12chapter 3 Results and Discussion

CHAPTER 3 RESULTS AND DISCUSSION
3.0 RESULTS AND DISCUSSION
3.1 CHARACTERIZATION OF TENOFOVIR

3.1.1 Melting point determination
The melting point of Tenofovir was found to 279ºC. The reported melting point was 278 -
281ºC.
3.1.2 Characterization by Infrared spectroscopy
Figure 3.1: IR Spectra of Tenofovir
Structure of Tenofovir
From the IR spectra of Tenofovir different function groups were found and these are
given in following table 3.1.
Sinhgad Institute of Pharmacy, Narhe, Pune.Page 52

Table 3.1: Functional groups of Tenofovir
Sr.No. Functional group Wavenumber Reported wavenumber(cm-1)

(cm-1)
1 N-H symm. 3345-3325 3221
stretching
2 C-H stretching 3000-3049 3049
3 C-H symm. 2880-2860 2989

stretching
4 C-H stretching 2970-2950 2938
5 N-H (s) 1650-1590 1680
3.2 DEVELOPMENT OF HPLC METHOD TO MONITOR DEGRADATION OF

TENOFOVIR
3.2.1 Selection of Chromatographic Method

Considering wide applicability of reverse phase chromatographic separations to
pharmaceutical compounds, it was decided to carry out work by using RP-HPLC.
3.2.2. Selection of Stationary Phase

The preferred brand of HPLC column should be selected primarily based on the long
term stability and lot-to-lot reproducibility. Preliminary development trials have
performed with various octadecyl columns (C18 columns) of different types and
dimensions from different manufacturers were tested for the peak shape and the number
of theoretical plates of Tenofovir raw material at 100 μg/mL concentration. Finally by
switching to Kromasil column (150 mm x 4.6 mm, 5μ) column there was a substantial
increase in the theoretical plates (~50000) with a significant improvement in the peak
shapes with 1.14 tailing factor. It also produced adequate resolution for Tenofovir.
Therefore Kromasil column was selected as stationary phase.

3.2.3 Selection of Wavelength for Analysis
The proper wavelength was needed to determine maximum detector response. The first
step was to run a UV-VIS spectrum (from 190-400 nm) using an HPLC system equipped
with the Photo Diode Array Detector, from the spectrum it is clear that Tenofovir absorbs
maximum light between 269.2 nm to 260 nm.
Figure 3.2: UV spectrum of Tenofovir (10μg/ml in Methanol)
The wavelength of 260 nm was selected since it produces less noise, which minimizes
problems that may exhibit around the active ingredient when attempting to quantify
Tenofovir. The spectra of diluted solutions of the Tenofovir in mobile phase (at 10 ppm
concentration) were recorded separately on UV spectrophotometer. The spectra of
Tenofovir, from both the concentrations showed absorption maximum at 260 nm. Also,
the solvents in mobile phase/ diluents was shows no interference at the same wavelength
and hence it is selected as detection wavelength.
3.2.4 Selection and Optimization of Mobile Phase for Monitoring Degradation of

Tenofovir
Here the aim was to develop simple RP-HPLC method which can be used to monitor
degradation pattern of Tenofovir under different stress conditions.

Various trials were carried out for developing a method and results of respective trial are
depicted in following chromatograms.
In first trial, mobile phase ratio was set to 30:70 v/v i.e 10 mM Ammonium Formate pH:
Methanol adjusted to 3.5 with o- phosphoric acid. Result of the trial is depicted in
following figure (Figure 3.3).
Figure 3.3: Chromatogram of Tenofovir (100μg/mL) for trial 1

[Flow rate 1 ml/min; Wavelength: 260 nm; Column: Kromasil C18 (150 X 4.6 mm , 5
µm)]
In this trial, retention time of analyte was found to be 2.744 min with tailing factor 1.188.
Next trial was carried out by changing mobile phase composition. In this trial, solvent A
(Ammonium acetate) was changed to sodium acetate and mobile phase ratio was set to
70:30 Methanol: 10mM Sodium Acetate Buffer pH adjusted to 3.5 with Acetic acid.
Result of the trial is depicted in following figure. (Figure 3.4)


[Flow rate 1 mL/min; Wavelength: 260 nm; Column: Kromasil C18 (150 X 4.6 mm ,
5µm)]
In this trial retention time 4.674 min with tailing factor 1.4. The retention time of peak
was less hence next trial was carried out.
In the next trial Mobile phase ratio was set to (70:30 v/v) Methanol: 10 mM Ammonium
Acetate Buffer pH adjusted to 8.5 with Acetic acid. Result of the trial is depicted in
following figure. (Figure 3.5)

5µm)]
Both retention time and peak shape was found as desired for analysis. The retention time
of analyte was found to be 4.689 min. with tailing factor 1.6


5µm)]
In the last trial Mobile phase ratio was set to (60:40 v/v) Methanol: 10mM Ammonium
Acetate Buffer pH adjusted to 8.5 with Acetic acid. Result of the trial is depicted in
following figure. Very sharp peak was obtained with good retention time. Hence this
method was selected for further studies. (Figure 3.6)
Optimized Chromatographic Conditions:

Column: Kromasil C18 (150 X 4.6 mm, 5µm)
Mobile Phase: Methanol: 10mM Ammonium Acetate Buffer pH adjusted to
8.5 adjusted with Triethylamine
Mobile phase ratio: 60:40 v/v
Wavelength: 260 nm
Column Temperature: Ambient
Flow Rate: 1 mL / min
Injector loop Size: 20 μL
Instrument: Shimadzu SPD-M20A, Photo Diode array detector,
Software- LC-Solution
3.3 FORCED DEGRADATION STUDIES

Stability of the stock solution as well as working standard solution was checked and it
was found that Tenofovir is stable if kept at 8-16°C for several weeks.
Results of analysis of samples kept under different stress conditions are as follows
Table: 3.2 Samples injected under different stress conditions
Samples Hydrolysis Oxidative Thermal Photo Deg

Deg. Deg.
Acid Alkaline Neutral
Blank stored under
√ √ √ √ ------- --------
normal condition
Blank subjected to
√ √ √ √ ------- --------
stress condition
Drug / drug
solution stored
√ √ √ √ √ √
under normal
condition
Drug / drug
solution subjected
√ √ √ √ √ √
to stress
condition*
* To get desired degradation initially degradation was carried out at room temperature
and if necessary samples were subjected to higher temperature or strength of stress
reagent was increased.
Results of analysis of samples kept under different stress conditions are as follows.

3.3.1 Acid Hydrolysis

The drug was treated with 0.1N HCl for 24 h. Three degradants were observed at 2.550,
2.951 and 3.730 min as shown in figure 3.7 and it indicates 76.92% degradation of drug.
Figure 3.7: Chromatogram of Tenofovir (100μg/mL) treated with 0.1 N HCl at

Room temperature for 24 h
(Mobile Phase: Methanol: 10mM Ammonium Acetate Buffer pH - 8.5; 60:40 v/v, Flow
rate 1 mL/min, Wavelength: 260 nm; Column: Kromasil C18 (150 X 4.6 mm, 5µm).
To achieve desired % degradation, drug solution was treated with 0.005N HCl and kept at
room temperature for 1 h 12.99%. The drug underwent acid hydrolysis and produced
three degradation products which were eluted at 2.54, 2.93, 3.13 and 4.93.
Figure 3.8: Chromatogram of Tenofovir (100μg/ml) treated with 0.005 N HCl at


The diode array detector (DAD) spectra provided information in terms of wavelength
maxima and peak purity (from fig. 3.9 to fig 3.11) for degradation product and Tenofovir.
The details are depicted as follows.
Figure 3.9: UV spectra, wavelength maxima and peak purity of Acid degradation
product 1
Figure 3.10: UV spectra, wavelength maxima and peak purity of Acid degradation
product 2

Figure 3.11: UV spectra, wavelength maxima and peak purity of Tenofovir
3.3.2 Alkali hydrolysis

The drug degraded significantly under basic condition. The degradation products were
eluted at 2.073, 2.538 and 2.934 min when drug exposed to 0.1 N NaOH for 24h. Almost
83.46 % degradation obtained (Figure 3.12)
Figure 3.12: Chromatogram of Tenofovir (100μg/mL) treated with 0.1 N NaOH at

To achieve desired % degradation, drug solution was treated with 0.001N NaOH and kept
at room temperature for 1 h 17.37%drug degradtion observed along with three degradants
at 2.56, 2.94 and 3.76.

Figure 3.13: Chromatogram of Tenofovir (100μg/mL) treated with 0.001 N NaOH at

rate 1 mL/min, Wavelength: 260 nm; Column: Kromasil C18 (150 X 4.6 mm, 5µm)
The spectra recorded by diode array detector (DAD) in terms of wavelength maxima and
peak purity for alkali degradation products and Tenofovir are depicted as follows. (Figure
3.14 to 3.17)
Figure 3.14: UV spectra, wavelength maxima and peak purity of Alkali degradation
product 1

product 2
product 3
Figure 3.17: UV spectra, wavelength maxima and peak purity of Tenofovir

3.3.3 Oxidative Degradation

Initially oxidative degradation of Tenofovir was carried out using 5% v/v, 15% v/v and
30% v/v hydrogen peroxide. Drug was treated with 30% v/v hydrogen peroxide for 24h.
One degradant is observed at 2.936 min with 63% degradation. (Figure 3.18)
Figure 3.18: Chromatogram of Tenofovir (100μg/mL) treated with 30% hydrogen

peroxide at room temperature for 24 h
rate 1 mL/min, Wavelength: 260 nm; Column: Kromasil C18 (150 X 4.6 mm, 5µm)
To obtain desired range of degradation drug was allowed to react with 3% hydrogen
peroxide at room temperature for 1 h and subjected for HPLC analysis. One degradant is
observed under this stress condition at 2.939
Figure 3.19: Chromatogram of Tenofovir (100μg/mL) treated with 3% hydrogen

peroxide at room temperature for 1 h

rate 1 ml/min, Wavelength: 260 nm; Column: Kromasil C18 (150 X 4.6 mm, 5µm)
peak purity for alkali degradation products and Tenofovir are depicted as follows. (Figure
3.20 and 3.21)
Figure 3.20: UV spectra, wavelength maxima and peak purity of Oxidation

degradation product
Figure 3.21: UV spectra, wavelength maxima and peak purity of Etravirine

3.3.4 Neutral Stress

The drug was allowed to react with water at room temperature for 2 h and subjected for
HPLC analysis. One degradant are observed under this stress condition at 2.94 min with
16.24% degradation.
Figure 3.22: Chromatogram of Tenofovir (100μg/mL) treated with water (Neutral)

at room temperature for 1 h
peak purity for Neutral degradation products and Tenofovir are depicted as follows.
(Figure 3.23 and 3.24)
Figure 3.23: UV spectra, wavelength maxima and peak purity of Neutral

degradation product I

Figure 3.24: UV spectra, wavelength maxima and peak purity of Neutral

degradation product
3.3.5 Thermal Degradation

The drug was kept in sealed glass ampoule in oven at temperature of 70 0C, 800C, 1000C
and 120 0C for 1, 7, 12 and 24 h. A control sample was also maintained in another oven
without exposure to heat. The HPLC analysis did not show significant reduction in the
peak area of drug as compared to control. No additional peak other than peak for
Tenofovir was observed. It indicated that Tenofovir was stable to dry heat.
Figure 3.25: Chromatogram of Tenofovir (100μg/mL) treated with water (Neutral)

at 100ºC for 24 h
3.3.6 Photo Degradation

To check photo stability of the drug in solution form, drug solution prepared in methanol
in volumetric flask was exposed to direct sun light up to 5 days. Same way control was
also maintained by wrapping another volumetric solution with aluminum foil. Tenfovir
exposed to ICH recommended dose of light( 1.2million lux h and 200 W h/m2 ) in photo
stability chamber. No additional peak emerged in control sample. Drug was stable to
photolytic condition.
3.4 Optimization of Forced degradation studies
In present study, stress studies were carried out as per trials suggested by software and %
degradation obtained in various stress conditions are as follows:
a) Acid Stress (HCl)
For acid stress, software suggested following trials. They were practically performed and
% degradation is observed as below.
Table 3.3 Data of % degradation in Acid stress
Run Factor 1: Strength Factor 2 : Time Response: Degradation (%)

(N) (h)
1 0.005 7 54.35
2 0.01 24 78.25
3 0.01 7 58.08
4 0.1 7 61.42
5 0.005 24 80.11
6 0.01 1 14.62
7 0.005 1 12.99
8 0.1 24 76.92
9 0.1 1 15.68
Following equation is obtained:

%Degradation = 86.39+0.87*A+31.57*B-1.44*AB-5.27*A2-34.78*B2
From above equation, factor A (Strength) and factor B (Time) are having positive sign,
factor AB (strength*Time), factor A2 Strength2 and factor B2 Time2
Fig 3.27: Graph of % degradation in alkali stress.
Fit summary: Quadratic model was suggested by the software.
Model Quadratic
P-value 0.0004
F- value 251.73
r2 0.9976
Values of "Prob > F" (p- value) less than 0.0500 indicate model terms are significant. The
Model F-value of 251.73 implies the model is significant. There is only a 0.04% chance
that an F-value this large could occur due to noise. The "Pred R-Squared" of 0.9493 is in
reasonable agreement with the "Adj R-Squared" of 0.9937; i.e. the difference is less than
0.2.

Design-Expert® Software
Factor Coding: Actual
%Degradation (%)
Design points above predicted value
Design points below predicted value
80.11
12.99
100
%Degradation (%)
X1 = A: Strength
X2 = B: Time
80
60
40
20
24 0.1
19.4 0.081
14.8 0.062
10.2 0.043
B: Time (h) 5.6 0.024 A: Strength (N)
1 0.005
Fig 3.28: Graph of % degradation for acid stress.
 Desirability
In the stress study, the response ie; % degradation was desired to be in the range of 5-30
%, and it was inserted in the software as desirability range, A list of solutions was
displayed by software indicating various strength and exposure time and the selected trial
(trail 9) was further practically performed.
Table 3.04 Desirability Data of Acid stress to get desired % Degradation
Sr. No. Strength (N) Time (h) % degradation Desirability

1. 0.027 17.930 91.878 1.000
2. 0.010 7.000 57.724 1.000
3. 0.100 7.000 59.617 1.000
4. 0.100 1.000 17.072 1.000
5. 0.005 1.000 12.462 1.000
6. 0.005 7.000 56.509 1.000
7. 0.100 24.000 77.331 1.000
8. 0.005 24.000 78.479 1.000
9. 0.010 1.000 13.756 1.000
10. 0.054 3.823 42.823 1.000

11. 0.028 7.257 62.584 1.000

12. 0.066 11.755 84.060 1.000
13. 0.063 10.167 78.555 1.000
14. 0.098 12.120 81.345 1.000
15. 0.067 19.957 91.719 1.000
16. 0.098 12.329 81.977 1.000
Desirability
1.000
1.000
0.000
Desirability = 1.000
Std # 1 Run # 7
X1 = A: Strength = 0.005
X2 = B: Time = 1
1.200
Desirability
1.000 1
0.800
0.600 5.6
0.400
0.200
0.000 10.2
0.1
14.8 B: Time (h)
0.081
0.062 19.4
0.043
0.024
0.005 24
A: Strength (N)
Fig 3.29: desirability graph of selected trial (9)
Table 3.05: % Prediction error of selected sample in acidic stress.
Predicted Actual ( % degradation) % Prediction error

(%degradation)
13.756 12.984 0.77
b) Alkali Stress (NaOH)
For alkali stress, software suggested following trials. They were practically performed on
HPLC and % degradation in different conditions is obtained as below.
Table 3.06: Data of % degradation in Alkali stress

Run Factor 1: Strength Factor 2 : Time Response: Degradation (%)

(N) (h)
1 0.001 24 72.63
2 0.001 7 35.11
3 0.1 7 44.6
4 0.1 24 83.46
5 0.01 7 40.12
6 0.01 1 20.61
7 0.001 1 17.37
8 0.1 1 24.25
9 0.01 24 78.41
After inserting the above data, following equation was generated by the software
% Degradation = 66.56+4.64*A+28.89*B+0.66*AB-11.65*A2-5.47*B2
factor AB (strength*Time), factor A2 Strength2 and factor B2 Time2
Factor Coding: Actual %degradation (%)
%degradation (%) 24
Design Points
83.46
17.37
19.4
80
X1 = A: Strength
X2 = B: Time
B: Time
14.8
60
10.2
5.6
40
20
1
0.001 0.012 0.023 0.034 0.045 0.056 0.067 0.078 0.089 0.1
A: Strength
Fig 3.30: Graph of % degradation in alkali stress.

Model Quadratic
P-value 0.0001
2
r 0.9996
F value 1687.09
The Model F-value of 1687.09 implies the model is significant. There is only a 0.01%
chance that an F-value this large could occur due to noise. Values of "P > F" less than
0.0500 indicate model terms are significant.
%degradation (%)
83.46
17.37
%degradation (%)
100
X1 = A: Strength
X2 = B: Time
80
60
40
20
24 0.1
0.089
19.4 0.078
14.8 0.067
0.056
10.2 0.045
0.034
B: Time 5.6 0.023
0.012 A: Strength
1 0.001
Fig 3.31: Graph of % degradation for Alkali stress.
 DESIRABILITY:
In order to get the desired response (%degradation), the desired range (5-30%) is inserted
in the software then the list of solutions is displayed as below
Table 3.07: List of solutions suggested by software for alkali stress.

1. 0.045 13.203 67.575 1.000
2. 0.100 1.000 24.524 1.000
3. 0.010 7.000 40.154 1.000
4. 0.001 1.000 16.565 1.000

5. 0.001 7.000 35.515 1.000

6. 0.100 24.000 83.625 1.000
7. 0.001 24.000 73.030 1.000
8. 0.100 7.000 44.161 1.000
9. 0.010 24.000 77.845 1.000
10. 0.065 18.481 80.556 1.000
11. 0.023 13.332 62.528 1.000
12. 0.049 10.498 61.195 1.000
13. 0.010 8.743 45.149 1.000
14. 0.086 19.239 79.277 1.000
15. 0.004 13.834 55.365 1.000
16. 0.018 2.609 30.010 1.000
The selected trial was further practically performed and % prediction error is
calculated.
Desirability
1.000
0.000
X1 = A: Strength
X2 = B: Time
1.200
Desirability
1.000
0.800
0.600
0.400
0.200
0.000
1.000 0.1
24 0.089
0.078
19.4 0.067
0.056
14.8
0.045
10.2 0.034
0.023 A: Strength
5.6
B: Time 0.012
1 0.001
Fig 3.32: 3D graph of selected trial (4)
Table 3.08: % Prediction error of selected trial.

Predicted (%degradation) Actual (%degradation) %Prediction Error
16.565 15.483 1.08
c) Oxidation
For oxidation stress, software suggested following trials were practically performed and
% degradation in different conditions is obtained as below.
Table 3.9 Data of % degradation in oxidation stress
Run Factor 1 : Strength (N) Factor 2 : Time (h) Response 1 :

(%Degradation)
1 15 1 21.41
2 30 24 63
3 3 7 25.58
4 30 1 49.2
5 3 1 12.97
6 15 24 45.4
7 30 7 56.4
8 15 7 34.62
9 3 24 37.88
After inserting the above data following equation was obtained along with the 3D graph.
% Degradtion = 42.94+14.94*A+10.35*B-2.65*AB+5.39*A2-7.20*B2
factor AB (strength*Time), factor A2 Strength2 and factor B2 Time2 , while interaction

between A&B has antagonistic effect . These effects can be well interpreted with the
help of response surface graphs.
Factor Coding: Actual % degradation (%)
% degradation (%) 24
Design Points
63
12.97
19.4
X1 = A: Strength
X2 = B: Time
60
B: Time
14.8
40 50
10.2
30
5.6
20
1
3 6 9 12 15 18 21 24 27 30
A: Strength (%)
Fig 3.33: Graph of % degradation in oxidation stress
Values of "Prob > F" (p- value) less than 0.0500 indicate model terms are significant
Model Quadratic
P-value 0.0007
r2 0.9964
F value 165.25
The Model F-value of 165.25 implies the model is significant. There is only a 0.07%
chance that an F-value this large could occur due to noise. Values of "Prob > F" less than
0.0500 indicate model terms are significant.

% degradation (%)
% degradation (%)
63
12.97
70
X1 = A: Strength
X2 = B: Time 60
50
40
30
20
10
24 30
27
19.4 24
14.8 21
18
10.2 15
12
B: Time 5.6 9 A: Strength (%)
6
1 3
Fig 3.34: Graph of % degradation for Oxidation
 DESIRABILITY:
In oxidation % degradation was desired to be in the range of 5-30% it is inserted in

software and a list of solution is displayed as below.
Table 3.10 Desirability Data of oxidation stress to get % Degradation in 5- 30%

1. 13.340 19.302 43.221 1.000
2. 30.000 7.000 57.449 1.000
3. 26.625 22.850 58.376 1.000
4. 30.000 1.000 47.879 1.000
5. 15.000 24.000 44.302 1.000
6. 15.000 7.000 34.118 1.000
7. 15.000 1.000 23.009 1.000
8. 3.000 24.000 38.705 1.000
9. 5.700 3.300 19.359 1.000
10. 22.997 15.765 52.884 1.000
11. 27.061 16.527 59.449 1.000
12. 11.212 14.231 38.978 1.000
13. 29.677 12.582 62.219 1.000

The selected solution was trialed on RP-HPLC and following values were observed,
along with graphical representation.
Table 3.11: % prediction error of selected trial
Predicted (% Actual (% degradation) % Prediction error

degradation)
23.009 20.102 2.90
Desirability
1.000
0.000
Desirability = 0.000
Std # 1 Run # 5
X1 = A: Strength = 3
X2 = B: Time = 1
0.000
Desirability
0.200
0.400
0.600
0.800
1.000 24
30 19.4
27 24 14.8
21 18 10.2
15
1.000 12 9 6 5.6 B: Time
A: Strength (%) 3 1
Fig 3.35: 3D graph of desirability in oxidation stress (7)

3.7 VALIDATION OF THE DEVELOPED STABILITY-INDICATING METHOD
3.7.1 Linearity and Range

The representative linearity graph is depicted in following figure.
Linearity of Tenofovir
350000
300000
f(x) = 1472.85571428571 x + 135353.666666667
250000 R² = 0.99955125075308
Peak Area
200000
150000
100000
50000
0
0 20 40 60 80 100 120 140
Concentrations ( µ/mL)
Figure: 3.39 Calibration curve for Tenofovir
Linearity range of Tenofovir was observed at 20, 40, 60, 80, 100 and 120 µ /mL. The
equation of linearity for Tenofovir y = 1472x+135354.Correlation coefficient (r2) was
found to be 0.999.
Table 3.18: Linearity data of Tenofovir for concentration 20-120 µ/mL
Sr. Conc. Sample Sample Sample Sample Sample

Average
No (µg/mL) 1 2 3 4 5
1. 20 163289 163386 163262 163389 163483 163361.8
2. 40 195432 195421 195513 195231 195398 195399
3. 60 224135 224186 224283 224108 224201 224183.2
4. 80 252914 252910 253898 252799 253901 253284.4
5. 100 283170 283190 283200 284160 285183 283780.6
6. 120 310814 310186 310193 312168 310201 310712.4
3.7.2 Precision

Table 3.19: Precision data obtained during intra-day (n=3) studies
Sr.No. Morning Afternoon Evening

Concentration 20 (µg/mL)
1 163262 163564 164562
2 164562 163568 163589
3 163216 165236 163566
Mean 163680 164122.7 163905.7
Avg. Conc.
(µg/mL) 19.93 19.45 19.22
SD 623.9509 787.2472 464.1927
% RSD 0.381202 0.47967 0.283207
1 195513 195462 195142
2 195456 192563 191956
3 194562 192356 191236
Mean 195177 193460.3 192778
Avg. Conc.
(µg/mL) 39.94 40.15 40.01
SD 435.4928 1417.913 1697.247
% RSD 0.223127 0.732922 0.880415
1 224286 223516 223564
2 223586 225643 225622
3 223651 225648 225623
Mean 223841 224935.7 224936.3
Avg. Conc.
(µg/mL) 59.87 60.09 59.90
SD 315.7795 1003.858 970.3863
% RSD 0.141073 0.446287 0.431405

Table 3.20: Precision data obtained during inter-day (n=3) studies
Sr.No. Day 1 Day 2 Day 3

1 163262 163564 164562
2 164582 162456 164523
3 165623 164568 165312
Mean 164489 163529.3 164799
Avg. Conc. 19.94 19.86 19.54
(µg/mL)
SD 966.1149 862.5688 363.095
% RSD 0.587343 0.52747 0.220326
1 195623 193561 195623
2 194568 194756 195652
3 195623 194568 199561
Mean 195271.3 194295 196945.3
Avg. Conc.
(µg/mL) 40.10 39.90 39.95
SD 497.3318 524.6605 1849.594
% RSD 0.254688 0.270033 0.939141
1 224586 235614 233568
2 224582 232456 234523
3 225623 234568 235312
Mean 224930.3 234212.7 234467.7
Avg. Conc.
(µg/mL) 59.84 60.06 59.91
SD 489.792 1313.504 713.0593
% RSD 0.217753 0.560817 0.304118

It was found that the precision results were found satisfactory with respect to % Relative
standard deviation (% RSD) for all levels which were within limit (NMT than 2). The
developed method was precise for determination of Tenofovir.
3.7.3 Recovery
Table 3.21: Recovery studies (n = 3)
Actual concentration Measured concentration Recovery % Mean

(µg/mL) (µg/ml) ; %R.S.D (%) recovery
80 78.41 ; 0.70 98.01
100 98.09 ; 0.69 98.09 98.65
120 119.84 ; 0.20 99.86
The mixture of stressed sample spiked with the drug concentrations of 80, 100 1nd 120
µg/mL. The % Recovery of Tenofovir was found to be 98.65 %. The result was found
satisfactory % mean recovery was within accepted range (98% to 120%).
3.7.4 Robustness
As per ICH guideline, small but deliberate variations, by altering the pH, wavelength
range or flow rate were made to check the capacity of the method. The method was found
to be unaffected by changing the pH. It also does not show any significant changes due to
alteration of flow rate and wavelength range.
Table 3.22: Robustness results of Tenofovir (20 µg/mL)
Change Parameter Peak area Avg. Peak area %R.S.D

258 nm 283170
Wavelength 260 nm 286452
287026 1.45
262 nm
291456
0.9 mL/min 286458
Flow rate 1.0 mL/min 295641 290554.3 1.60

1.1 mL/min 289564
8.4 283654
pH 8.5
286351 284553 0.54
8.6 283654
The method was found to be unaffected by changing the wavelength, flow rate and pH.
The % RSD within limit (NMT 2.0). The Developed method was found to be robust.
3.7.5 LOD and LOQ:
Table 3.22 limit of detection and limit of Quantification
Sr. Parameter Results

No.
1 Limit of Detection (LOD) 0.463 µg/mL
2 Limit of Quantification (LOQ) 1.405 µg/mL
Limit of detection and limit of quantification were calculated by using slope method (3.3
x SD/S and 10 x SD/S respectively) and found to be 0.463 μg/mLand 1.405 μg/mL
respectively.
3.7.6 System Suitability:
Table 3.17: System suitability parameters

Parameter Tenofovir
Peak area (100µg/mL) 283170
No. of Theoretical plate 4066.886
Tailing Factor 1.212

12chapter 3 Results and Discussion

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12chapter 3 Results and Discussion

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CHAPTER 3 RESULTS AND DISCUSSION

3.0 RESULTS AND DISCUSSION

3.1 CHARACTERIZATION OF TENOFOVIR

3.1.2 Characterization by Infrared spectroscopy

Figure 3.1: IR Spectra of Tenofovir

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 52

Table 3.1: Functional groups of Tenofovir

Sr.No. Functional group Wavenumber Reported wavenumber(cm-1)

3 C-H symm. 2880-2860 2989

5 N-H (s) 1650-1590 1680

3.2 DEVELOPMENT OF HPLC METHOD TO MONITOR DEGRADATION OF

3.2.1 Selection of Chromatographic Method

3.2.2. Selection of Stationary Phase

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 53

3.2.3 Selection of Wavelength for Analysis

Figure 3.2: UV spectrum of Tenofovir (10μg/ml in Methanol)

3.2.4 Selection and Optimization of Mobile Phase for Monitoring Degradation of

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 54

Figure 3.3: Chromatogram of Tenofovir (100μg/mL) for trial 1

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 55

Figure 3.4: Chromatogram of Tenofovir (100μg/mL) for trial 2

Figure 3.5: Chromatogram of Tenofovir (100μg/mL) for trial 3

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 56

Figure 3.6: Chromatogram of Tenofovir (100μg/mL) for trial 4

Optimized Chromatographic Conditions:

3.3 FORCED DEGRADATION STUDIES

Table: 3.2 Samples injected under different stress conditions

Samples Hydrolysis Oxidative Thermal Photo Deg

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 58

3.3.1 Acid Hydrolysis

Figure 3.7: Chromatogram of Tenofovir (100μg/mL) treated with 0.1 N HCl at

Figure 3.8: Chromatogram of Tenofovir (100μg/ml) treated with 0.005 N HCl at

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 59

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 60

Figure 3.11: UV spectra, wavelength maxima and peak purity of Tenofovir

3.3.2 Alkali hydrolysis

Figure 3.12: Chromatogram of Tenofovir (100μg/mL) treated with 0.1 N NaOH at

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 61

Figure 3.13: Chromatogram of Tenofovir (100μg/mL) treated with 0.001 N NaOH at

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 62

Figure 3.17: UV spectra, wavelength maxima and peak purity of Tenofovir

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 63

3.3.3 Oxidative Degradation

Figure 3.18: Chromatogram of Tenofovir (100μg/mL) treated with 30% hydrogen

Figure 3.19: Chromatogram of Tenofovir (100μg/mL) treated with 3% hydrogen

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 64

Figure 3.20: UV spectra, wavelength maxima and peak purity of Oxidation

Figure 3.21: UV spectra, wavelength maxima and peak purity of Etravirine

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 65

3.3.4 Neutral Stress

Figure 3.22: Chromatogram of Tenofovir (100μg/mL) treated with water (Neutral)

Figure 3.23: UV spectra, wavelength maxima and peak purity of Neutral

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 66

Figure 3.24: UV spectra, wavelength maxima and peak purity of Neutral

3.3.5 Thermal Degradation

Figure 3.25: Chromatogram of Tenofovir (100μg/mL) treated with water (Neutral)

3.3.6 Photo Degradation

Sinhgad Institute of Pharmacy, Narhe, Pune.Page 67

3.4 Optimization of Forced degradation studies

a) Acid Stress (HCl)

Table 3.3 Data of % degradation in Acid stress

Run Factor 1: Strength Factor 2 : Time Response: Degradation (%)

Following equation is obtained: