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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.
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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
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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
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
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
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).
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
Preparation of stock solution:
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).
Determination of λmax:
1 ml of the stock solution was transferred to a 10 ml volumetric flask and diluted with
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.
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 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
Table 4.5.2.1 showed the mean absorbance values of the solutions along with the
standard deviation values. As shown in Figure 4.5.2.2, Felodipine follows Beer-
Lambert’s law in the range of 10-45 μg/ml. The high value of regression coefficient
(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:
Preparation of stock solution:
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
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).
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 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.
Preparation of calibration curve:
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
Table 4.6.1.1 showed the mean absorbance values of the solutions along with the
standard deviation values. As shown in Figure 4.6.1.2, Valsartan follows Beer-
Lambert’s law in the range of 75-225 μg/ml. The high value of regression coefficient
(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.
Low values of standard deviation also indicate the reproducibility of the analytical
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
Preparation of stock solution:
Accurately weighed 100 mg of Valsartan was transferred to 100 ml volumetric flask.
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).
Determination of λmax:
1 ml of the stock solution was transferred to a 10 ml volumetric flask and diluted with
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
Preparation of calibration curve:
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.
Table 4.6.2.1 showed the mean absorbance values of the solutions along with the
standard deviation values. As shown in Figure 4.6.2.2, Valsartan follows Beer-
Lambert’s law in the range of 5-25 μg/ml. The high value of regression coefficient
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
concentration (µg/ml)
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.
Low values of standard deviation also indicate the reproducibility of the analytical
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
Preparation of stock solution:
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
and finally volume was made up to the mark with methanol 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 methanol to make stock solution (100 µg/ml).
Preparation of calibration curve:
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 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
Table 4.6.4.1 showed the mean absorbance values of the solutions along with the
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
concentration (µg/ml)
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
Regression coefficient
(R2)
232 Methanol 5-25µg/ml y = 0.001x - 0.001 0.993
Calibration curve of Valsartan in PBS pH 6.8 at 232nm
Preparation of stock solution:
Accurately weighed 100 mg of Valsartan was transferred to 100 ml volumetric flask.
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).
Preparation of calibration curve:
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 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
Table 4.6.4.3 showed the mean absorbance values of the solutions along with the
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
Regression coefficient
(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
concentration (µg/ml)
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.