chapter chapter –––vivi diazotization...
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CHAPTER CHAPTER CHAPTER CHAPTER ––––VIVIVIVI
Diazotization Method for the Assay Diazotization Method for the Assay Diazotization Method for the Assay Diazotization Method for the Assay
of Selected Drugs in Pharmaceutical of Selected Drugs in Pharmaceutical of Selected Drugs in Pharmaceutical of Selected Drugs in Pharmaceutical
FormulationsFormulationsFormulationsFormulations
Various spectrophotometric, chromatographic and HPLC methods are
available in the literature for the estimation of selected drugs. HPLC method and
chromatographic methods are time consuming and expensive. These instruments
are not within the reach of many laboratories. Usually spectrophotometric
technique is simple and less expensive. The selectivity and sensitivity of the
spectrophotometric methods depends only on the nature of chemical reactions
involved in colour development and not on the sophistications of the experiment.
UV and visible spectrophotometric methods are highly versatile,
sensitive and reproducible. The author has therefore intended to develop new
spectrophotometric methods for the estimation of selected drugs in pharmaceutical
preparations.
Diazotisation method is used in pharmaceutical analysis for estimating
drugs containing amino group. The process of converting a primary aromatic
amine into a diazonium salt is known as diazotization. In the diazotisation reaction,
the temperature is kept between 0 to 5oC. The diazonium salts condense with
aromatic amine containing amino group in weak acidic medium and with hydroxyl
group of aromatic compounds in a weak alkaline medium to form highly coloured
derivatives known as azo compounds. The process is called coupling and it usually
takes place at para-position to the amino or phenolic group, in case the para-
position is blocked, coupling occurs at the ortho-position.
Various spectrophotometric methods are available in the literature for
estimation of drugs by diazotization and coupling reaction. The reagents such as
acetylacetone, benzoyl acetone, dibenzyl methane, 1-naphthyl ethylene diamine,
1:1 ammonia: water solution, 2-napthol, 3-amino phenol, etc., are used for the
estimation of drugs by diazotization method. But all have certain limitations. In
these methods more steps are involved, heating is necessary; the colour
development is not instant and not reproducible values. The recently proposed
method using 1:1 ammonia: water solution is less sensitive, time consuming and
involves several steps.
No method is reported in the literature for estimation of the selected
drugs by using β-naphthol as the coupling reagent. Hence, it is proposed to use β-
naphthol as coupling reagent for the estimation of the selected drugs by
spectrophotometry. The method is simple, rapid, reproducible, precise, and needs
no extraction or heating, colour development is instantaneous, and the colour is
stable for more than 24 hours. Further, the controlling of experimental conditions
is minimum.
The proposed general procedure: The drug containing amino group is treated
with cold solution of sodium nitrite in acidic medium at 0-5oC temperature. The
resultant solution is allowed to stand for five minutes for the diazotization to
complete. Then the drug is treated with β-naphthol to produce coloured species.
The absorbance of the coloured species is measured at the wavelength of
maximum absorbance for each drug against the reagent blank (prepared in a
similar manner devoid of drug solution) and the amount of drug is determined from
the calibration curve made between the absorbance and the amount of drug.
Section (i)6.1: Diazotisation and coupling reaction of mesalamine with
β-naphthol
Mesalamine, chemically known as 5-aminosalicylic acid is used for its local
effects in the treatment of inflammatory bowel disease, including ulcerative colitis
and Crohn’s disease. The amino group in mesalamine is diazotised with sodium
nitrite and hydrochloric acid at 0oC temperature. After diazotisation, the diazonium
salt is coupled with β-naphthol. The orange coloured chromogen formed in the
method is stable for more than 24 hours. The orange coloured chromogen is used
to determine the mesalamine spectrophotometrically.
Mesalamine could be readily diazotized in acid medium and the
resultant diazonium cation would then react with coupling reagent β-naphthol by
electrophilic substitution at the position ortho to the phenolic hydroxyl group and
β-naphthol results in the formation of the coloured product.
6.1.(a) Spectrum of diazotized mesalamine treated with β-naphthol:
The wavelength of maximum absorbance of the diazotised drug treated
with β-naphthol solution is ascertained by the following procedure.
1 ml of mesalamine solution (100 µg/ml) is transferred into a 10 ml
volumetric flask. To this, 2.0 ml of 0.1N hydrochloric acid and 1.0 ml of cold 0.1N
sodium nitrite solution are added. The resultant solution is well mixed, and then
allowed to stand for five minutes at 0-5oC temperature for diazotization. To this
solution 1.0 ml of 1% urea solution is added and shaken frequently for nitrogen gas
to escape. Then 1.0 ml of 0.5N sodium carbonate and 1.0 ml of 1% β-naphthol
solution are added and the volume is made to 10 ml with methanol. The
absorbance of the orange colour formed is measured in the wavelength range of
440 to 560 nm, against the reagent blank. The spectrum is given in fig.6.1.1.
Fig:6.1.1: Spectrum of diazotized mesalamine treated with β-naphthol
From fig 6.1.1, it is clear that the diazotised drug treated with β- naphthol
solution has maximum absorbance at 510 nm. Hence, all further studies are made
at 510 nm.
The optimal conditions for the determination of mesalamine are arrived at by
the following steps.
Spectrum of mesalamine
0
0.1
0.2
0.3
0.4
0.5
400 450 500 550 600 650 700
Wavelength
Ab
sorb
ance
6.1.(b). Effect of concentration of hydrochloric acid on the diazotization
and coupling reaction
The stability of the colour species depends on the concentration of
hydrochloric acid. The effect of hydrochloric acid on the absorbance is studied by
varying the volume of hydrochloric acid (0.1N) and measuring the absorbance at
510 nm. The data is presented in table. 6.1.1.
Table. 6.1.1.
Effect of concentration of hydrochloric acid
solution on absorbance
The data in table.6.1.1 show that 2.0 ml of hydrochloric produces maximum
absorbance and hence the same concentration is maintained throughout the
experimental work.
6.1.(c). Effect of concentration of sodium nitrite on the absorbance of
coupling reaction is studied by the following procedure.
Volume of
HCl (ml)
Absorbance at
510 nm.
1.0 0.236
1.5 0.325
2.0 0.408
2.5 0.405
In a series of 10 ml volumetric flasks containing 1.0 ml of
(100 µg/ml) mesalamine, 2.0 ml of 0.1N hydrochloric acid, 1.0 ml of β-
naphthol solution, 1.0 ml of 1% urea solution, 1.0 ml of 0.5N sodium carbonate
solution are taken and varying amounts of sodium nitrite are added. The contents
are made up to the mark and set aside for 5 minutes for completion of the reaction.
The absorbance of the resultant solutions is measured at 510nm and the data are
presented in table.6.1.2.
Table.6.1.2:
Effect of concentration of sodium nitrite
The data in table 6.1.2 indicate that 1.0 ml of sodium nitrite is necessary for
achieving maximum absorbance and hence maintained through out the
experimental studies.
6.1.(d). Effect of concentration of β-naphthol on the coupling reaction
is studied by the following procedure.
Volume of
Sodium nitrite
(ml)
Absorbance at
510 nm.
0.5 0.243
1.0 0.402
1.5 0.403
2.0 0.403
In a series of 10 ml volumetric flasks containing 1.0 ml of
(100 µg/ml) mesalamine, 2.0 ml of 0.1N hydrochloric acid, 1.0 ml of 0.1N sodium
nitrite solution, 1.0 ml of 1% urea solution, 1.0 ml of 0.5N sodium carbonate
solution are taken and varying amounts of β-naphthol are added. The contents are
made up to the mark and set aside for 5 minutes for completion of the reaction. The
absorbance of the resultant solutions is measured at 510 nm and the data are
presented in table.6.1.3.
Table.6.1.3:
Effect of concentration of β-naphthol
.
The data in table.6.1.3 indicate that 1.0 ml of 1% β-naphthol is necessary for
achieving maximum absorbance and hence maintained through out the
experimental studies
6.1.(e) Assay Procedure:
Volume of
β-naphthol(ml)
Absorbance at
510 nm.
0.5 0.245
1.0 0.405
1.5 0.403
2.0 0.401
To study the effect of drug concentration on the absorbance of the coupling
reaction under optimal conditions now arrived is studied by the following method
to know the suitability of the method for the assay of mesalamine.
Various aliquots of the standard mesalamine solution ranging from 0.2-1.0
ml are transferred into a series of 10 ml volumetric flasks. To each flask, 2.0 ml of
0.1N hydrochloric acid solution and 1.0 ml of cold 0.1N sodium nitrite solution are
added. The resultant solution in each flask is well shaken and allowed to stand for
five minutes at 0-50C temperature for diazotization to complete. 1.0 ml of 1% urea
solution is added to each flask and the solution is shaken frequently to allow
nitrogen gas to escape. Then 1.0 ml of 0.5N sodium carbonate solution and 1.0 ml
of 1% β-naphthol solution are added and the volume in each flask is made up to 10
ml with methanol. An orange colour is formed. The maximum absorbance of the
orange coloured solution is measured at 510 nm against the reagent blank.
Calibration graph is obtained by plotting absorbance values against the
concentration of mesalamine solution. The calibration curve is found to be linear
over a concentration range of 20 to 100 µg/ml of mesalamine. The amount of
mesalamine present in the sample is estimated from the calibration graph. The
results are presented in fig.6.1.2
Fig: 6.1.2 Calibration curve of mesalamine
6.1.(f) Assay of mesalamine in pharmaceutical formulations:
The proposed procedure for the assay of mesalamine is applied for its
determination in commercial tablets.
Preparation of the sample solution:
Powdered tablet equivalent to 50 mg of the drug is weighed accurately and
transferred into a 50 ml beaker and mixed well with 30 ml of methanol. The
solution is filtered and transferred into a 50 ml volumetric flask and the volume is
made up to 50 ml with methanol. The concentration of the drug solutions is now
Calibration curve of mesalamine
0
0.1
0.2
0.3
0.4
0.5
0 20 40 60 80 100 120
Amount of drug in micrograms
Ab
sorb
ance
Y=0.0044X+0.0024
R2= 0.9998
1mg/ml. This stock solution is further diluted to obtain the working concentration
of 200 µg/ml.
The pharmaceutical preparation as prepared above is analysed by the
following procedure.
6.1.(g) Assay Procedure: Known volumes of the drug formulation prepare as
above are transferred into a series of 10 ml volumetric flasks and 2 ml of 0.1N
hydrochloric acid solution, 1.0 ml of 0.1N sodium nitrite solution are added. The
resultant solution in each flask is shaken well and allowed to stand for five minutes
at 0-50C temperature for diazotization. Then 1.0 ml of 1% urea solution, 1 ml of
0.5N sodium carbonate and 1.0 ml of 1% β-naphthol solution are added and the
volume is made up to 10 ml with methanol. The absorbance of the resultant
solution is measured at 460 nm. The amount of mesalamine in the pharmaceutical
formulation is evaluated from the predetermined calibration plot. The results are
presented in table. 6.1.5.
Table. 6.1.4:
Optical characteristics of proposed method
parameters Proposed method
λmax (nm) 510
Beer’s law limit (µg/ml) 20-100
Molar absorptivity (l mole-1 cm-1) 7.01x102
Sandell’s sensitivity
(µg cm-2 / 0.001 absorbance unit)
1.425
Regression equation (Y = a + bC) Y=0.0044+0.0024
Slope (b) 0.0044
Intercept (a) 0.0024
Correlation coefficient (r) 0.9997
Table. 6.1.5:
Assay of mesalamine in tablets
*A
ver
age
of
five determination based on the label claim
6.1.(h) Results and discussion:
Mesalamine undergoes diazotisation when treated with sodium nitrite and
hydrochloric acid. The excess nitrous acid during the diazotisation is removed by
the addition of urea solution. The solution was shaken frequently to allow the
nitrogen gas to escape. The diazonium cation reacts with the coupling reagent β-
S.No Sample
(mg)
*Amount
Found(mg)
±S.D*
Percentage of
Label claim
%RSD*
*tcal
1 250 250.03±0.10 100.01 1082 0.6211
2 250 249.8±0.43 99.92 0.1721 0.1040
3 250 249.92±0.44 99.96 0.1798 0.3998
naphthol by electrophilic substitution at the o-position of the coupling agent to
produce an orange azo product. This orange colour product shows maximum
absorbance at 510 nm. The colour of the product is stable for more than 24 hours.
The calibration curve (concentration vs absorbance) is linear over the range of 40-
200 µg/ml of mesalamine. The optical characteristics of the proposed method such
as absorption maxima, Beer´s law limits, molar absorptivity and Sandell´s
sensitivity are presented in Table 6.1.4. The molar absorptivity and Sandell´s
sensitivity values show that method is sensitivity. The regression analysis using
method of least squares was made for the slope (b), intercept (a) and correlation (r)
obtained from different concentrations and results are summarized in the Table
6.1.4. The value of correlation coefficient was 0.999, which indicated the good
linearity of calibration lines. The percent relative standard deviation calculated
from the five measurements of mesalamine shown in Table 6.1.5. The % RSD is
less than 2, which indicates that the method has good reproducibility. The values of
standard deviation, coefficient of variation values are low, indicates high accuracy
and reproducibility of the method. The‘t’ calculated values are compared well with
the theoretical value of 2.78 there by indicating that the precision of the method is
good. There no effect of additives and excipients such starch, calcium lactose and
glucose in the concentrations those present in general pharmaceutical preparations.
The proposed method is found to be simple, precise, accurate and time
saving, reproducible and can be conveniently adopted for routine analysis of
estimation of mesalamine in bulk drugs samples and pharmaceutical formulations.
Section (ii): Diazotisation and coupling reaction of darunavir
with β-naphthol:
The amino group in darunavir is diazotised with sodium nitrite and
hydrochloric acid at 0oC temperature. After diazotisation, the diazonium salt is
coupled with β-naphthol. The orange coloured chromogen formed in the method is
stable for more than 24 hours. The orange red coloured chromogen is used to
determine the darunavir spectrophotometrically.
Darunavir could be readily diazotized in acid medium and the resultant
diazonium cation would then react with coupling reagent β-naphthol by
electrophilic substitution at the position ortho to the phenolic hydroxyl group and
results in the formation of the coloured product.
6.2.(a) Spectrum of diazotized darunavir treated with β-naphthol:
The wavelength of maximum absorbance of the diazotised drug treated
with β-naphthol solution is ascertained by the following procedure.
1.0 ml of darunavir solution (100 µg/ml) is transferred into a 10 ml
volumetric flask. To this, 2.0 ml of 0.1N hydrochloric acid and 1.0 ml of cold 0.1N
sodium nitrite solution are added. The resultant solution is well mixed, and then
allowed to stand for five minutes at 0-5oC temperature for diazotization. To this
solution 1 ml of 1% urea solution is added and shaken frequently for nitrogen gas
to escape. Then 1.0 ml of 0.5N sodium carbonate and 1ml of 1% β-naphthol
solution are added and the volume is made to 10 ml with methanol. The
absorbance of the orange coloured form is measured in the wavelength range of
400 to 650 nm, against the reagent blank. The spectrum is given in fig.6.2.1.
Fig.6.2.1: Spectrum of diazotized darunavir treated with β-naphthol
From fig 6.2.1, it is clear that diazotised drug treated with β-naphthol
solution has maximum absorbance at 490 nm. Hence, all further studies are made
at 490 nm. The optimal conditions for the determination of darunavir are arrived at
by the following steps.
Spectrum of darunavir
0
0.1
0.2
0.3
0.4
0.5
0.6
400 450 500 550 600 650 700
Wavelength
Ab
sorb
ance
6.2(b). Effect of concentration of hydrochloric acid on the diazotization and
coupling reaction
The stability of the colour species depends on the concentration of
hydrochloric acid. The effect of hydrochloric acid on the absorbance is studied by
varying the volume of hydrochloric acid (0.1N) and measuring the absorbance at
490 nm. The data are presented in table. 6.2.1.
Table.6.2.1.
Effect of concentration of hydrochloric acid solution on absorbance
Volume of
HCl (ml)
Absorbance at
490 nm.
1.0 0.221
1.5 0.315
2.0 0.489
2.5 0.491
The data in table.6.2.1 shows that 2.0 ml of hydrochloric produces maximum
absorbance and hence the same concentration is maintained throughout the
experimental work.
6.2.(c). Effect of concentration of sodium nitrite on the coupling reaction
is studied by the following procedure.
In a series of 10 ml volumetric flasks containing 1.0 ml of (100 µg/ml)
darunavir, 2.0 ml of 0.1N hydrochloric acid, 1.0 ml of 1% β-naphthol, 1.0 ml of
1% urea solution, 1.0 ml of 0.5N sodium carbonate solution are taken and varying
amounts of sodium nitrite are added. The contents are made up to the mark and set
aside for 5 minutes for completion of the reaction. The absorbance of the resultant
solutions is measured at 450 nm and the data are presented in table.6.2.2.
Table.6.2.2:
Effect of concentration of sodium nitrite
Volume of
Sodium nitrite
(ml)
Absorbance at
490 nm.
0.5 0.320
1.0 0.487
1.5 0.485
2.0 0.452
The data in table.6.2.2 indicate that 1.0 ml of sodium nitrite is necessary for
achieving maximum absorbance and hence maintained throughout the
experimental studies.
6.2(d). Effect of concentration β-naphthol on the coupling
reaction is studied by the following procedure.
In a series of 10 ml volumetric flasks containing 1.0 ml of (100 µg/ml)
darunavir , 2.0 ml of 0.1N hydrochloric acid, 1.0 ml of 0.1N sodium nitrite
solution, 1.0 ml of 1% urea solution, 1.0 ml of 0.5N sodium carbonate solution are
taken and varying amounts of β-naphthol are added. The contents are made up to
the mark and set aside for 5 minutes for completion of the reaction. The
absorbance of the resultant solutions is measured at 450 nm and the data are
presented in table.6.2.3.
Table6.2.3:
Effect of concentration of β-naphthol
Volume of
β-naphthol(ml)
Absorbance at
490 nm.
0.5 0.375
1.0 0.486
1.5 0.487
2.0 0.481
The data in table 6.2.3 indicate that 1.0 ml of 1% β-naphthol is necessary for
achieving maximum absorbance and hence maintained through out the
experimental studies.
6.2(e) Assay Procedure:
To study the effect of drug concentration on the absorbance of the coupling
reaction under optimal conditions now arrived is studied by the following method
to know the suitability of the method for the assay of darunavir.
Various aliquots of the standard darunavir solution ranging from 0.2-1.0 ml
are transferred into a series of 10 ml volumetric flasks. To each flask, 2.0 ml of
0.1N hydrochloric acid solution and 1.0 ml of cold 0.1N sodium nitrite solution are
added. The resultant solution in each flask is well shaken and allowed to stand for
five minutes at 0-5oC temperature for diazotization to complete. 1.0 ml of 1% urea
solution is added to each flask and the solution is shaken frequently to allow
nitrogen gas to escape. Then 1.0 ml of 0.5N sodium carbonate solution and 1.0 ml
of 1% β-naphthol solution are added and the volume in each flask is made up to 10
ml with methanol. An orange colour is formed. The maximum absorbance of the
orange coloured solution is measured at 490 nm against the reagent blank.
Calibration graph is obtained by plotting absorbance values against the
concentration of darunavir solution. The calibration curve is found to be linear over
a concentration range of 20 to 100 µg/ml of darunavir. The amount of darunavir
present in the sample is estimated from the calibration graph. The results are
presented in fig. 6.2.2
Fig. 6.2.2: Calibration curve of darunavir
6.2.(f) Assay of darunavir in pharmaceutical formulations:
The proposed procedure for the assay of darunavir is applied for its
determination in commercial tablets.
Preparation of the sample solution:
Powdered tablet equivalent to 50 mg of the drug is weighed accurately and
transferred into a 50 ml beaker and mixed well with 30 ml of methanol. The
solution is filtered and transferred into a 50 ml volumetric flask and the volume is
made up to 50 ml with methanol. The concentration of the drug solutions is now
1mg/ml. This stock solution is further diluted to obtain the working concentration
of 100 µg/ml.
Calibration curve of darunavir
0
0.1
0.2
0.3
0.4
0.5
0.6
0 20 40 60 80 100 120
Amount of drug in micrograms
Ab
so
rban
ce
Y=0.0084X+0.0017
R2= 0.9998
The pharmaceutical preparation as prepared above is analysed by the
following procedure.
6.2.(g) Assay Procedure: Known volumes of the drug formulation prepare as
above are transferred into a series of 10 ml volumetric flasks and 2 ml of 0.1N
hydrochloric acid solution, 1.0 ml of 0.1N sodium nitrite solution are added. The
resultant solution in each flask is shaken well and allowed to stand for five minutes
at 0-50C temperature for diazotization. Then 1.0 ml of 1% urea solution, 1.0 ml of
0.5N sodium carbonate and 1.0 ml of 1% β-naphthol solution are added and
volume is made up to 10 ml with methanol. The absorbance of the resultant
solution is measured at 490 nm. The amount of darunavir in the pharmaceutical
formulation is evaluated from the predetermined calibration plot. The results are
presented in table 6.2.5.
Table. 6.2.4.
The optical characteristics of the proposed method
parameters Proposed method
λ max (nm) 490
Beer’s law limit (µg/ml) 20-100
Molar absorptivity (l mole-1 cm-1) 2.384x103
Sandell’s sensitivity
(µg cm-2 / 0.001 absorbance unit)
0.351
Regression equation (Y = a + bC) Y=0.0084x+0.017
Slope (b) 0.0084
Intercept (a) 0.017
Correlation coefficient (r) 0.9998
Table6.2.4:
Assay of darunavir in pharmaceutical formulations
Sample
Labelled
amount
(mg)
*Amount
found
±S.D*
% of
Label claim
*%RSD
*tcal
Tablet 1
400 399.96 ±0.28 99.99 0.071 0.3333
Tablet 2 400 399.99 ±0.3 99.99 0.0167 0.0729
Tablet 3 400 399.92±0.48 99.98 0.1204 0.3715
*Average of five determinations based on the label claim
6.2(h) Results and discussion:
Darunavir undergoes diazotisation when treated with sodium nitrite and
hydrochloric acid. The excess nitrous acid during the diazotisation is removed by
the addition of urea solution. The solution was shaken frequently to allow the
nitrogen gas to escape. The diazonium cation reacts with the coupling reagent,
β-naphthol by electrophilic substitution at the o-position of the coupling agent to
produce an orange azo product. This orange red product shows maximum
absorbance at 490 nm. The colour of the product is stable for more than 24 hours.
The calibration curve (concentration vs absorbance) is linear over the range of 20-
100 µg/ml of darunavir. The optical characteristics of the proposed method such as
absorption maxima, Beer´s law limits, molar absorptivity and Sandell´s sensitivity
are presented in Table 6.2.4. The molar absorptivity and Sandell´s sensitivity
values show that method is sensitivity. The regression analysis using method of
least squares was made for the slope (b), intercept (a) and correlation (r) obtained
from different concentrations and results are summarized in the Table 6.2.4. The
value of correlation coefficient was 0.999, which indicated the good linearity of
calibration lines. The percent relative standard deviation calculated from the five
measurements of darunavir shown in Table 6.2.5. The % RSD is less than 2,
which indicates that the method has good reproducibility. The standard deviation
values are low indicates high accuracy and reproducibility of the method. The‘t’
calculated values are compares well with the theoretical value of 2.78 there by
indicating that the precision of the method is good.
The proposed method is simple, rapid and accurate and precise and can be
used for routine analysis of darunavir from tablet formulations
Section (iii): Diazotisation and coupling reaction of mosapride
with β-naphthol
The amino group in mosapride is diazotised with sodium nitrite and
hydrochloric acid at 0oC temperature. After diazotisation, the diazonium salt is
coupled with β-naphthol. The orange coloured chromogen formed in the method is
stable for more than 24 hours. The orange coloured chromogen is used to
determine the mosapride spectrophotometrically.
Mosapride could be readily diazotized in acid medium and the resultant
diazonium cation would then react with coupling reagent β- Naphthol by
electrophilic substitution at the position ortho to the phenolic hydroxyl group ( β-
naphthol) and results in the formation of the coloured product.
6.3. (a) Spectrum of diazotized mosapride treated with β-naphthol:
The wavelength of maximum absorbance of the diazotised drug treated
with β-naphthol solution is ascertained by the following procedure.
1.0 ml of mosapride solution (100µg/ml) is transferred into a 10 ml
volumetric flask. To this, 2.0 ml of 0.1N hydrochloric acid and 1.0 ml of cold 0.1N
sodium nitrite solution are added. The resultant solution is well mixed, and then
allowed to stand for five minutes at 0-5oC temperature for diazotization. To this
solution 1 ml of 1% urea solution is added and shaken frequently for nitrogen gas
to escape. Then 1.0 ml of 0.5N sodium carbonate and 1ml of 1% β-naphthol
solution are added and the volume is made to 10 ml with methanol. The
absorbance of the orange coloured form is measured in the wavelength range of
400 to 560 nm, against the reagent blank. The spectrum is given in fig.6.3.1.
Fig.6.3.1: Spectrum of diazotized mosapride treated with
β-naphthol
Spectrum of mosapride
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
400 450 500 550 600 650 700
Wavelength
Ab
sorb
ance
From fig 6.3.1, it is clear that the diazotised drug treated with β-naphthol
solution has maximum absorbance at 500 nm. Hence, all further studies are made
at 500 nm.
The optimal conditions for the determination of mosapride are arrived to by
the following steps.
6.3.(b). Effect of concentration of hydrochloric acid on the
diazotization and coupling reagent
The stability of the colour species depends on the concentration of
hydrochloric acid. The effect of hydrochloric acid on the absorbance is studied by
varying the volume of hydrochloric acid (0.1N) and measuring the absorbance at
500 nm. The data are presented in table. 6.4.1.
Table. 6.3.1.
Effect of concentration of hydrochloric acid solution
on absorbance
Volume of
Hcl (ml)
Abs., at
500 nm.
1.0 0.298
1.5 0.468
2.0 0.645
2.5 0.643
The data in table.6.4.1 shows that 2.0 ml of hydrochloric produces maximum
absorbance and hence the same concentration is maintained throughout the
experimental work.
6.3.(c). Effect of concentration of sodium nitrite on the coupling
reaction is studied by the following procedure.
In a series of 10 ml volumetric flasks containing 1.0 ml of (100 µg/ml)
mosapride, 2.0 ml of 0.1N hydrochloric acid, 1.0 ml of 1% urea solution, 1.0 ml of
0.5N sodium carbonate solution, 1.0 ml of 1% β-naphthol are taken and varying
amounts of sodium nitrite are added. The contents are made up to the mark and set
aside for 5 minutes for completion of the reaction. The absorbance of the resultant
solutions is measured at 500 nm and the data are presented in table.6.3.2.
Table.6.3.2:
Effect of concentration of sodium nitrite
Volume of
sodium nitrite
(ml)
Absorbance at
500 nm.
0.5 0.278
1.0 0.642
1.5 0.645
2.0 0.643
The data in table.6.3.2 indicate that 1.0 ml of sodium nitrite is necessary for
achieving maximum absorbance and hence maintained throughout the
experimental studies.
6.3.(d). Effect of concentration β-naphthol on the coupling reaction is
studied by the following procedure.
In a series of 10 ml volumetric flasks containing 1.0 ml of (100 µg/ml)
mosapride, 2.0 ml of 0.1N hydrochloric acid, 1.0 ml of 0.1N sodium nitrite
solution, 1.0 ml of 1% urea solution, 1.0 ml of 0.5N sodium carbonate solution are
taken and varying amounts of β-naphthol are added. The contents are made up to
the mark and set aside for 5 minutes for completion of the reaction. The
absorbance of the resultant solutions are measured at 500nm and the data are
presented in table.6.3.3
Table.6.3.3:
Effect of concentration of β-naphthol
Volume of
β-naphthol(ml)
Absorbance at
500 nm.
0.5 0.314
1.0 0.645
1.5 0.642
2.0 0.640
The data in table.6.3.3 indicate that 1.0 ml of 1% β-naphthol is necessary for
achieving maximum absorbance and hence maintained throughout the
experimental studies.
6.3.(e) Assay Procedure:
To study the effect of drug concentration on the absorbance of the coupling
reaction under optimal conditions now arrived is studied by the following method
to know the suitability of the method for the assay of mosapride.
Various aliquots of the standard mosapride solution ranging from 0.2-1.0 ml
are transferred into a series of 10 ml volumetric flasks. To each flask, 2.0 ml of
0.1N hydrochloric acid solution and 1.0 ml of cold 0.1N sodium nitrite solution are
added. The resultant solution in each flask is well shaken and allowed to stand for
five minutes at 0-50C temperature for diazotization to complete. 1.0 ml of 1% urea
solution is added to each flask and the solution is shaken frequently to allow
nitrogen gas to escape. Then 1.0 ml of 0.5N sodium carbonate solution and 1.0 ml
of 1% β-naphthol solution are added and the volume in each flask is made up to 10
ml with methanol. An orange colour is formed. The maximum absorbance of the
orange coloured solution is measured at 500 nm against the reagent blank.
Calibration graph is obtained by plotting absorbance values against the
concentration of mosapride solution. The calibration curve is found to be linear
over a concentration range of 20 to 100 µg/ml of mosapride. The amount of of
mosapride present in the sample is estimated from the calibration graph. The
results are presented in fig. 6.3.2
Fig. 6.3.2: Calibration curve of mosapride
6.3.(f) Assay of mosapride in pharmaceutical formulations:
The proposed procedure for the assay of mosapride is applied for its
determination in commercial tablets.
Preparation of the sample solution:
Calibration curve of mosapride
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 20 40 60 80 100 120
Amount of drug in micrograms
Ab
sorb
ance
Y=0.0045X+0.0068
R2 = 0.9995
Powdered tablet equivalent to 50 mg of the drug is weighed accurately and
transferred into a 50 ml beaker and mixed well with 30 ml of methanol. The
solution is filtered and transferred into a 50 ml volumetric flask and the volume is
made up to 50 ml with methanol. The concentration of the drug solutions is now
1mg/ml. This stock solution is further diluted to obtain the working concentration
of 100 µg/ml.
The pharmaceutical preparation as prepared above is analysed by the
following procedure.
6.3.(g)Assay Procedure: Known volumes of the drug formulation prepare as
above are transferred into a series of 10 ml volumetric flasks and 2.0 ml of 0.1N
hydrochloric acid solution, 1.0 ml of 0.1N sodium nitrite solution are added. The
resultant solution in each flask is shaken well and allowed to stand for five minutes
at 0-50C temperature for diazotization. Then 1.0 ml of 1% urea solution, 1 ml of
0.5N sodium carbonate and 1.0 ml of 1% β-naphthol solution are added and the
volume is made to 10 ml with methanol. The absorbance of the resultant solution is
measured at 500 nm. The amount of mosapride in the pharmaceutical formulation
is evaluated from the predetermined calibration plot. The results are presented in
table 6.3.5.
Table.6.3.4:
Optical characteristics of proposed method
parameters Proposed method
λ max (nm) 470
Beer’s law limit (µg/ml) 20-100
Molar absorptivity (l mole-1 cm-1) 2.766x103
Sandell’s sensitivity
(µg cm-2 / 0.001 absorbance unit)
0.3614
Regression equation (Y = a + bC) Y=0.045x+0.0068
Slope (b) 0.045
Intercept (a) 0.0068
Correlation coefficient (r) 0.9995
Table 6.3.5:
Assay of mosapride in pharmaceutical formulations
Sample
Labelled
amount
(mg)
*Amount
Found
±S.D*
% of
Label
claim
%RSD
*tcal
Tablet 1
5 4.99 ±0.02 99.8 0.5231 0.7874
Tablet 2 5 4.98 ±0.04 99.6 0.8947 0.9808
Tablet 3 5 5.02±0.05 100.4 0.0116 0.7692
*Average of five determinations based on the label claim
6.3.(h) Results and discussion:
Mosapride undergoes diazotisation when treated with sodium nitrite and
hydrochloric acid. The excess nitrous acid during the diazotisation is removed by
the addition of urea solution. The solution was shaken frequently to allow the
nitrogen gas to escape. The diazonium cation reacts with the coupling reagent, β-
naphthol by electrophilic substitution at the o-position of the coupling agent to
produce an orange azo product. This wine red product shows maximum
absorbance at 500 nm. The colour of the product is stable for more than 24 hours.
The calibration curve (concentration vs absorbance) is linear over the range of 20-
100 µg/ml of mosapride. The optical characteristics of the proposed method such
as absorption maxima, Beer´s law limits, molar absorptivity and Sandell´s
sensitivity are presented in Table 6.4.4. The molar absorptivity and Sandell´s
sensitivity values show that method is sensitivity. The regression analysis using
method of least squares was made for the slope (b), intercept (a) and correlation (r)
obtained from different concentrations and results are summarized in the Table
6.4.4. The value of correlation coefficient was 0.999, which indicated the good
linearity of calibration lines. The percent relative standard deviation calculated
from the five measurements of mosapride shown in Table 6.4.5. The % RSD is
less than 2, which indicates that the method has good reproducibility. The standard
deviation values are low indicates high accuracy and reproducibility of the method.
The‘t’ calculated values are compares well with the theoretical value of 2.78 there
by indicating that the precision of the method is good. There no effect of additives
and excipients such starch, calcium lactose and glucose in the concentrations those
present in general pharmaceutical preparations.
The proposed method are found to be simple, sensitive, selective, accurate, precise,
and economical, and can be used in the determination of mosapride in bulk drug
and its pharmaceutical dosage forms tablets in a routine manner.
Section (iv)6.4: Diazotisation and coupling reaction of abacavir with
β-naphthol
The amino group in abacavir is diazotised with sodium nitrite and hydrochloric
acid at 0oC temperature. After diazotisation, the diazonium salt is coupled with β-
naphthol. The orange coloured chromogen formed in the method is stable for more
than 24 hours. The orange coloured chromogen is used to determine the abacavir
spectrophotometrically.
Abacavir could be readily diazotized in acid medium and the resultant
diazonium cation would then react with coupling reagent β-naphthol by
electrophilic substitution at the position ortho to the phenolic hydroxyl group (β-
naphthol) and results in the formation of the coloured product.
6.4. (a) Spectrum of diazotized abacavir treated with β-naphthol:
The wavelength of maximum absorbance of the diazotised drug treated
with β-naphthol solution is ascertained by the following procedure.
1.0 ml of abacavir solution (100 µg/ml) is transferred into a 10 ml
volumetric flask. To this, 2.0 ml of 0.1N hydrochloric acid and 1.0 ml of cold 0.1N
sodium nitrite solution are added. The resultant solution is well mixed, and then
allowed to stand for five minutes at 0-5oC temperature for diazotization. To this
solution 1 ml of 1% urea solution is added and shaken frequently for nitrogen gas
to escape. Then 1.0 ml of 0.5N sodium carbonate and 1ml of 1% β-naphthol
solution are added and the volume is made to 10 ml with methanol. The
absorbance of the orange colour formed is measured in the wavelength range of
400 to 560 nm, against the reagent blank. The spectrum is given in fig.6.4.1.
Fig.6.4.1: Spectrum of diazotized abacavir treated with β- naphthol
From fig 6.3.1, it is clear that the diazotised drug treated with β-naphthol
solution has maximum absorbance at 470 nm. Hence, all further studies are made
at 470 nm.
The optimal conditions for the determination of Abacavir are arrived to by
the following steps.
6.4.(b). Effect of concentration of hydrochloric acid on the
diazotization and coupling reagent
Spectrum of abacavir
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
400 450 500 550 600 650 700
Wavelength
Ab
sorb
ance
The stability of the colour species depends on the concentration of
hydrochloric acid. The effect of hydrochloric acid on the absorbance is studied by
varying the volume of hydrochloric acid (0.1N) and measuring the absorbance at
400 nm. The data are presented in table. 6.4.1.
Table. 6.4.1.
Effect of concentration of hydrochloric acid solution
on absorbance
Volume of
HCl (ml)
Abs., at
470 nm.
1.0 0.574
1.5 0.854
2.0 1.053
2.5 1.051
The data in table.6.3.1 shows that 2.0 ml of hydrochloric produces maximum
absorbance and hence the same concentration is maintained through out the
experimental work.
6.4.(c). Effect of concentration of sodium nitrite on the coupling
reaction is studied by the following procedure.
In a series of 10 ml volumetric flasks containing 1.0 ml of (100 µg/ml)
abacavir, 2.0 ml of 0.1N hydrochloric acid, 1.0 ml of 1% urea solution, 1.0 ml of
0.5N sodium carbonate solution, 1.0 ml of 1% β-naphthol are taken and varying
amounts of sodium nitrite are added. The contents are made up to the mark and set
aside for 5 minutes for completion of the reaction. The absorbance of the resultant
solutions is measured at 470 nm and the data are presented in table.6.42.
Table.6.4.2:
Effect of concentration of sodium nitrite
Volume of
sodium nitrite
(ml)
Absorbance at
470 nm.
0.5 0.982
1.0 1.043
1.5 1.041
2.0 1.041
The data in table.6.4.2 indicate that 1.0 ml of sodium nitrite is necessary for
achieving maximum absorbance and hence maintained through out the
experimental studies.
6.4.(d). Effect of concentration β-naphthol on the coupling
reaction is studied by the following procedure.
In a series of 10 ml volumetric flasks containing 1.0 ml of (100 µg/ml)
abacavir, 2.0 ml of 0.1N hydrochloric acid, 1.0 ml of 0.1N sodium nitrite solution,
1.0 ml of 1% urea solution, 1.0 ml of 0.5N sodium carbonate solution are taken and
varying amounts of β-naphthol are added. The contents are made up to the mark
and set aside for 5 minutes for completion of the reaction. The absorbance of the
resultant solutions are measured at 470 nm and the data are presented in table.6.4.3
Table.6.4.3:
Effect of concentration of β-Naphthol
Volume of
β-naphthol(ml)
Absorbance at
470 nm.
0.5 0.745
1.0 1.055
1.5 1.053
2.0 1.052
The data in table.6.3.3 indicate that 1.0 ml of 1% β-naphthol is necessary for
achieving maximum absorbance and hence maintained through out the
experimental studies.
6.4.(e) Assay Procedure:
To study the effect of drug concentration on the absorbance of the coupling
reaction under optimal conditions now arrived is studied by the following method
to know the suitability of the method for the assay of abacavir.
Various aliquots of the standard abacavir solution ranging from 0.2-1.0 ml
are transferred into a series of 10 ml volumetric flasks. To each flask, 2.0 ml of
0.1N hydrochloric acid solution and 1.0 ml of cold 0.1N sodium nitrite solution are
added. The resultant solution in each flask is well shaken and allowed to stand for
five minutes at 0-50C temperature for diazotization to complete. 1.0 ml of 1% urea
solution is added to each flask and the solution is shaken frequently to allow
nitrogen gas to escape. Then 1.0 ml of 0.5N sodium carbonate solution and 1.0 ml
of 1% β-naphthol solution are added and the volume in each flask is made up to 10
ml with methanol. An orange colour is formed. The maximum absorbance of the
orange coloured solution is measured at 470 nm against the reagent blank.
Calibration graph is obtained by plotting absorbance values against the
concentration of abacavir solution. The calibration curve is found to be linear over
a concentration range of 20 to 100 µg/ml of abacavir. The amount of abacavir
present in the sample is estimated from the calibration graph. The results are
presented in fig. 6.4.2
Fig. 6.3.2: Calibration curve of abacavir
6.4.(f) Assay of abacavir in pharmaceutical formulations:
The proposed procedure for the assay of abacavir is applied for its
determination in commercial tablets.
Preparation of the sample solution:
Powdered tablet equivalent to 50 mg of the drug is weighed accurately and
transferred into a 50 ml beaker and mixed well with 30 ml of methanol. The
Calibration curve of abacavir
0
0.2
0.4
0.6
0.8
1
1.2
0 20 40 60 80 100 120
Amount of drug in micrograms
Ab
sorb
ance
Y=0.0087X+0.0047
R2= 0.9992
solution is filtered and transferred into a 50 ml volumetric flask and the volume is
made up to 50 ml with methanol. The concentration of the drug solutions is now
1mg/ml. This stock solution is further diluted to obtain the working concentration
of 100 µg/ml.
The pharmaceutical preparation as prepared above is analysed by the
following procedure.
6.4.(g)Assay Procedure: Known volumes of the drug formulation prepare as
above are transferred into a series of 10 ml volumetric flasks and 2.0 ml of 0.1N
hydrochloric acid solution, 1.0 ml of 0.1N sodium nitrite solution are added. The
resultant solution in each flask is shaken well and allowed to stand for five minutes
at 0-50C temperature for diazotization. Then 1.0 ml of 1% urea solution, 1 ml of
0.5N sodium carbonate and 1.0 ml of 1% β-naphthol solution is added. The
absorbance of the resultant solution is measured at 470 nm. The amount of
abacavir in the pharmaceutical formulation is evaluated from the predetermined
calibration plot. The results are present in table 6.4.5.
Table.6.4.4:
Optical characteristics of proposed method
parameters Proposed method
λmax (nm) 470
Beer’s law limit (µg/ml) 20-100
Molar absorptivity (l mole-1 cm-1) 4.28x103
Sandell’s sensitivity
(µg cm-2 / 0.001 absorbance unit)
0.233
Regression equation (Y = a + bC) Y=0.0087x+0.0047
Slope (b) 0.0087
Intercept (a) 0.0047
Correlation coefficient (r) 0.9992
Table 6.3.5:
Assay of abacavir in pharmaceutical formulations
Labelled
amount (mg)
*Amount
Found
±S.D*
% of
Label claim
%RSD
**tcal
300 300.01 ±0.34 100.00 0.1148 0.0644
300 300.05 ±0.38 100.01 0.1278 0.2912
300 300.09±0.36 100.03 0.1203 0.5572
6.4(h) Results and discussion:
Abacavir undergoes diazotisation when treated with sodium nitrite and
hydrochloric acid. The excess nitrous acid during the diazotisation is removed by
the addition of urea solution. The solution was shaken frequently to allow the
nitrogen gas to escape. The diazonium cation reacts with the coupling reagent, β-
naphthol by electrophilic substitution at the o-position of the coupling agent to
produce an orange azo product. This orange product shows maximum absorbance
at 400 nm. The colour of the product is stable for more than 24 hours. The
calibration curve (concentration vs absorbance) is linear over the range of 20-100
µg/ml of abacavir. The optical characteristics of the proposed method such as
absorption maxima, Beer´s law limits, molar absorptivity and Sandell´s sensitivity
are presented in Table 6.3.4. The molar absorptivity and Sandell´s sensitivity
values show that method is sensitivity. The regression analysis using method of
least squares was made for the slope (b), intercept (a) and correlation (r) obtained
from different concentrations and results are summarized in the Table 6.4.4. The
value of correlation coefficient was 0.999, which indicated the good linearity of
calibration lines. The percent relative standard deviation calculated from the five
measurements of abacavir shown in Table 6.4.5. The % RSD is less than 2, which
indicates that the method has good reproducibility. The standard deviation values
are low indicates high accuracy and reproducibility of the method. The‘t’
calculated values are compared well with the theoretical value of 2.78 there by
indicating that the precision of the method is good. There no effect of additives and
excipients such starch, calcium lactose and glucose in the concentrations those
present in general pharmaceutical preparations.
The proposed method is simple, accurate, precise and selective for the
estimation of abacavir in bulk and in capsule dosage forms.