CHAPTER CHAPTER –––VIVI Diazotization …shodhganga.inflibnet.ac.in/bitstream/10603/18365/11/11...of Selected Drugs in Pharmaceutical FormulationsFormulations Various spectrophotometric,

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


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


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:



1.0 ml of darunavir solution (100 µg/ml) is transferred into a 10 ml




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


absorbance of the orange coloured form is measured in the wavelength range of


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


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




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


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:


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


6.2(d). Effect of concentration β-naphthol on the coupling

reaction is studied by the following procedure.


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


Table6.2.3:


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



6.2(e) Assay Procedure:



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



five minutes at 0-5oC temperature for diazotization to complete. 1.0 ml of 1% urea







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








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


Ab

so

rban

ce

Y=0.0084X+0.0017

R2= 0.9998



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



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


λ max (nm) 490





0.351

Regression equation (Y = a + bC) Y=0.0084x+0.017

Slope (b) 0.0084

Intercept (a) 0.017


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



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



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:



1.0 ml of mosapride solution (100µg/ml) is transferred into a 10 ml







absorbance of the orange coloured form is measured in the wavelength range of


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


at 500 nm.

The optimal conditions for the determination of mosapride are arrived to by


6.3.(b). Effect of concentration of hydrochloric acid on the

diazotization and coupling reagent





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



experimental work.

6.3.(c). Effect of concentration of sodium nitrite on the coupling



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



solutions is measured at 500 nm and the data are presented in table.6.3.2.

Table.6.3.2:


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




6.3.(d). Effect of concentration β-naphthol on the coupling reaction is

studied by the following procedure.


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:


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







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











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



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


Ab

sorb

ance

Y=0.0045X+0.0068

R2 = 0.9995






of 100 µg/ml.



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




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:



λ max (nm) 470





0.3614


Slope (b) 0.045



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



nitrogen gas to escape. The diazonium cation reacts with the coupling reagent, β-


produce an orange azo product. This wine red product shows maximum



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:



1.0 ml of abacavir solution (100 µg/ml) is transferred into a 10 ml







absorbance of the orange colour formed is measured in the wavelength range of


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


at 470 nm.

The optimal conditions for the determination of Abacavir are arrived to by


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





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


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



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



solutions is measured at 470 nm and the data are presented in table.6.42.

Table.6.4.2:


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




6.4.(d). Effect of concentration β-naphthol on the coupling



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







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











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





Calibration curve of abacavir

0

0.2

0.4

0.6

0.8

1

1.2

0 20 40 60 80 100 120


Ab

sorb

ance

Y=0.0087X+0.0047

R2= 0.9992




of 100 µg/ml.



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




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:



λmax (nm) 470





0.233


Slope (b) 0.0087



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



nitrogen gas to escape. The diazonium cation reacts with the coupling reagent, β-


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.

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CHAPTER CHAPTER –––VIVI Diazotization …shodhganga.inflibnet.ac.in/bitstream/10603/18365/11/11...of Selected Drugs in Pharmaceutical FormulationsFormulations Various spectrophotometric,