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4. Experimental

4. Experimental

Akshay R. Koli 104

Contents

4. Experimental ............................................................................................ 107

4.1 Research Methodology ................................................................... 107

4.2 List of equipments .......................................................................... 110

4.3 List of Materials .............................................................................. 111

4.4 Identification and Characterization of Drugs ................................... 113

4.4.1 Identification and Characterization by FTIR absorption

spectroscopy .................................................................................. 113

4.4.2 Identification and Characterization by UV absorption

spectroscopy .................................................................................. 116

4.5 Estimation of Felodipine by UV-visible spectroscopy ...................... 118

4.5.1 Preparation of Calibration Curve of Felodipine in Methanol...... 118

4.5.2 Preparation of calibration curve of Felodipine in PBS pH 6.8 ..... 121

4.5.3 Interference Study of Felodipine with Excipients ........................ 125

4.6 Estimation of Valsartan by UV-visible spectroscopy ........................ 126

4.6.1 Preparation of calibration curve of Valsartan in Methanol: ....... 126

4.6.2 Preparation of calibration curve of Valsartan in PBS pH 6.8 ...... 129

4.6.3 Interference Study of Valsartan with excipients ......................... 132

4.6.4 Derivative Method ......................................................................... 133

4.7 References ...................................................................................... 137

4. Experimental

Akshay R. Koli 105

List of Figures

Figure 4.4.1.1: FTIR Spectra of sample Felodipine ........................................ 114

Figure 4.4.1.2 FTIR spectra of reference Felodipine ..................................... 114

Figure 4.4.1.3 FTIR spectra of sample Valsartan ........................................... 115

Figure 4.4.1.4 FTIR Spectra of reference Valsartan ....................................... 115

Figure 4.4.2.1 UV spectra of sample Felodipine in Methanol ....................... 116

Figure 4.4.2.2 UV spectra of sample Valsartan in Methanol ......................... 117

Figure 4.5.1.1: UV spectrum of 10 µg/ml of Felodipine in methanol ........... 119

Figure 4.5.1.2: Calibration curve of Felodipine in methanol at 360.50 nm ... 120

Figure 4.5.2.1: UV Spectrum of 10 µg/ml of Felodipine in PBS pH 6.8

containing 1% Tween80 at 360.50 nm .................................... 122

Figure 4.5.2.2: Calibration curve of Felodipine in PBS pH 6.8 containing 1%

Tween 80 at 360.50 nm .......................................................... 123

Figure 4.5.3.1: UV Spectrum of 10 µg/ml Felodipine solution in absence and

presence of excipients in PBS pH 6.8 containing 1% Tween 80 125

Figure 4.6.1.1: UV spectrum of 10 µg/ml of Valsartan in methanol .............. 127

Figure 4.6.1.2: Calibration curve of Valsartan in methanol at 250 nm .......... 128

Figure 4.6.2.1: UV spectrum of Valsartan in pH 6.8 phosphate buffer .......... 129

Figure 4.6.2.2: Calibration curve of Valsartan in PBS pH 6.8 at 250nm. ........ 130

Figure 4.6.3.1: Zero Crossing Point of Tween 80 .......................................... 132

Figure 4.6.4.1: Calibration Curve of Valsartan in methanol at 232nm ......... 134

Figure 4.6.4.2: Calibration Curve of Valsartan in PBS pH 6.8 at 232nm ........ 136

4. Experimental

Akshay R. Koli 106

List of Tables

Table 4.2.1.1: List of instruments required for the research work ................ 110

Table 4.3.1.1: List of materials required for the research work ................... 111

Table 4.3.1.2: List of reagents required for the research work. .................... 112

Table 4.5.1.1: Calibration curve of Felodipine in methanol. ......................... 120

Table 4.5.1.2: Calibration data of Felodipine in methanol ............................ 121

Table 4.5.2.1: Calibration curve of Felodipine in PBS pH 6.8 containing 1%

Tween 80 ................................................................................ 123

Table 4.5.2.2: Calibration data of Felodipine in PBS pH 6.8 containing 1%

Tween 80 ................................................................................ 124

Table 4.6.1.1: Calibration curve of Valsartan in methanol ........................... 128

Table 4.6.1.2: Calibration data of Valsartan in methanol.............................. 129

Table 4.6.2.1: Calibration curve of Valsartan in PBS pH 6.8 .......................... 130

Table 4.6.2.2: Calibration data of Valsartan in PBS pH 6.8 ............................ 131

Table 4.6.4.1: Calibration curve of Valsartan in methanol at 232nm ............ 134

Table 4.6.4.2: Calibration data of Valsartan in methanol at 232nm .............. 135

Table 4.6.4.3: Calibration curve of Valsartan in PBS pH 6.8 at 232nm .......... 135

Table 4.6.4.4: Calibration data of Valsartan in PBS pH 6.8 at 232nm ............ 136

4. Experimental

Akshay R. Koli 107

4. Experimental

4.1 Research Methodology

To achieve the objectives of present work, the experimental work was performed as

per the following steps.

1. Identification and characterization of the selected drugs (Felodipine and

Valsartan) by Melting point study, FTIR spectroscopy and UV spectroscopy.

2. Establishment of analytical method of the selected drugs Felodipine and

Valsartan.

3. Selection of suitable oil phase based on solubility study of drugs in oils.

Solubility of drugs was determined in different oils (such as capmul MCM,

Capryol 90, Capmul MCM C8, Capmul MCM C10, Captex 200P, Captex

355, Isopropyl myristate, Soyabean oil, Castor oil)

4. Selection of suitable surfactants and co-surfactants based on solubility study:

Surfactants such as such as Tween 20, Tween 80, Labrasol, Plurol oleique,

Cremophore EL were used. Co-surfactants such as Transcutol P, PEG 400,

Propylene Glycol, Labrafil were used.

5. Drug and Surfactant compatibility study.

i. Physical compatibility includes precipitation/crystallization, phase

separation and color change in the drug surfactant solution during

course study.

ii. Chemical compatibility was regarded as the chemical stability of the

drug in a surfactant solution.

6. Optimization of surfactant: co-surfactant ratio by pseudo-ternary phase

diagram for microemulsion and SMEDDS. The existence of microemulsions

regions were determined using pseudo-ternary phase diagrams at different

weight ratio of surfactants and co-surfactants by water titration method. Also

the effect of drug loading on phase diagram of the selected systems was

studied.

7. Formulation development of microemulsions of selected drugs by phase

titration (water titration) method.

4. Experimental

Akshay R. Koli 108

i. Felodipine Microemulsion System prepared using:

Oil: Capmul MCM

Surfactant: Tween 20

Co-surfactant: PEG 400

ii. Valsartan self Microemulsifying drug delivery system prepared using:

Oil: Capmul MCM

Surfactants: Tween 80, Labrasol

Co-surfactants: Transcutol P, PEG 400.

8. Characterization and optimization of Microemulsion and SMEDDS:

i. Appearance

ii. Clarity

iii. Thermodynamic stability

iv. Dispersibility test

v. Droplet size and zeta potential analysis

vi. Polydispersivity index

vii. Dye solubility test (Felodipine Microemulsion)

viii. Conductivity measurement (Valsartan SMEDDS)

ix. Assay

x. pH

xi. Viscosity

xii. Dynamic surface tension (Felodipine Microemulsion)

xiii. Transmission electron microscopy (Felodipine Microemulsion)

9. The effect of drug loading and pH of the dispersion medium on droplet size of

microemulsion was studied.

10. In-vitro drug release study

i. In-vitro dissolution study

ii. In-vitro intestinal permeability study.

11. In-vivo absorption studies of oral microemulsion of Felodipine and

comparison with plain drug suspension.

12. Development of Solid SMEDDS of Valsartan from optimized liquid

SMEDDS by adsorption on solid carriers.

4. Experimental

Akshay R. Koli 109

13. Characterization and Optimization of Solid SMEDDS of Valsartan:

i. Angle of Repose

ii. In-vitro dissolution study of solid SMEDDS dosage form and

comparision with optimized liquid SMEDDS and marketed drug

formulation.

iii. Reconstitution properties of solid SMEDDS

iv. Morphological analysis of solid SMEDDS

v. Solid state characterization of Solid SMEDDS

14. Stability studies of optimized microemulsion, SMEDDS and Solid SMEDDS:

i. Robustness to dilution

ii. Physical Stability

iii. Chemical Stability

4. Experimental

Akshay R. Koli 110

4.2 List of equipments

The list of equipments used for our entire research in order of their utilization is

provided in Table 4.2.1.1

Table 4.4.1.1: List of instruments required for the research work

Sr.

No.

Equipment Model & Make of the

Equipment/Instrument

1. Digital weighing balance Shimadzu electronic analytical

balance

2. FT IR FTIR– Brucker alpha-E model

3. UV spectrophotometer

UV-1800 spectrophotometer,

Shimadzu

4. Magnetic Stirrer MS500, Remi Equipments

5. Malvern zetasizer

Nano ZS LU-227, Malvern

Instruments,

6. Digital pH meter

Systronic, 361-micro pH meter

7. Brookfield Viscometer DVII

LVDVII+PRO, Brookfield, USA

8. Bubble Tensiometer BPA-800P

9. Transmission electron microscope TEM-JEM-100SX, JEOL, Tokyo

10. Dissolution apparatus Electrolab TDT-80L

11. Organ Bath

SE 1 AW Orchid Scientifics

India

12.

HPLC with UV-Visible Detector

and HPLC packed column- C18 SPD-10A, Shimadzu

13. Vortex Mixer Remi Equipments

14. Mono ocular Electron Microscope Olympus, 220 V

15. Centrifuge RM 12 C, Remi Equipments

4. Experimental

Akshay R. Koli 111

4.3 List of Materials

The list of materials used for our entire research as per their category is provided in

Table 4.3.1.1

Table 4.4.1.1: List of materials required for the research work

Sr.

no.

Name Category Supplier of material

1. Felodipine IP, BP, USP API SPARC Vadodara

2. Valsartan IP, USP API Torrent Pharma

3. Capmul MCM Oil Abitec Corporation, USA

4. Capmul MCM C8 Oil Abitec Corporation, USA

5. Capmul MCM C10 Oil Abitec Corporation, USA

6. Captex 200 Oil Abitec Corporation, USA

7. Captex 200P Oil Abitec Corporation, USA

8. Captex 355 Oil Abitec Corporation, USA

9. Capryol 90 Oil Gattefosse,France

10. Castor oil IP Oil Suvidhinath Lab

11. Olive oil IP Oil Sous Cuetora

12. Isopropyl myristate IP Oil S.D fine Chem

13. Soyabin oil Oil S.D fine Chem

14. Tween 80 IP Surfactant S.D fine Chem

15. Tween 20 IP Surfactant S.D fine Chem

16. Labrasol Surfactant Gattefosse,France

17. Plurol Oleique Surfactant Gattefosse,France

18. Peceol Co-surfactant Gattefosse,France

19. Transcutol P Co-surfactant Gattefosse,France

20. Polyethylene Glycol 400(PEG

400) IP

Co-surfactant Suvidhinath Lab

21. Aerosil 200 IP, USP Adsorbent Evonik Deggussa

22. Avicel PH 102 BP, USP Adsorbent SD fine chem. Ltd.

4. Experimental

Akshay R. Koli 112

Table 4.4.1.2: List of reagents required for the research work.

Sr. No. Reagents

1. 0.05M Phosphate buffer pH 6.8

2. 0.1 M Hydrochloric Acid (HCl)

3. 0.02 M NaOH

4. Methanol

5. 0.02M Potassium Dihydrogen Phosphate

4. Experimental

Akshay R. Koli 113

4.4 Identification and Characterization of Drugs

The drugs selected for present investigation were Felodipine and Valsartan.

Felodipine was selected for formulation and development of microemulsion and

Valsartan was selected for formulation and development of Solid SMEDDS. The

sample of Felodipine was a yellowish white crystalline powder. The sample of

Valsartan was a white crystalline powder. The melting point of Felodipine and

Valsartan were determined by Open Capillary Method and the uncorrected melting

point was found to be 140-1450C and 105-110

0C respectively. To prevent

photodegradation of Felodipine, all the experimental work was carried out under

light protected conditions.

4.4.1 Identification and Characterization by FTIR absorption

spectroscopy

The IR Spectrum of the drug samples were recorded using FTIR– Brucker alpha-E

model. The peaks observed for Felodipine and Valsartan are shown in Fig 4.4.1.1 and

Fig.4.4.1.3 respectively.

4. Experimental

Akshay R. Koli 114

Figure 4.4.1.1: FTIR Spectra of sample Felodipine

Figure 4.4.1.2 FTIR spectra of reference Felodipine[1, 2]

The IR spectrum of the pure drug Felodipine used in the present study shows

characteristic absorption bands at 3366(N-H Stretching), 2980(Aromatic C-H

stretching), 2945(C-H stretching of CH2 and CH3 Groups), 1688 (C=O stretching),

1642 (N-H Bending), 1620, 1494, 1461 (C=C ring stretching), 1096 (C-O-C

stretching), 726, 801 (Substituted benzene ring), 564 (Cl stretching) cm-1

respectively.

The FTIR spectrum of the sample was compared with the reference spectrum as

shown in Fig 4.4.1.2. It was concluded that sample of Felodipine obtained was pure.

4. Experimental

Akshay R. Koli 115

Figure 4.4.1.3 FTIR spectra of sample Valsartan

Figure 4.4.1.4 FTIR Spectra of reference Valsartan

From the FTIR studies, the characteristic bands for important functional group of pure

Valsartan were identified as shown in Figure 4.1.1.3. The following peaks were

observed in both spectrum of Valsartan; 3100 cm -1

( N – H stretching), 2961.13 cm-1

(C – H streaching), 1273.68 and 1204.31 cm-1

due to C–N stretching, 1667 cm-1

due

to C = O stretching, 1512 cm-1

( N=N bond), 1353 cm-1

(C=N bond). FTIR spectrum

showed that the characteristics bands of sample of Valsartan were similar to that of

reference spectrum of Valsartan found in Indian Pharmacopoeia 2010 as shown in Fig

4.4.1.4. The sample obtained of Valsartan was found to be pure.

4. Experimental

Akshay R. Koli 116

4.4.2 Identification and Characterization by UV absorption

spectroscopy

Accurately weighed 25 mg Felodipine was transferred to 25 ml volumetric flask.

Small quantity of methanol was added to ensure complete dissolution of Felodipine

and finally volume was made up to the mark with methanol (1 mg/ml solution). From

the above solution, 5ml of solution was withdrawn accurately with the help of pipette

and transferred to 50ml volumetric flask. Volume was made up to the mark with

methanol to make stock solution (100 µg/ml). 1 ml of this solution was transferred to

a 10 ml volumetric flask and diluted with methanol to make up the volume. Then the

prepared solution of Felodipine (10 µg/ml) was scanned using Shimadzu double beam

UV-visible spectrophotometer from wavelength 200-400 nm range using methanol as

blank. Absorption maximum (λmax) was obtained at 360 nm as shown in Fig. 4.4.2.1.

Figure 4.4.2.1 UV spectra of sample Felodipine in Methanol

Accurately weighed 100 mg of Valsartan was transferred to 100 ml volumetric flask.

Small quantity of methanol was added to ensure complete dissolution of Valsartan




4. Experimental

Akshay R. Koli 117

methanol to make stock solution (100 µg/ml).1 ml of this solution was transferred to a

10 ml volumetric flask and diluted with methanol to make up the volume. Then the

prepared solution of Valsartan(10 µg/ml) was scanned using Shimadzu double beam

UV-visible spectrophotometer from wavelength 200-400 nm range using methanol as

blank. Absorption maximum (λmax) was obtained at 250 nm as shown in Fig. 4.4.2.2.

It complies with IP’2010.

Figure 4.4.2.2 UV spectra of sample Valsartan in Methanol

The absorption maximum wavelength (λmax) obtained for both drugs were in

compliance with λmax of both drugs as reported in literature. The above results

confirmed that both samples of drugs were pure as sharp and satisfactory peaks were

observed.

4. Experimental

Akshay R. Koli 118

4.5 Estimation of Felodipine by UV-visible spectroscopy

In this investigation, microemulsion system for Felodipine was prepared for

bioavailability enhancement. The analytical methods used for the estimation of drug

content, the developed formulation, and for the purpose of ex-vivo studies were based

on the reported UV spectrophotometric methods using methanol or sodium

hydroxide[1]

. In the present study, the solvent used for estimation of drug content in

the formulation was methanol and for ex-vivo studies was Phosphate Buffer Solution

(PBS) pH 6.8 containing 1% Tween-80. Tween 80 was used in order to maintain sink

conditions as Felodipine has very low solubility in PBS 6.8.

4.5.1 Preparation of Calibration Curve of Felodipine in Methanol

Preparation of stock solution:

Accurately weighed (25 mg) Felodipine was transferred to 25 ml volumetric flask.

Small quantity of methanol was added to ensure complete dissolution of Felodipine




methanol to make stock solution (100 µg/ml).

Determination of λmax:

1 ml of the stock solution was transferred to a 10 ml volumetric flask and diluted with

methanol to make up the volume. Then the prepared solution of Felodipine (10 µg/ml)

was scanned in the range of 200 nm-400 nm using methanol as blank.

4. Experimental

Akshay R. Koli 119

Figure 4.5.1.1: UV spectrum of 10 µg/ml of Felodipine in methanol

As shown in Figure 4.5.1.1, Felodipine in methanol showed maximum absorbance at

360.50 nm, this was thus selected as the analytical wavelength.

Preparation of calibration curve:

From the stock solution, aliquots of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 ml were accurately

withdrawn with the help of pipette and transferred to separate 10ml volumetric flasks

and the volume was made up to the mark with methanol to give final concentration of

10, 15, 20, 25, 30, 35, 40, 45 µg/ml. The absorbance of all the prepared solutions was

then measured at the absorption maxima, using methanol as blank. The readings were

recorded in triplicate.

4. Experimental

Akshay R. Koli 120

Table 4.5.1.1: Calibration curve of Felodipine in methanol.

Conc. (µg/ml)

Absorbance*

(*Mean ± SD, n = 3)

0 0

10 0.165 ± 1.24

15 0.255 ± 1.14

20 0.349 ± 0.55

25 0.443 ± 0.55

30 0.519 ± 0.89

35 0.616 ± 1.56

40 0.701 ± 0.77

45 0.804 ± 0.89

Figure 4.5.1.2: Calibration curve of Felodipine in methanol at 360.50 nm

Table 4.5.1.1 showed the mean absorbance values of the solutions along with the

standard deviation values. As shown in Figure 4.5.1.2, Felodipine follows Beer-

Lambert’s law in the range of 10-45 μg/ml. The high value of regression coefficient

(0.999) in methanol indicates that the absorbance and concentration of drug are

linearly related. Optical characteristics of Felodipine are summarized in Table 4.5.1.2.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 10 20 30 40 50

Ab

sorb

an

ce

Conc. (µg/ml)

Calibration plot of Felodipine in methanol

4. Experimental

Akshay R. Koli 121

Low values of standard deviation also indicate the reproducibility of the analytical

method.

Table 4.5.1.2: Calibration data of Felodipine in methanol

λmax

(nm)

Solvent

used

Conc.

range

Regression

equation

Regression coefficient

(R2)

360.50 Methanol 10-45µg/ml y = 0.017x – 0.008 0.999

4.5.2 Preparation of calibration curve of Felodipine in PBS pH 6.8


Accurately weighed (25 mg) Felodipine was transferred to 25 ml volumetric flask.

Small quantity (1-2 ml) of methanol was added to ensure complete dissolution of

Felodipine and finally volume was made up to the mark with PBS 6.8 containing 1%

Tween 80 (1 mg/ml solution). From the above solution, 5ml of solution was

withdrawn accurately with the help of pipette and transferred to 50ml volumetric

flask. Volume was made up to the mark with PBS 6.8 containing 1% Tween 80 to

make stock solution (100 µg/ml).



PBS 6.8 containing 1% Tween 80 to make up the volume. The prepared solution of

Felodipine (10 µg/ml) was scanned in the range of 200 nm-400 nm using PBS 6.8

containing 1% Tween 80 as blank.

4. Experimental

Akshay R. Koli 122

Figure 4.5.2.1: UV Spectrum of 10 µg/ml of Felodipine in PBS pH 6.8 containing

1% Tween80 at 360.50 nm

As shown in Figure 4.5.2.1, Felodipine in PBS pH 6.8 containing 1% Tween 80

showed maximum absorbance at 360.50 nm, which was thus selected as the analytical

wavelength.


From the stock solution, aliquots of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 ml were accurately

withdrawn with the help of pipette and transferred to separate 10 ml volumetric flasks

and the volume was made up to the mark with PBS 6.8 containing 1% Tween 80 to

give final concentration of 10, 15, 20, 25, 30, 35, 40, 45 µg/ml. The absorbance of all

the prepared solutions was then measured at the absorption maxima, using PBS 6.8

containing 1% Tween 80 as blank. The readings were recorded in triplicate. Results

are shown in Table 4.5.2.1.

4. Experimental

Akshay R. Koli 123

Table 4.5.2.1: Calibration curve of Felodipine in PBS pH 6.8 containing 1%

Tween 80

Conc. (µg/ml) Absorbance

Mean ± SD, n = 3

0 0

10 0.189 ± 1.56

15 0.284 ± 2.34

20 0.373 ± 0.56

25 0.466 ± 0.76

30 0.565 ± 0.56

35 0.653 ± 1.24

40 0.753 ± 1.89

45 0.840 ± 0.55

Figure 4.5.2.2: Calibration curve of Felodipine in PBS pH 6.8 containing 1%

Tween 80 at 360.50 nm


standard deviation values. As shown in Figure 4.5.2.2, Felodipine follows Beer-


(0.999) in PBS pH 6.8 containing 1% Tween 80 indicates that the absorbance and

concentration of drug were linearly related. Optical characteristics of Felodipine are

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 10 20 30 40 50

Ab

sorb

ance

Conc. (µg/ml)

Calibration curve in Felodipine in PBS pH 6.8

containing 1% tween 80

4. Experimental

Akshay R. Koli 124

summarized in Table 4.5.2.2. Low values of standard deviation also indicate the

reproducibility of the analytical method.

Table 4.5.2.2: Calibration data of Felodipine in PBS pH 6.8 containing 1%

Tween 80

λmax

(nm) Solvent used

Conc.

range

Regression

equation

Regression

coefficient (R2)

360.50 PBS 6.8 containing

1% Tween 80

10-

45µg/ml y = 0.018x + 0.001 0.999

4. Experimental

Akshay R. Koli 125

4.5.3 Interference Study of Felodipine with Excipients

In order to ascertain the non-interference of the excipients in estimation of Felodipine,

solutions containing known concentration of each excipient were prepared in

methanol and PBS 6.8 containing 1% Tween 80. The prepared solutions were scanned

in the UV region between 200-400 nm using the respective blank. Also, to study the

effect in presence of drug, Felodipine solutions (10 µg/ml) in methanol as well as in

PBS 6.8 containing 1% Tween 80 was spiked with known concentrations of each

excipient (capmul MCM, Tween 20 and PEG 400) and scanned in the UV region

between 200 nm-400 nm.

Figure 4.5.3.1: UV Spectrum of 10 µg/ml Felodipine solution in absence and

presence of excipients in PBS pH 6.8 containing 1% Tween 80

Figure 4.5.3.1 showed spectrum of 10 µg/ml Felodipine solution in PBS pH 6.8

containing 1% Tween 80 alone and with excipients, almost overlapping, indicates that

they are not interfering the estimation of Felodipine in diffusion medium.

4. Experimental

Akshay R. Koli 126

4.6 Estimation of Valsartan by UV-visible spectroscopy

In this investigation, SMEDDS for Valsartan was prepared for bioavailability

enhancement. The analytical methods used for the estimation of drug content, the

developed formulation, and for the purpose of ex-vivo studies were based on the

reported UV spectrophotometric methods using methanol at 250nm. In the present

study, the solvent used for estimation of drug content in the formulation was methanol

and for ex-vivo studies was Phosphate Buffer Solution (PBS) pH 6.8 containing 1%

Tween-80[4]

. Tween 80 was used in order to maintain sink conditions as Valsartan has

very low solubility in PBS 6.8[3]

.

4.6.1 Preparation of calibration curve of Valsartan in Methanol:







methanol to make stock solution (100 µg/ml).



methanol to make up the volume. Then the prepared solution of Valsartan(10 µg/ml)

was scanned in the range of 200 nm-400 nm using methanol as blank.

4. Experimental

Akshay R. Koli 127

Figure 4.6.1.1: UV spectrum of 10 µg/ml of Valsartan in methanol

As shown in Figure 4.6.1.1, Valsartan in methanol showed maximum absorbance at

250 nm, this was thus selected as the analytical wavelength.


Secondary stock solution with concentration of 50 g/mL was prepared by diluting 5

ml of primary stock solution (100 g/mL) to 10 mL with methanol. Aliquots of the

secondary stock solutions of valsartan ranging from 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5

were transferred into separate 10ml volumetric flasks and volumes were made up to

10 ml using methanol to obtain final concentrations of 7.5, 10, 12.5, 15, 17.5, 20 and

22.5 μg/ml.

The absorbance of all the prepared solutions was then measured at the absorption

maxima, using methanol as blank. The readings were recorded in triplicate. Results

are shown in Table 4.6.1.1.

4. Experimental

Akshay R. Koli 128

Table 4.6.1.1: Calibration curve of Valsartan in methanol

Sr No. Concentration

(mcg/ml)

Absorbance at 250nm

Mean ± SD, n = 3

1 7.5 0.249 ± 0.002

2 10 0.345 ± 0.002

3 12.5 0.439 ± 0.001

4 15 0.505 ± 0.001

5 17.5 0.586 ± 0.001

6 20 0.671 ± 0.001

7 22.5 0.751 ± 0.001

Figure 4.6.1.2: Calibration curve of Valsartan in methanol at 250 nm


standard deviation values. As shown in Figure 4.6.1.2, Valsartan follows Beer-


(0.998) in methanol indicates that the absorbance and concentration of drug are

linearly related. Optical characteristics of Valsartan are summarized in Table 4.6.1.2.


method.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 5 10 15 20 25

Ab

sorb

ance

concentration (µg/ml)

4. Experimental

Akshay R. Koli 129

Table 4.6.1.2: Calibration data of Valsartan in methanol

λmax

(nm)

Solvent

used Conc. range

Regression

equation

Regression

coefficient (R2)

250 Methanol 75-225µg/ml y =0.0033x + 0.012 0.998

4.6.2 Preparation of calibration curve of Valsartan in PBS pH 6.8



Small quantity of PBS pH 6.8 was added to ensure complete dissolution of Valsartan

and finally volume was made up to the mark with PBS pH 6.8 to obtain 1mg/ml stock

solution. From the above solution, 5ml of solution was withdrawn accurately with the

help of pipette and transferred to 50ml volumetric flask. Volume was made up to the

mark with PBS 6.8 to make stock solution (100 µg/ml).



PBS 6.8 to make up the volume. The prepared stock solution of Valsartan (10 µg/ml)

was scanned in the range of 200 nm-400 nm using PBS pH 6.8 as blank.

As shown in Figure 4.6.2.1, Valsartan in phosphate buffer pH 6.8 showed maximum

absorbance at 250 nm, this was thus selected as the analytical wavelength.

Figure 4.6.2.1: UV spectrum of Valsartan in pH 6.8 phosphate buffer

4. Experimental

Akshay R. Koli 130


From the stock solution, aliquots of 0.5, 1, 1.5, 2, 2.5 ml were accurately withdrawn

with the help of pipette and transferred to separate 10 ml volumetric flasks and the

volume was made up to the mark with PBS pH 6.8 containing 1% Tween 80 to give

final concentration of 5, 10, 15, 20, 25 µg/ml. The absorbance of all the prepared

solutions was then measured at the absorption maxima, using PBS 6.8 as blank. The

readings were recorded in triplicate. Results are shown in Table 4.6.2.1.

Table 4.6.2.1: Calibration curve of Valsartan in PBS pH 6.8

Sr No. Concentration

(mcg/ml)

Absorbance

Mean ± SD, n = 3

1 0 0

2 5 0.174 ± 0.002

3 10 0.321 ± 0.001

4 15 0.461 ± 0.003

5 20 0.615 ± 0.002

6 25 0.770 ± 0.001

Figure 4.6.2.2: Calibration curve of Valsartan in PBS pH 6.8 at 250nm.


standard deviation values. As shown in Figure 4.6.2.2, Valsartan follows Beer-


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 5 10 15 20 25 30

Ab

sorb

ance


4. Experimental

Akshay R. Koli 131

(0.999) in PBS pH 6.8 indicates that the absorbance and concentration of drug are

linearly related. Optical characteristics of Valsartan are summarized in Table 4.6.2.2.


method.

Table 4.6.2.2: Calibration data of Valsartan in PBS pH 6.8

λmax

(nm)

Solvent

used

Conc.

range Regression equation

Regression

coefficient (R2)

250 PBS pH 6.8 5-25µg/ml y = 0.0297x + 0.0222 0.999

4. Experimental

Akshay R. Koli 132

4.6.3 Interference Study of Valsartan with excipients

In order to ascertain the non-interference of the excipients in estimation of Valsartan,

solutions containing known concentration of each excipients were prepared in

methanol and PBS pH 6.8.The prepared solutions were estimated in the UV region

between 200-400 nm using the respective blank. Also, to study the effect in presence

of drug, Valsartan solutions (10 µg/ml) in methanol as well as PBS pH 6.8 was spiked

with known concentrations of each excipient (Capmul MCM, Tween 80 and PEG

400) and scanned in the UV region between 200 nm-400 nm.

During study solution containing known concentration of drug with Tween 80 showed

shifting of peak which indicates interference of Tween 80 in estimation of drug. So

first order derivative method was used for further estimation, which was carried out at

zero crossing point of Tween 80 that is 232nm (ZCP). It is shown in Figure 4.6.3.1.

Figure 4.6.3.1: Zero Crossing Point of Tween 80

232 nm

4. Experimental

Akshay R. Koli 133

4.6.4 Derivative Method

As there is an interference of Tween 80 in drug estimation, the further study was

carried out at Zero Crossing Point of Tween 80 at 232nm by first order derivatization

method.

Mode : Spectrum

Scan speed : Medium

Wavelength range : 200-400 nm

Derivative order : 1

Scan pitch: 0.1

Scaling factor: 5

The derivative spectra were recorded by using digital differentiation (Convolution

method) with a derivative wavelength difference (Δλ (N)) of 5 nm in the range of

200-400 nm[5]

.

Calibration curve of Valsartan in Methanol at 232nm




and finally volume was made up to the mark with methanol to obtain 1mg/ml stock



mark with methanol to make stock solution (100 µg/ml).




volume was made up to the mark with methanol containing 1% Tween 80 to give

final concentration of 5, 10, 15, 20, 25 µg/ml. The absorbance of all the prepared

solutions was then measured at the absorption maxima of derivative spectra i.e. 232

nm, using methanol as blank. The readings were recorded in triplicate. Results are

shown in Table 4.6.4.1 and Figure 4.6.4.1.

4. Experimental

Akshay R. Koli 134

Table 4.6.4.1: Calibration curve of Valsartan in methanol at 232nm

Sr no. Concentration

( µg/ml)

Absorbance

(Mean + SD)

1 5 0.005± 0.001

2 10 0.013± 0.001

3 15 0.018± 0.001

4 20 0.024± 0.001

5 25 0.032± 0.001

Correlation coefficient R2=0.999, (*Mean ± SD, n = 3)

Figure 4.6.4.1: Calibration Curve of Valsartan in methanol at 232nm


standard deviation values. The high value of regression coefficient (0.999) in

methanol indicates that the absorbance and concentration of drug are linearly related.

Optical characteristics of Valsartan are summarized in Table 4.6.4.2. Low values of

standard deviation also indicate the reproducibility of the analytical method.

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0 5 10 15 20 25 30

Ab

sorb

ance


4. Experimental

Akshay R. Koli 135

Table 4.6.4.2: Calibration data of Valsartan in methanol at 232nm

λmax

(nm)

Solvent

used

Conc.

range

Regression

equation


(R2)

232 Methanol 5-25µg/ml y = 0.001x - 0.001 0.993

Calibration curve of Valsartan in PBS pH 6.8 at 232nm



Small quantity of PBS pH 6.8 was added to ensure complete dissolution of Valsartan

and finally volume was made up to the mark with PBS pH 6.8 to obtain 1mg/ml stock



mark with PBS 6.8 to make stock solution (100 µg/ml).




volume was made up to the mark with PBS 6.8 containing 1% Tween 80 to give final

concentration of 5, 10, 15, 20, 25 µg/ml. The absorbance of all the prepared solutions

was then measured at the absorption maxima of derivative spectra i.e. 232 nm, using

PBS 6.8 as blank. The readings were recorded in triplicate. Results are shown in

Table 4.6.4.3 and calibration curve in Figure 4.6.4.2.

Table 4.6.4.3: Calibration curve of Valsartan in PBS pH 6.8 at 232nm

Sr no. Concentration

( µg/ml)

Absorbance*

Mean ± SD, n = 3

1 5 0.007 ± 0.001

2 10 0.014 ±0.001

3 15 0.020 ±0.001

4 20 0.027 ±0.000

5 25 0.034 ±0.001

4. Experimental

Akshay R. Koli 136

Figure 4.6.4.2: Calibration Curve of Valsartan in PBS pH 6.8 at 232nm


standard deviation values. The high value of regression coefficient (0.999) in PBS pH

6.8 indicates that the absorbance and concentration of drug are linearly related.

Optical characteristics of Valsartan are summarized in Table 4.6.4.4. Low values of

standard deviation also indicate the reproducibility of the analytical method.

Table 4.6.4.4: Calibration data of Valsartan in PBS pH 6.8 at 232nm

λmax

(nm)

Solvent

used

Conc.

range Regression equation


(R2)

232 PBS pH 6.8 5-25µg/ml y = 0.0013x + 0.0003 0.999

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0 5 10 15 20 25 30

Ab

sorb

an

ce


4. Experimental

Akshay R. Koli 137

4.7 References

1. Gedil, F., et al., Quantitative determination of felodipine in pharmaceuticals

by high pressure liquid chromatography and uv spectroscopy. Turkish J.

Pharm. Sci, 2004. 1(2): p. 65-76.

2. http://fda.gov/cder.com.

3. Shafiq, S., et al., Development and bioavailability assessment of ramipril

nanoemulsion formulation. European journal of pharmaceutics and

biopharmaceutics, 2007. 66(2): p. 227-243.

4. Li, P., et al., Effect of combined use of nonionic surfactant on formation of

oil-in-water microemulsions. International journal of pharmaceutics, 2005.

288(1): p. 27-34.

5. Gupta, K., A. Wadodkar, and S. Wadodkar, UV-Spectrophotometric methods

for estimation of Valsartan in bulk and tablet dosage form. Inter J ChemTech

Res, 2010. 2(2): p. 985-989.

http://fda.gov/cder.com

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