Chapter: make up the volume with mobile phase .From

 

Chapter:
8.0 RESULTS AND DISCUSSION

 

8.1. Solubility Studies

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These
studies are carried out at 25 0 C

CANDESARTAN:

Freely soluble in ethanol and methanol,
and slightly soluble in acetone and isopropanol and
very slightly soluble in water.

HYDROCHLOROTHIAZIDE:

Freely soluble in methanol and water.

8.2. Determination
Of  Working Wavelength (?max)

In simultaneous estimation of  two drugs isobestic wavelength is used.
Isobestic point is the wavelength where the molar absorptivity is the same for
two substances that are interconvertible. So this wavelength is used in
simultaneous estimation to estimate both drugs accurately.

8.2.1. Preparation of standard stock solution of CANDESARTAN

10
mg of CANDESARTAN
was weighed and transferred in to 100ml volumetric
flask and dissolved in methanol and then make up to the mark with methanol and
prepare 10 µg /ml of solution by diluting 1ml to 10ml with methanol.

8.2.2. Preparation of standard stock solution of HYDROCHLOROTHIAZIDE

 10 mg of HYDROCHLOROTHIAZIDE was
weighed in to 100ml volumetric flask and dissolved in Methanol and then dilute
up to the mark with methanol and prepare 10 µg /ml of solution by diluting 1ml
to 10ml with methanol.

8.2.3. Results

           The wavelength of maximum absorption
(?max) of the drug, 10 ?g/ml solution of the drugs in methanol were
scanned using UV-Visible spectrophotometer within the wavelength region of
200–400 nm against methanol as blank. The resulting spectra are shown in the
fig. no. 8.1and The isobestic point was found to be 228 nm for the combination.

 

 

 

 

 

Fig. 8.1: UV-VIS spectrum of  CANDESARTAN and HYDROCHLOROTHIAZIDE and the isosbestic point was 228 nm

Observation:
The Isobestic point was found to be 228 nm for CANDESARTAN and HYDROCHLOROTHIAZIDE in
combination and was shown in figure 8.3

8.3.
METHOD DEVELOPMENT OF CANDESARTANand
HYDROCHLOROTHIAZIDE

 

Trial
– 1

Chromatographic conditions

Mobile phase               :  NAH2PO4+K2HPO4
: METHANOL

pH                               : 3.0

Ratio                           :  40:60

Column                       :
Inertsil ODS 3V (250×4.6× 5µ)

wavelength                  : 228 nm

Flow rate                     : 1ml/min

Preparation
of mixed standard solution

weigh accurately 10mg of CANDESARTAN
and 10mg of HYDROCHLOROTHIAZIDE  in 100 ml of volumetric flask and dissolve in
100ml of mobile phase and make up the volume with mobile phase .From above
stock solution 16 µg/ml of CANDESARTAN and 12.5
µg/ml of of HYDROCHLOROTHIAZIDE  is prepared by diluting  2.85ml of above stock solution into  10ml volumetric flask make up with mobile
phase. This solution is used for recording chromatogram.

 

             Fig. 8.3.1:
Chromatogram of HYDROCHLOROTHIAZIDE and CANDESARTAN
by using mobile phase

 

Observation:

·        
The peak response of   HYDROCHLOROTHIAZIDE   was
very less.

·        
Assymetry factor more
than 2

·        
Hence it was not taken
for optimization.

 

 

 

 

Trial-
2:

Chromatographic
conditions

Mobile
phase             : Phosphate Buffer : Methanol

pH                               :  4.0

Ratio                           :   60:40

Column                       :
InertsilODS 3V, (250×4.6× 5µ)

Wavelength                 :
228nm

Flow rate                     : 1ml/min

Preparation
of mixed standard solution

weigh accurately 10mg of CANDESARTAN
and 10mg of HYDROCHLOROTHIAZIDE  in 100 ml of volumetric flask and dissolve in
100ml of mobile phase and make up the volume with mobile phase .From above
stock solution 16 µg/ml of CANDESARTAN and 12.5
µg/ml of of HYDROCHLOROTHIAZIDE  is prepared by diluting  2.85ml of above stock solution into  10ml volumetric flask make up with mobile
phase. This solution is used for recording chromatogram.

 

Fig.
8.3.3: Chromatogram of HYDROCHLOROTHIAZIDE and CANDESARTAN
by using mobile phase

 

 

Observation:

·        
Efficiency of
hydrochlorothiazide was more.

·        
The resolution was not
good .

·        
The details are given
in the table figure 8.3.3,
hence it was not taken for optimization.

 

 

Trial-
3:

Chromatographic conditions

 

Mobile
phase              :  NA2PO4 buffer : acn

 pH                              :    4.0

 Ratio                          :   60:40

Column                       :
InertsilODS , (250×4.6× 5µ)

Wavelength                 : 
228nm

Flow rate                     :  1ml/min

Preparation
of mixed standard solution

weigh accurately 10mg of CANDESARTAN
and 10mg of HYDROCHLOROTHIAZIDE  in 100 ml of volumetric flask and dissolve in
100ml of mobile phase and make up the volume with mobile phase .From above
stock solution 16 µg/ml of CANDESARTAN and 12.5
µg/ml of of HYDROCHLOROTHIAZIDE  is prepared by diluting  2.85ml of above stock solution into  10ml volumetric flask make up with mobile
phase. This solution is used for recording chromatogram.

 

Fig.
8.3.4: Chromatogram of HYDROCHLOROTHIAZIDE and CANDESARTAN
by using mobile  phase

 

Observation:

·        
Efficiency of CANDESARTAN
were very low as compared with that of HYDROCHLOROTHIAZIDE.

·        
Assymetry of more than
2.

·        
The details are given
in the table figure 8.3.4,
hence it was not taken for optimization.

 

Trial-
4:

Chromatographic conditions

 

Mobile
phase              :  phosphate buffer : Acetonitrile

 Ratio                          :   55:45

Column                       : Inertsil ODS , (250×4.6× 5µ)

Wavelength                 :  228 nm

Flow rate                     :  1ml/min

 

Preparation
of mixed standard solution

weigh accurately 10mg of CANDESARTAN
and 10mg of HYDROCHLOROTHIAZIDE  in 100 ml of volumetric flask and dissolve in
100ml of mobile phase and make up the volume with mobile phase .From above
stock solution 16 µg/ml of CANDESARTAN and 12.5
µg/ml of of HYDROCHLOROTHIAZIDE  is prepared by diluting  2.85ml of above stock solution into  10ml volumetric flask make up with mobile
phase. This solution is used for recording chromatogram.

 

 

Fig.
8.3.5: Chromatogram of HYDROCHLOROTHIAZIDE and CANDESARTAN
by using mobile phase

OBSERVATION:

EFFICIENCY
WAS LESS

 

Trial-
5:

Chromatographic conditions

 

Mobile
phase              :  Methanol: Acetonitrile : Water

 Ratio                          :   40:40:20

Column                       : Inertsil ODS , (250×4.6× 5µ)

Wavelength                 :  228 nm

Flow rate                     :  1ml/min

 

Preparation
of mixed standard solution

weigh accurately 10mg of CANDESARTAN
and 10mg of HYDROCHLOROTHIAZIDE   in 100 ml of volumetric flask and dissolve in
100ml of mobile phase and make up the volume with mobile phase .From above
stock solution 16 µg/ml of CANDESARTAN and 12.5
µg/ml of of HYDROCHLOROTHIAZIDE  is prepared by diluting  2.85ml of above stock solution into  10ml volumetric flask make up with mobile
phase. This solution is used for recording chromatogram.

Fig.
8.3.6: Chromatogram of HYDROCHLOROTHIAZIDE and CANDESARTAN
by using mobile phase

 

 

 

 

Observation:

·        
All the system
suitability requirements were met.

·        
The peak Asymmetry
factor was less than 2 for both HYDROCHLOROTHIAZIDE and CANDESARTAN.

·        
The efficiency was more
than 2000 HYDROCHLOROTHIAZIDE and CANDESARTAN.

·        
Resolution between two
peaks >1.5.

·        
The details are given
in the figure 8.3.6, hence this method was for optimized.

  

Table 8.3.8: Optimized
chromatographic conditions

Mobile phase

METHANOL:
ACN:WATER

Ph

——

Column

Inertsil ODS 3V column,C18(150×4.6 ID)
5µm

Flow rate

1.0 ml/min

Column temperature

Room temperature(20-25oC)

Sample temperature

Room temperature(20-25oC)

Wavelength

228

Injection volume

20 µl

Run time

6 min

Retention time

About
2.947 min for CANDESARTAN and 4.413 min for HYDROCHLOROTHIAZIDE.

 

 

 

 

 

 

 

8.4. Assay

Preparation of samples
for Assay

Preparation
of mixed standard solution

weigh
accurately 10mg of CANDESARTAN
and 10mg
of HYDROCHLOROTHIAZIDE  in 100 ml of volumetric flask and dissolve in
100ml of mobile phase and make up the volume with mobile phase .From above
stock solution 16 µg/ml of CANDESARTAN
and 12.5
µg/ml of of HYDROCHLOROTHIAZIDE  is prepared by diluting  2.85ml of above stock solution into  10ml volumetric flask make up with mobile
phase. This solution is used for recording chromatogram.

Tablet  sample

     10 Tablets (each capsule contains HYDROCHLOROTHIAZIDE-
12.5mgmg
CANDESARTAN- 16 mg) were weighed
and   taken into a mortar and crushed to
fine powder and uniformly mixed. Tablet  stock solutions of HYDROCHLOROTHIAZIDE and CANDESARTAN (?g/ml)
were prepared by dissolving weight equivalent to 10 mg of HYDROCHLOROTHIAZIDE
and
 CANDESARTAN and dissolved in
sufficient mobile phase. After that filtered the solution using 0.45-micron syringe
filter and Sonicated for 5 min and dilute to 100ml with mobile phase. Further
dilutions are prepared in 5 replicates of 12.5?g/ml of HYDROCHLOROTHIAZIDE
and
 16 ?g/ml  of CANDESARTAN was made by adding 2.85
ml of stock solution to 10 ml of mobile phase.

 

 

Calculation

The
amount of HYDROCHLOROTHIAZIDE and CANDESARTANpresent
in the formulation by using the formula given below, and results shown in above
table:

 

 

Where,

AS: 
Average peak area due to standard preparation

AT: 
Peak area due to assay preparation

WS: Weight of HYDROCHLOROTHIAZIDE /CANDESARTANin
mg

WT: Weight of sample in assay
preparation

DT: Dilution of assay preparation

 

 

 

 

Fig. 8.4.1: Chromatogram of Assay standard preparation-1

 

 

 

Fig. 8.4.2: Chromatogram of Assay standard preparation-2

 

 

Fig. 8.4.3: Chromatogram of Assay standard preparation-3

 

 

 

 

 

 

 

Fig. 8.4.4: Chromatogram of Assay standard preparation-4

 

 

 

       Fig. 8.4.5: Chromatogram of Assay standard preparation-5

 

Fig. 8.4.6: Chromatogram of Assay sample preparation-1

 

 

Fig. 8.4.7: Chromatogram of Assay sample preparation-2

 

 

Fig. 8.4.8: Chromatogram of Assay sample preparation-3

 

Fig. 8.4.9: Chromatogram of Assay sample preparation-4

 

Fig. 8.4.10: Chromatogram of Assay sample preparation-5

 

 

 

 

 

 

 

 

 

Table
No.8.4.9.2: Assay Results

CANDESARTAN

HYDROCHLOROTHIAZIDE

 

Standard
Area

Sample
Area

Standard
Area

Sample
Area

Injection-1

349.708

350.179

3467.353

3414.145

Injection-2

368.638

349.044

3391.872

3421.661

Injection-3

357.983

349.699

3403.268

3469.736

Injection-4

348.496

356.853

3410.801

3476.752

Injection-5

353.975

370.141

3415.216

3408.756

Average Area

355.760

355.760

3417.702

3438.21

Assay(%purity)

99.80
 

100.30
 

 

 

 

Observation

The amount of CANDESARTAN
and HYDROCHLOROTHIAZIDE present in the taken dosage form was found to be   99.80% and 100.30 % respectively.

x

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