I.
Experimental work
1. Study of solubility profile of Cabotegravir
The solubility of Cabotegravir was studied in different solvents including methanol, ethanol, acetone,
chloroform, diethyl ether and dimethyl sulfoxide.
2.Melting point determination
Melting point was determined by digital melting point apparatus. Capillary was sealed from one side
and small quantity of drug was administered from another side of the sealed capillary. Capillary was
placed in capillary holder of digital melting point apparatus. Melting point was observed from eyepiece
of the apparatus.
3.Sample preparation
i)Stock solution A
Accurately 10mg of drug was weighed and transferred in thoroughly washed and dried volumetric flask
of 10ml. Volume was made up to 10ml mark with methanol to produce 1000mg per ml (1000µg/mL).
ii)Stock solution B
From stock solution A, 1 ml was pipetted out with a micropipette and transferred in 10 ml volumetric
flask. Volume was made up to 10 ml to produce 100 mg per ml (100µg/mL). Spectrum was observed for
this solution with UV-Spectroscopy.
iii)Working solution
In Solvent Methanol From stock solution B, 0.1, 0.2, 0.3 and 0.4ml was pipetted out with a
micropipette and transferred in 10 ml volumetric flask separately. Volume was made up to 10 ml with
methanol to produce (1, 2, 3, 4 and 5 µg/ml).
In Solvent Distill Water From stock solution B, 0.1, 0.2, 0.3 and 0.4 was pipetted out using a
micropipette and transferred to 10 ml volumetric flask separately. Volume was made up to 10 ml mark
with distill water (1, 2, 3, 4 and 5 µg/ml).
In Solvent potassium phosphate buffer pH 6.8 From stock solution B, 0.1, 0.2, 0.3 and 0.4 were
pipetted out and transferred to 10 ml volumetric flask. The volume was made up to 10 ml using
phosphate buffer (1, 2, 3, 4 and 5 µg/ml).
iv)Preparation of potassium phosphate buffer pH 6.8 - 7.2 g of disodium hydrogen phosphate and
2.8g of potassium di-hydrogen phosphate were weighed and transferred in 250 mL volumetric flask
volume was made up with distilled water. Homogeneous mixture was made and pH was checked using
digital pH meter.
4. FT-IR analysis of drug
FTIR spectra was recorded on a Shimadzu model named IRSpirit-T. Drug was placed under prism of
FT-IR. The spectra collected from the range of 400-4500cm-1.
5. Saturation solubility study
For saturation study, 5 ml of distill water and phosphate buffer pH 6.8 was measured and transferred to
volumetric flask separately.1 mg of drug was weighed and transferred to flasks containing water and
potassium phosphate buffer. Volumetric flask was sonicated for 5 mins. To make the solution saturated,
again 1 mg of drug was transferred to both tubes and sonicated for 5 mins. The solution in the volumetric
flask was transferred to centrifugation tubes and these tubes were kept on rotatory shaker for 48 hrs. at
400 rpm. After completion of 48 hrs. solutions were filtered with whatman filter paper. Absorbance of
filtrate was estimated at 258 nm with UV-visible spectrometer.
6. Phase Solubility analysis
Phase solubility analysis was carried out to determine the amount of drug soluble in Molar concentration
of the Hydrogen acceptor (Alanine) and Hydrogen bond doner (Lactic acid, Ethylene glycol and
Propylene glycol). 0.01M, 0.02M, 0.03M and 0.04 M of each HBA and HBD was transferred to a
mixture of 1: 2 (methanol: distill water) separately. Excess amount of drug was weighed and transferred
to mixtures containing HBA and HBD, to make the mixture saturated. these mixtures were kept on
rotatory shaker for 48 hrs. After completion of 48hrs. mixtures were filtered using whatman filter paper.
From this filtrate, 1 ml was pipetted out and transferred to volumetric flask. volume was made up to 10
ml using distill water. Absorbance was estimated by UV- visible spectroscopy at 258 nm.
7. Prediction of Solubility using COSMO-RS
COSMO-RS is a theory (i.e. a set of equations) used to calculate the chemical potentials of liquids. It
is based on quantum chemistry to determine chemical potentials.
II. Results
1. Solubility profile of drug Cabotegravir
Solvent Solubility
Methanol Soluble
Ethanol Not soluble
Acetone Not soluble
Chloroform Soluble
Diethyl ether Not soluble
Dimethyl sulfoxide Soluble
Dimethyl formamide Not soluble
2.Melting point determination
Melting point (Observed value) Melting point (Average Melting point (Reported
value) value)
251°C 251.1°C 248-251°C
250.8°C
251.5°C
3.UV-visible spectroscopy analysis
i) Calibration cure of Cabotegravir in Methanol
Concentration (µg/mL) Absorbance
1 0.058
2 0.104
3 0.163
4 0.228
5 0.275
y = 0.0938x - 0.624
Calibration curve of Cabotegravir in methanol
R² = 0.9995
0.5
0.45
0.4
0.35
0.3
Absorbance
0.25
0.2
0.15
0.1
0.05
0
0 1 2 3 4 5 6
Concentartion(mg/mL)
Fig. no. 1. Calibration curve of Cabotegravir in methanol.
ii)Calibration curve of Cabotegravir in potassium phosphate buffer.
Concentration Absorbance
(µg/mL)
1 0.03
2 0.14
3 0.28
4 0.42
5 0.56
y = 0.134x - 0.116
Calibration curve of Cabotegravir in pH 6.8 buffer R² = 0.998
0.6
0.5
0.4
Absorbance
0.3
0.2
0.1
0
0 1 2 3 4 5 6
Concentration (mg/mL)
Fig. no. 2 Calibration curve of Cabotegravir in potassium phosphate buffer pH 6.8
iii)Calibration of Cabotegravir in distil water
Concentration(µg/mL) Absorbance
1 0.039
2 0.116
3 0.158
4 0.250
5 0.299
y = 0.0648x - 0.228
Callibration curve of Cabotegravir in distil water
R² = 0.9984
0.35
0.3
0.25
Absorbance
0.2
0.15
0.1
0.05
0
0 1 2 3 4 5 6
Concentration
Fig. no. 3 Calibration curve of Cabotegravir in distil water
4)Saturation solubility study
Solvent Absorbance
Distil water 0.217
Potassium phosphate buffer pH 6.8 0.312
Calculation of Molar absorptivity
For water,
C=0.4 mg/ml
A=0.217
ϵ = ?
A = A 1%1cm ꞏbꞏ c
0.217= A 1%1cm. 1 ꞏ 0.4
0.217
A 1%1cm= 0.4
ꞏ 1
A 1%1cm= 0.542
ϵ = A 1%1cm ꞏ Mol. wt./10
ϵ = 21.951
For Potassium phosphate buffer pH 6.8,
C = 0.4 µg/ml
A = 0.312
ϵ = ?
A = A 1%1cm ꞏbꞏ c
0.312= A 1%1cm. 1 ꞏ 0.4
0.312
A 1%1cm= ꞏ 1
0.4
A 1%1cm= 78
ϵ = A 1%1cm ꞏ Mol. wt./10
ϵ = 3,159
To determine concentration,
For water,
C=?
A = 0.217
ϵ = 21.951
𝑉𝑖
Dilution factor=𝑉𝑓
Where, Vi = Volume of stock transferred
Vf= Final volume
A= ϵꞏ lꞏ C
𝐴
C = ϵ ꞏ lꞏ D.F
0.217
C = 21.95 × 1×10
C = 0.0988 µg/ml
For potassium phosphate buffer pH 6.8
C =?
A = 0.312
ϵ = 31.95
𝑉𝑖
Dilution factor=𝑉𝑓
Where, Vi = Volume of stock transferred
Vf= Final volume
A= ϵꞏl ꞏ C
𝐴
C = ϵ ꞏ lꞏ D.F
0.312
C= 31.59 ×1×10
C= 0.1421 g/ml
5)Prediction of solubility using COSMO-RS
Fig. no. 4 Prediction of solubility in DES containing Alanine and lactic acid in various combination
using COSMO-RS
Fig. no. 5 Prediction of solubility in DES containing Alanine and Ethylene glycol in various
combination using COSMO-RS
Fig. no. 6 Prediction of solubility in DES containing Alanine and Propylene glycol in various
combination using COSMO-RS
6)Phase solubility analysis
i)For Ethylene glycol
Concentration (in Moles) Absorbance
0.1 0.445
0.2 0.496
0.3 0.545
0.4 0.601
y = 0.0517x + 0.392
Phase solubility curve of Ethylene glycol R² = 0.9992
0.7
Absorbance of Cabotegravir
0.6
0.5
0.4
0.3
0.2
0.1
0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Molar concentration of Ethylene glycol
Fig. no. 7 Phase solubility curve of Ethylene glycol.
y=0.0517x + 0.3925
R2=0.9992
For 0.1 M: Absorbance=0.445
0.445=0.0517x+0.392
∴ x= (0.445-0.39)/0.0517
x =1.06 mg/ml
For 0.2M: Absorbance=0.496
0.496=0.0517x+0.0392
∴ x= (0.496-0.039)/0.0517
x=2.07mg/ml
For 0.3M: Absorbance=0.545
0.545=0.0517x+0.392
∴ x= (0.496-0.39)/0.0517
x=3.03mg/ml
For 0.4M: Absorbance=0.601
0.601=0.0517x+0.392
∴ x= (0.601-0.39)/0.0517
x=4.08mg/ml
ii)Phase solubility Propylene glycol
Concentration (in Moles) Absorbance
0.1 0.480
0.2 0.576
0.3 0.712
0.4 0.819
Phase solubility curve for Propylene Glycoly = 0.0733x + 0.3585
R² = 0.996
0.9
0.8
Absorbance of Cabotegravir
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Molar concentration of Propylene Glycol
Fig. no. 8 Phase solubility curve of Propylene glycol.
For 0.1 M: Absorbance = 0.480
0.480=0.0733x+0.3585
∴ x= (0.480-0.3585)/0.0733
x=2.4 mg/ml
For 0.2 M: Absorbance=0.576
0.576=0.0733x+0.3585
∴ x= (0.480-0.3585)/0.0733
x=3.1 mg/ml
For 0.3 M: Absorbance=0.712
0.576=0.0733x+0.3585
∴ x= (0.712-0.3585)/0.0733
x=4.8 mg/ml
For 0.4M: Absorbance=0.819
0.819=0.0733x+0.3585
∴ x= (0.819-0.3585)/0.0733
x=6.2 mg/ml
iii)For Lactic acid
Concentration (in moles) Absorbance
0.1 0.768
0.2 0.877
0.3 0.971
0.4 1.029
Calibration curve for Lactic acid y = 0.0994x + 0.692
R² = 0.9828
1.2
Absorbance of Cabotegravir
0.8
0.6
0.4
0.2
0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Molar concentration of Lactic acid
Fig. no. 9 Phase solubility curve for Lactic acid
For 0.1M: Absorbance=0.768
y=0.0994x+0.692
R2=0.9828
For 0.1M: Absorbance=0.768
0.768=0.0994x+0.692
∴ x= (0.768-0.692)/0.0994
x=1.86mg/ml
For 0.2M: Absorbance=0.867
0.877=0.0994x+0.692
∴ x= (0.877-0.692)/0.0994
x=3.07mg/ml
For 0.3 M: Absorbance =0.971
0.971=0.0994x+0.692
∴ x= (0.971-0.692)/0.0867
x=4.13mg/ml
For 0.4M: Absorbance=1.029
1.029=0.0994x+0.692
∴ x= (1.029-0.692)/0.0867
x=4.77mg/ml
iv)For Alanine
Concentration (in Absorbance
moles)
0.1 0.694
0.2 0.794
0.3 0.882
0.4 0.996
Phase solubility curve for Alanine y = 0.0877x + 0.593
R² = 0.9976
1.2
Absorbance of Cabotegravir
0.8
0.6
0.4
0.2
0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Molar concentration of Alanine
y=0.0877x+0.593
R2=0.9976
For 0.1M: Absorbance=0.694
0.694=0.0877x+0.593
∴ x= (0.694-0.593)/0.0877
x=2.45mg/ml
For 0.2 M: Absorbance= 0.794
0.794=0.0877x+0.593
∴ x= (0.794-0.593)/0.0877
x=3.67mg/ml
For 0.3M: Absorbance =882
0.882=0.0877x+0.593
∴ x= (0.882-0.593)/0.0877
x=4.77mg/ml
For 0.4 M: Absorbance=0.996
0.996=0.0877x+0.593
∴ x= (0.996-0.593)/0.0877
x=5.65mg/ml
FTIR analysis
Functional group(cm-1) Observed frequency (cm-1) Reported frequency (cm-1)
-OH 3736 3406.8
-NH 3075 3318
-C=0 1675 1725
-C-N 1500.5, 1280 1051.7, 1288
