48
0.11
0.03
0.15
0.015
0.013
0.034
0.24
0.96
1.43
0.04
0.01
0.06
0.0056
0.0049
0.0128
0.090
0.36
0.54
2.18
0.32
1.12
1.51
0.65
0.83
2.33
3.13
2.85
±2.17
±0.31
±1.11
±1.50
±0.64
±0.82
±2.24
±3.01
±2.75
Spectrophotometry
Kinetic method
*In titrimetry, CPH taken/found, range, SD, SDM are in mg and in the other two methods the said quantities are in μg mL-1 . RE.
relative error; SD. Standard deviation; SDM. Standard deviation of the mean; RSD. Relative standard deviation and ROE. Range of
error.
* * At 95% confidence level for six degrees of freedom.
BASAVAIAH: ASSAY METHODS FOR CYPROHEPTADINE IN PHARMACEUTICALS
365
Table 2⎯ Result of assay of CPH tablets by the proposed methods
Brand name of Formulation*
Label claim,
Practin
tabletsa
4mg/tablet
Ciplactin
syrupb
2mg/5mL
Found** (% of label claim ±SD)
Titrimetry
Spectrophotometry
Kinetic method
Reference method
100.9±0.84
t=1.91
F=1.16
97.58±1.22
t=2.96 F=2.21
99.69±1.85
t=3.24
F=5.62
98.12±1.28
t=1.89
F=2.43
100.9±1.66
t=1.26
F=4.53
98.46±1.78
t=0.73
F=4.7
101.6±0.78
99.05±0.82
** Mean value of five determinations
* Marketed by : a. Merind b. Cipla India Ltd.
Table 3⎯ Results of recovery experiment
Formulation
studied
Practin
tablets
(4 mg)
Ciplactin
Syrup
(2 mg/5mL)
Titrimetry
Spectrophotometric method
Kinetic method
CPH
Pure Total Pure CPH CPH in
Pure Total Pure CPH CPH in
Pure Total Pure CPH
informulation, CPH found, recovered* formulation, CPH found recovered* formulation, CPH found recovered*
mg
added, mg
%
µg
added, µg
%
µg mL-1
added, µg
%
mg
µg
µg mL-1 mL-1
5.05
5.05
5.05
4.88
4.88
4.88
2.0
5.0
10.0
2.0
5.0
10.0
7.12
10.19
15.08
6.77
9.57
14.64
103.6
102.8
100.3
94.66
93.73
97.58
9.97
9.97
9.97
9.81
9.81
9.81
5
10
20
5
10
20
15.30
20.80
30.67
14.65
19.25
29.76
106.7
108.3
103.5
96.76
94.38
99.75
19.62
19.62
19.62
19.69
19.69
19.69
10
20
30
10
20
30
30.07
40.88
51.90
29.44
39.15
47.99
104.5
106.3
107.6
95.73
97.28
94.33
*Mean value of three determinations
Indian Pharmacopoeial method6 by applying the
Student’s t-test and F-test at the 95% confidence
level. The calculated Student’s t-test and F-values did
not exceed the tabulated values for four degrees of
freedom suggesting that there was no significant
difference between the proposed methods and the
reference method in respect of accuracy and
precision.
The accuracy and validity of the methods were
further ascertained by performing recovery
experiments via standard–addition technique. When
the tablet powder or syrup (pre-analysed) spiked with
known amounts of pure CPH was analysed, the
recoveries of pure drug added to the tablet were
quantitative (Table 3), revealing that common
additives and excipients such as talc, starch,
gumacacia, lactose, sodium alginate, magnesium
stearate,
calcium
gluconate,
calcium
dihydrogenorthophosphate etc, did not interfere in the
determination.
Conclusions
The assay results demonstrate that it is possible to
use bromate-bromide mixture and methyl orange as
quantitative reagents for the determination of
cyproheptadine in authentic samples. The methods
allow determination of as small an amount as 5 μg
and as large an amount as 15 mg of CPH with a fair
degree of accuracy and precision. The methods can be
rapidly carried out, are simple to perform, and do not
involve any specific/special sample treatment or do
not require any expensive experimental set-up. The
titrimetric method is the first ever assay procedure
reported for cyproheptadine and is applicable over a
wide dynamic range. The spectrophotometric method
is easier to perform than all the currently available
methods which involve liquid-liquid extraction step
and use of organic solvents. The method is the most
sensitive ever developed for cyproheptadine and its
sensitivity comparable with that of many HPLC
procedures reported earlier. Even the kinetic method
is reasonably sensitive and does not require any
sophisticated equipment to measure the bleaching
time but can be followed visually. All the three
methods use eco-friendly chemicals, are fairly
accurate and hence can be used for routine quality
control.
Acknowledgements
The author gratefully acknowledges the receipt of
pure cyproheptadine from Cipla India Ltd., Mumbai,
India.
366
INDIAN J. CHEM. TECHNOL., JULY 2006
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