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1. CONTENTS ()
Introduction
Literature survey
Drug Profile
Objective and plan of work
Materials and Method
Method development
Method validation
Forced degradation studies
Results and discussion
Conclusion
References

2. Introduction of UPLC
Chromatography is a non-destructive procedure for resolving a multi-component mixture of traces,
minor or constituents into individual fractions. It is a method of separating a mixture of components
into individual components through a porous medium under the influence of solvent.
UPLC refers to Ultra Performance Liquid Chromatography. UPLC brings dramatic improvements in
sensitivity, resolution and speed of analysis can be calculated. It has instrumentation that operates at
high pressure than that used in HPLC & in this system uses fine particles (less than 2.5µm) & mobile
phases at high linear velocities decreases the length of column, reduces solvent consumption & saves
time.
Advantages : 1. Decrease run time and increase the sensitivity.
2. Provides selectivity , sensitivity and maintain resolution performance
3. Fast resolving power quickly quantifies related and unrelated compounds
4. Less solvent consumption and operational cost is less
5. High speed , high resolution performance

3. Instrumentation of UPLC
 Instrumentation
 The instruments in UPLC technique work with high speed, good resolution and great sensitivity.
 The basic instrumentation comprises:
 Binary solvent manager
 Sample manager including the column heater
 Optional sample manager
 Pumps
 Detector
 Principle:
 The UPLC is based on the principal of use of stationary phase consisting of particles less than 2.5 μm (while HPLC
columns are typically filled with particles of 3 to 5 μm). The underlying principles of this evolution are governed by the
Van Deemter equation, which is an empirical formula that describes the relationship between linear velocity (flow rate)
and plate height (HETP or column efficiency). H=A+B/v+Cv Where; A, B and C are constants, v is the linear
velocity, the carrier gas flow rate.

4. S.N
O
AUTHOR WORK DONE
TITLE OF THE
JOURNAL
VOLUM
E
ISSU
E
PAGE
S
YEAR OF
PUBLICATI
ON
1 Satinder A, Dong
MW
Method development and validation pharmaceutical
analysis by HPLC reverse phase high performance liquid
chromatography .
New york: Elsevier 6 2 16-70 2005
2
Akram NMD,
Umamahesh
M,et.al.,
A New Validated RP-HPLC method for the
Determination of Emtricitabine and Tenofovir AF in its
bulk and pharmaceutical dosage forms
Journal of Chemical and
Pharmaceutical Sciences
10 4 54–59 2017
3
Badgujar BP,
Mahajan MP, et.
al.,
Developmentand Validation of RP-HPLC Method for the
SimultaneousEstimation of Tenofovir Alafenamide and
Emtricitabinein Bulk and tablet dosage form
International journal of
chemical technology
10 5
731-
739
2017
4
Mastanamma S,
Venkata Reddy D,
Saidulu P, et. al.,
Developmentand Validation of Stability Indicating RP-
HPLCMethod for the Simultaneous estimation of
Emtricitabine,Tenofovir Alafenamide bulk and their
combined dosage form
International journal of
pharmacy
9 9 70-80 2017
5
Joshi M, Nikalje
AP, Shahed M, et.
al.,
HPTLC methodfor the simultaneous estimation of
emtricitabine andtenofovir in tablet dosage form
Indian journal of
Pharmaceutical sciences
71 - 95-97 2009
LITERATURE REVIEW

5. S.N
O
AUTHOR WORK DONE
TITLE OF THE
JOURNAL
VOLUME ISSUE
PAGE
S
YEAR OF
PUBLICATIO
N
11
Venkateswara Rao
B, Vidyadhara S,
et. al.,
A Novel Stability Indicating RP-HPLC Method
DevelopmentAnd Validation For The Determination Of
Tenofovir Disoproxil Fumarate And Emtricitabine In
Bulk And Pharmaceutical formulations
International journal of
pharmaceutical sciences
and research
8 5
2168–
2176
2017
12
Yenumula BRR,
Singampalli MR,
et. al.,
Simultaneous Estimation of Emtricitabine and Tenofovir
DisoproxilFumarate in Tablet Dosage Form by Reverse
Phase Highperformance Liquid Chromatography
International Journal
of Analytical
Techniques
5 - 1-6 2015
13
Sharma R, Gupta
P, et. al.,
A Validated RP - HPLC method for Simultaneous
estimation of Emtricitabine and TenofovirDisoproxil
fumarate in a tablet dosage form.
Eurasian Journal of
analytical chemistry
4 3
276-
284
2009
14
Shelke A, Shinde
M, Mogal R, et. al
Application of UV Spectrophotometric methods for
simultaneous estimation of Emtricitabine and Tenofovir
alafenamidefumarate in bulk
Asian journal of
pharmacy and
technology
8 2
103-
107
2018
15
Tej Kumar K,
Suryakala.D, et. al
RP-HPLC method development and validation for the
estimation of Emtricitabine, Bictegravir and Tenofovir
alafenamide in bulk and pharmaceutical dosage form
Journal of Taibah
University for Science
1 2
1137-
1146
2019
16
Chinnalalaiah
R, Ravi Kumar P,
et. al
A Validated Stability Indicating RP-HPLC Method for the
Determination of Emtricitabine, Tenofovir Disoproxil
Fumarate, Elvitegravir and Cobicistat in Pharmaceutical
Journal of
chromatography science
54 5
759-
764
2016

6. S.N
O
AUTHOR WORK DONE
TITLE OF THE
JOURNAL
VOLUM
E
ISSU
E
PAGE
S
YEAR OF
PUBLICAT
ION
1
Chandni saha
The Development of A Validated Stability Indicating LC-
MS Method For The Determination Of Tenofovir
Disoproxil Fumarate Using Quality By Design Approach
International journal of
applied pharmaceutics
11 4
406-
417
2019
2
Varaprasad
Jagadab,et.al.,
Identification And Quantification Of Potential Impurities
Using LC-PDA Coupled With New Qda Mass Detector
In A New Single Tablet Regimen Containing
Dolutegravir, Emtricitabine And Tenofovir Disoproxil
Fumarate Tablets Used In Hiv-1 Prevention
International research
journal of pharmacy
10 3 91-104 2019
3
Chhaganbhai N
Patel, et. al.,
RP-HPLC method for simultaneous estimation of
tenofovir disoproxil fumarate, lamivudine, and efavirenz
in combined tablet dosage form
Pharmaceutical methods 3 2 73-78 2012
4
Muthyala Sneha
et. al.,
Stability indicating RP-HPLC method for simultaneous
estimation of emtricitabine, bictegravir and tenofovir
alafenamide in bulk and formulation
International journal of
pharmacy and analytical
research
8 3 70-80 2019
5
Joshi M, Nikalje
AP, Shahed M, et.
al.,
HPTLC methodfor the simultaneous estimation of
emtricitabine andtenofovir in tablet dosage form
Indian journal of
Pharmaceutical sciences
71 - 95-97 2009
LITERATURE REVIEW ( Method-II)

7. 1 Drug profile of Emtricitabine
 Structure:
 IUPAC Name: 4- amino-5-fluro-1-[(2R,5S)-2-(hydroxymethyl)1,3-oxathiolan-5-
yl}pyrimidine-2-one
 Molecular Weight: 247.24
 Molecular formula C8H10FN3O3S
 Drug Category:
 Prevention and treatment of HIV infection in adults and childrens
 Appearance: White to off-white powder
 Solubility: Emtricitabine is very soluble in water, soluble in methanol,.
 Pka: 2.65

8.  2. Drug profile of Tenofovir alafenamide
 Structure:
 IUPAC Name: Isopropyl (2S)-2-[[[(1R)-2-(6-aminopurin-9-yl)-1-methyl-ethoxy]methyl-
phenoxy-phosphoryl]amino]propanoate
 Molecular Weight: 476.466
 Molecular formula: C21H29N6O5P
 Drug Category:
 is a hepatitis B virus nucleotide reverse transcriptase inhibitor for the treatment of chronic
hepatitis B virus infection in adults
 Appearance: White to off-white crystalline powder
 Solubility: soluble in water, soluble in methanol, sparingly soluble in ethanol.
 Pka: 11.36

9.  2. Drug profile of Bictegravir
 Structure:
 IUPAC Name: (2R,5S,13aR)-8-hydroxy-7,9-dioxo-N-(2,4,6-trifluorobenzyl)-
2,3,4,5,7,9,13,13a-octahydro-2,5-methanopyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazepine-10-
carboxamide
 Molecular Weight: 449.38
 Molecular formula: C21H18F3N3O5
 Drug Category:
 studying the treatment of HIV-1 and HIV-2 infection
 Appearance: White to off-white crystalline powder
 Solubility: soluble in water, soluble in methanol, sparingly soluble in ethanol.
 Pka: 9.81

10. Aim & Objective:
.
To develop and Validate Ultra performance liquid chromatography(UPLC)- method for the
simultaneous estimation of Emtricitabine, Tenofovir Alafenamide and Bictegravir in
Bulk & pharmaceutical dosage form
Plan of Work
 Detection of maximum absorbance by UV-Visible spectroscopy for selection of wave
length
 Several Trials for the separation of analytes by UPLC.
 Optimization of Stability indication UPLC Method
 Method Validation the simultaneous estimation of Emtricitabine, Tenofovir Alafenamide
and Bictegravir in bulk & Pharmaceutical dosage form as per ICH Guidelines.

11.  Materials and Methods
 Instrumentation
The 1290 Series UPLC system (Agilent Technologies) with PDA detection. Data processing was
performed on EZCHROM Elite software package.
 Chromatographic conditions
Acquity BEH C18 130A° (100 × 2.1 mm, 1.7μ) column, Triethyl amine buffer pH 3.0.: Methanol
(45:55 v/v) mobile phase with a flow-rate of 0.5 mL/min. The column was placed at a temperature of
40OC. 20 μL of sample was injected into UPLC System. The retention time of Emtricitabine, Tenofovir
Alafenamide and Bictegravir were found to be 2.6 min, 4.3 min and 5.2 min.
 Drug Samples:
The standard samples of Emtricitabine, Tenofovir Alafenamide and Bictegravirwere procured from
Dr.reddy’s Laboratories, Hyderabad and Tablet dosage forms (Biktarvy 500 mg Emtricitabine, 200mg
TenofovirAlafenamide and 25mg of Bictegravir were purchased from local Market.
 Chemicals and Reagents:
Triethylamine(Make :Merck and Grade: AR)
Formic cid(Make:Rankem and Grade:AR)
Methanol (Make :Merck and Grade: HPLC)
Acetonitrile (Make :Merck and Grade: HPLC)

12.  Analytical Methodology:
 Working wavelength optimization:
 Preparation of Standard solutions:
Weighed accurately 10mg of each Emtricitabine in to 100mL volumetric flask and
dissolved with 70mL of Methanol by sonication and Volume made up with Methanol and
mixed well. Further diluted 1mL to 10mL this solution with methanol(10µg/mL of
Emtricitabine) Prepared 10µg/mL of Tenofovir alafenamide and Bictegravir solutions in
above preparation manner
Scanned these solutions in UV Spectrophotometer at 200nm to 400nm using methanol as
a Blank. Emitricitabine shown maximum absorbance at 258nm and Bictegravir shown
maximum absorbance at 256nm, however Tenofovir has very less absorbance at the
region of 256nm to 258nm hence 280nm selected as a Working wavelength. . The UV
graphs of the Emtricitabine, Bictegravir and Tenofovir were shown at 258 nm ,256nm and
280 nm and graphs were shown in Fig 4, Fig 5 and Fig. 6.

13. UV graph of Emtricitabine at 258 nm UV graph of Tenofovir Alafenamide at 285 nm
UV graph of Bictegravir at 256 nm

14.  Preparation of Standard stock:
Accurately weighed and transferred 200mg of Emitricitabine, 25mg of Tenofovir Alafenamide
and 50mg of Bictegravir in to 100mL Volumetric flask, added 70mL of Diluent then kept on
sonicator up to dissolved, Final volume made up to mark with diluents and mixed well.
(Emtricitabine 2000µg/mL, Tenofovir Alafenamide 250µg/mL and Bictegravir 500µg/mL)
 Preparation of Standard working solution:
Taken 5mL of above standard stock solution in to 50mL volumetric flask then diluted up to
mark with diluents and mixed well. (Emtricitabine 200µg/mL, Tenofovir Alafenamide
25µg/mL and Bictegravir 50µg/mL)
 Preparation of Sample stock solution:
Taken 20Tablets and weighed, Average weight of each table calculated from the 20tablets
weight then and transferred 20tablets in to mortar and pestle, crushed to fine powder.
Accurately weighed and transferred tablets fine powder equivalent to 200mg of Emitricitabine,
25mg of Tenofovir Alafenamide and 50mg of Bictegravir in to 100mL Volumetric flask, added
70mL of Diluent then kept on sonicator up to 30min with intermediate shaking by maintain
sonicator temperature 20°C to 25°C , Final volume made up to mark with diluents and mixed
well. Centrifuged this sample solution at 5000RPM for 10min.

15.  Preparation of Sample solution:
 Taken 5mL of supernatant of sample stock solution in to 50mL volumetric flask then
diluted up to mark with diluents and mixed well. Filtered through 0.45µ
PVDF(Polyvinylidene diflouride) filter
 Preparation of pH 3.0Buffer:
Accurately taken 2mL of Triethylamine and Transferred in to 1000mL of water, mixed
well and adjusted pH 3.00 with diluted formic acid. Filtered through 0.45µm Nylon
membrane filter
 Diluted formic acid Preparation:
Diluted 5mL of Formic acid 20mL with water and mixed well.
 Preparation of Mobile Phase:
Accurately taken 450mL of Buffer (TEA) and 550mL of Methanol in a 1000mL of
mobile phase bottle and mixed well. Degassed by soncation 5min.
 Optimized Conditions: the optimum chromatographic conditions were obtained with
several trail and error methods and the optimum chromatographic conditions were shown
in Table no.1 and the optimum chromatogram was shown in Fig.7

16. Method Development
Optimisation of Chromatographic condition
Table-Selection of Column
S.No LC Condition Experiment Conclusion
1
Acquity Phenyl Column130A°
(100 × 2.1 mm, 1.7μ) column,
Mobile Phase: pH 3.0 Phosphate Buffer
and Acetonitrile50:50v/v), flow rate 0.6
mL/min
UPLC parameter
optimization
Analytes were not
separated(Resolution
between analytes were
less) & Peak shapes
were not good
2 Acquity C8 Column130A°
(100 × 2.1 mm, 1.7μ) column,
Mobile Phase: pH 3.0 Phosphate Buffer
and Acetonitrile 50:50v/v), flow rate 0.6
mL/min
UPLC parameter
optimization
Emitricitabine and
Tenofovir Peaks were
not separated
3
Acquity C8 Column130A°
(100 × 2.1 mm, 1.7μ) column,
Mobile Phase: pH 3.0 Phosphate Buffer
and Methanol 50:50v/v), flow rate 0.5
mL/min
UPLC parameter
optimization
Analytes were separated
but Resolution less than
2.0, Peak shapes also not
good.

17. Column Acquity BEH C18 130A° (100 × 2.1 mm, 1.7μ)
Mobile Phase and Composition pH 3.0 Triethylamine Buffer:Methanol(45:55)
Flowrate 0.5mL/min
Column ovenTemperature 40 °C
Injection volume 5µL
Detection wavelength 285nm
Autosampler Temperature 25°C
Retention Times 2.60min of Emitricitabine, 4.27min for Tenofovir
5.25min for Bictegravir (Total Run time 8.0min)
Optimized Chromatographic conditions
Typical Optimized Chromatogram of EMT,TEN and BCT

18. Method Validation as per ICH Guidelines(Q2B R1)
The developed method was validated as per the ICH guidelines
 System suitability and system precision
 Specificity (Blank, Placebo Interferences and Forced degradation study)
 Method precision and Intermediate Precision(Ruggedness)
 Accuracy and Recovery
 Linearity and Range
 Robustness

19.  Method Validation:
 Analytical method validation as per ICH: Method validation is a process of
documenting/proving that an analytical method provides analytical data acceptable for the
intended use
 System Suitability and System Precision:
By preparing standard solutions of Emtricitabine(200ppm), Bictegravir(50ppm)and
Tenofovir alfanamide (25ppm) the system suitability parameters were determined the
solutions were injected six times and the parameters like peak tailing, resolution and the
USP theoretical plate count were assessed to check whether the results complies with
recommended limits.
For System Precision:
 %RSD for retention times and Area for the six replicate injections should not be more than
2.0 of each analyte .
 The results for the system suitability were summarized in Table 2 and the results for the
system precision were summarized in Table 3.

20.  System Suitability Results
Rt USP Retention
(min) Plate count time (min)
1 2.601 3501 1.48 4.277 3105 1.4 5.7 5.248 4238 1.2 3.4
2 2.601 3503 1.48 4.281 3108 1.39 5.7 5.246 4239 1.2 3.4
3 2.6 3501 1.49 4.285 3106 1.4 5.7 5.246 4200 1.2 3.4
4 2.6 3548 1.47 4.289 3105 1.38 5.7 5.244 4239 1.2 3.4
5 2.6 3502 1.48 4.291 3108 1.4 5.7 5.243 4240 1.2 3.4
6 2.599 3508 1.48 4.294 3107 1.4 5.7 5.242 4240 1.2 3.4
Plate count
Tailing
Factor Resolution
Table 2: SystemSuitability Results
EMT TEN BCT
S.No
Tailing
Factor Rt(min) Plate count
Tailing
Factor Resolution
Acceptance criteria :
Tailing factor should be less than 2.0
Theoritical plates should be more than 2000
Resolution should be more than 2.0

21.  Table 3: System Precision results
S.No
Rt
Peak
Area Rt
Peak
Area Rt
Peak
Area
1 2.601 1524.48 4.277 759.88 5.248 1157.69
2 2.601 1525.29 4.281 759.97 5.246 1157.47
3 2.6 1516.22 4.285 760.25 5.246 1156.93
4 2.6 1523.34 4.289 759.86 5.244 1156.77
5 2.6 1515.63 4.291 759.92 5.243 1161.6
6 2.599 1516.1 4.294 760.04 5.242 1156.09
AVG 2.6 1520.18 4.286 759.99 5.245 1157.75
%RSD 0.03 0.31 0.15 0.02 0.04 0.17
Table 3: Precision results
EMT TEN BCT
Acceptance criteria
%RSD for retension times and area for 6 replicants should be not more than2.0

22.  Specificity
Specificity is the ability of analytical method to measure the response of the analyte and
have no interference from other extraneous components and well resolved peaks are
obtained .
The blank and Placebo solution was injected into the UPLC system and the Blank and
placebo solution was not shown any interference at the retention time of the main peaks.
The blank and placebo chromatograms were shown in fig.8 and Fig 9. respectively
Fig.8 :Blank Chromatogram
Fig.9:Placebo Chromatogram

23.  Degradation study
 In these studies samples were treated with Acid (5N HCl/4Hrs/60°C), Base (5N
NaOH/4Hrs/60°C), Peroxide (30% Hydrogen Peroxide for 4Hrs at Bench top), Thermal
(80°C/24Hrs) and Photolytic condition.
 In base treatment, sample degradation occurred 28.6% for Emtricitabine, In
remaining all conditions treated samples have shown less degradation (below 5%). Peak
purity was also achieved for main analytes.
 The results for the degradation studies were conducted in different parameters and the
results for the all samples were shown in Table and the chromatographic graphs of the acid
treated , base treated, Peroxide treated, Photolytic treated and thermal treated samples were
shown in the following figures
Emtricitabine Tenofovir Bictegravir
Control Sample (As Such Sample) 99.8 98.8 100.1
Acid (5N HCl/4Hrs at 60°C) 97.5 98.2 99.2
Base (5N NaOH/4Hrs at 60°C) 72.4 97.6 97.8
Peroxide(30% H2O2 4Hrs/25°C) 100.2 97.2 98.4
Thermal (80°C/24Hrs 99.7 99.6 98.9
Photolytic (1.2m lux hours) 99.4 100.1 99.1
Degradation Condition
%Assay for Degradation Sample

24. Fig.10: Acid Treated Sample(5N HCl/4Hrs/60°C)
Fig.11: Base Treated Sample(5N NaOH/4Hrs/60°C)
Fig.12: Peroxide Treated Sample(5N NaOH/4Hrs/60°C) Fig.13: Photolytic Treated Sample

25. Fig.14: Thermal Treated Sample

26.  Method Precision:
Six sample solutions were prepared individually and injected into the UPLC
System, Calculated %Assay against average area of Six Standards of
Emtricitabine, Tenofovir and Bictegravir individually.
Name of the Sample %Assay of
Emtricitabine
%Assay of
Tenofovir
%Assay of
Bictegravir
Method Precision-01 102.0 99.4 100.3
Method Precision-02 100.3 98.5 99.4
Method Precision-03 100.5 98.6 99.5
Method Precision-04 100.0 98.7 99.6
Method Precision-05 100.5 98.7 99.6
Method Precision-06 100.1 98.8 99.7
Standard Deviation 0.720 0.322 0.340
Average 100.545 98.787 99.673
%RSD 0.72 0.33 0.34
Acceptance criteria:
% Assay should be 90-110% for EMT,TNF and BCT
%RSD for 6 replicants of assay should be NMT 2.0

27.  Accuracy:
 Three levels of accuracy samples were prepared in triplicate by standard addition method
injected each level of Accuracy and mean %Recovery was obtained 99.8% for Emitricitibine,
100.2% for Tenofovir and 100.7% for Bictegravir respectively. The Accuracy,Recovery and
%RSD of Emtricitabine, Tenofovir and Bictegravir were calculated and summarized in the
table.
Name of the Level Emtricitabine Tenofovir Bictegravir
50% Accuracy 100.9 100.6 100.6
100% Accuracy 99.7 100.4 101.2
150% Accuracy 98.7 99.5 100.2
Mean 99.8 100.2 100.7
%RSD 0.94 0.57 0.42
Acceptance criteria : % recovery should be between 98-102%

28. Emtricitabine Tenofovir Bictegravir
Conc. in µg/mL Peak Area Conc. in µg/mL Peak Area Conc. in µg/mL Peak Area
100 769.660 12.5 380.530 25 582.290
160 1225.960 20 611.190 40 934.520
200 1513.960 25 756.300 50 1155.200
240 1810.030 30 899.730 60 1380.190
300 2260.800 37.5 1129.990 75 1732.650
Correlation
coefficient
0.999
Correlation
coefficient
0.999
Correlation
coefficient
0.999
Calibration curve details of Emtricitabine,Tenofovir and Bictegravir
 Linearity
Five linearity solutions (50%, 80% 100%, 120% and 150%) were prepared from standard stock solution i.e., 100µg/mL to
300µg/mL for Emtricitabine, 12.5µg/mL to 37.5µg/mL for Tenofovir and 25µg/mL to 75µg/mL for Bictegravir.
Results
The Correlation coefficient was found 0.999 for Emtricitabine, Tenofovir Alafenamide and Bictegravir. The linearity of
different concentrations of the Emtricitabine,Tenofovir and Bictegravir were calculated and summarized in the table and
graphs were shown in Figures.

30. S. No. Condition
%RSD for
Emtricitabine
%RSD for
Tenofovir
%RSD for
Bictegravir
01 Flow Rate_0.4mL/min 0.3 0.2 0.2
02 Flow Rate_0.6mL/min 0.2 0.5 0.1
03 Wavelength (275nm) 0.7 0.4 0.6
04 Wavelength (285nm) 0.4 0.6 0.9
Table8: Robustness
Robustness conditions like flow (0.5mL/min±0.1mL) and Wavelength (280nm±5nm)
were maintained in the UPLC System and injected Six standards replicate injections in
each condition.
System suitability parameters were within the acceptance criteria.
Results
%RSD for area of six standard injections for within the limit.

31. Ruggedness
Acceptance criteria:
Mean %Assay was obtained between 90 to 110% individual preparation and %RSD for
%Assay between Analyst-I and Analyst-II Should be below 2.0%.
Results
The % RSD for % Assay of Analyst-I &II results within the acceptance criteria and results
were summarized in the Table
Analyst-I 100.5 98.8 99.7
Analyst-II 100.2 99.5 99.4
Average 100.4 99.1 99.5
%RSD 0.24 0.51 0.19
IntermediatePrecision(Ruggedness)

32. Results and Discussion
 To develop and establish a suitable UPLC method for simultaneous estimation of
Emtricitabin, Bictegravir and Tenofovir alfanamide in bulk and Tablet dosage forms,
different preliminary tests were performed and different chromatographic conditions were
tested and optimized chromatographic conditions were developed which were given in
table.The final analysis was performed by using 55% TEA:45% Methanol at a flow rate of
0.5 mL/min. Samples were analyzed at 280 nm detector wave length and at an injection
volume of .with 10 µL using Acquity BEH(Bridged Ethylene hybrid) C18 130A° (100mm ×
2.1 mm, 1.7μm) column 5 run time of 10 min. The proposed method was optimized to give
sharp peak with good resolution and minimum tailing effect for Emtricitabine, Bictegravir
and Tenofovir alfanamide, the optimized chromatogram was obtained as shown in figure.
 The retention times of the Emtricitabine, Tenofovir alafenamide and Bictegravir was found
to be 2.6 min,4.3 min and 5.2min. The linearity was found satisfactory for the drugs in the
range 50 to 250μg/ml for Emtricitabine, Tenofovir alafenamide 12.5µg/mL to 37.5µg/mL
and Bictegravir was 25µg/mL to 75µg/mL

33.  Precision of the method was studied by repeated injection of tablet solution and results
showed lower % RSD values. This reveals that the method is quite precise. The
percent recoveries of the drug solutions were studied at three different concentration
levels.
 The percent individual recovery and the % RSD at each level were within the
acceptable limits. This indicates that the method is accurate.
 The absence of additional peaks in the chromatogram indicates non-interference of the
commonly used excipients in the tablets and hence the method is specific.
 The deliberate changes in the method( wavelength, Flow) have not much affected the
peak tailing, theoretical plates and the percent assay. This indicates that the present
method is robust.
 The system suitability studies were carried out to check various parameters such as
theoretical plates and tailing factor.
 The solution stability studies indicate that both the drugs were stable up to 24 hours.
The forced degradation studies indicate that both the drugs were stable in stability
studies.

34.  CONCLUSION for Method-I:
The developed stability indicating technique was once validated efficaciously for
simultaneous estimation of Emtricitabine, Tenofovir and Bictegravir . The proposed
approach used to be examined for its accuracy, linearity, precision, robustness, and
degradation studies. The compelled degradation research had been carried out, and the
mentioned technique efficiently separates the drug supplies besides interference of
excipients and degradation products. Hence, it can be concluded that the developed
technique can be used for the pursuits evaluation of Emtricitabine, Tenofovir and
Bictegravir in the pharmaceutical dosage form

35.  CONCLUSION
 A specific, Reliable, Precise and Robust Stability indicating Assay Method was
developed for simultaneous estimation of Emtricitabine, Tenofovir Alafenamide and
Doultegravir in tablet dosage form by UPLC
 The proposed approach used to be examined for its accuracy, linearity, precision,
robustness, and degradation studies. The compelled degradation research had been
carried out, and the mentioned technique efficiently separates the drug supplies besides
interference of excipients and degradation products.
 Hence, it can be concluded that the developed technique can be used for the pursuits
evaluation of Emtricitabine, Tenofovir and Doultegravir in the pharmaceutical dosage
form

36. References
 Akram NMD, Umamahesh M. A New Validated RP-HPLC method for the Determination
of Emtricitabine and Tenofovir AF in its bulk and pharmaceutical dosage forms. Journal of
Chemical and Pharmaceutical Sciences.2017; 10(4):54–59.
 Badgujar BP, Mahajan MP, Sawant SD. Developmentand Validation of RP-HPLC
Method for the SimultaneousEstimation of Tenofovir Alafenamide and Emtricitabinein
Bulk and tablet dosage form. Int J Chem Tech Res.2017; 10(5):731–739.
 Satinder A, Dong MW. Method development and validation pharmaceutical analysis by
HPLC reverse phase high performance liquid chromatography Vol. 6. New york: Elsevier.
2005;16–70.
 Swartz ME, krull I. Analytical method development and validation. 1st ed. New York:
Marcel Dekker.2009;17–80.
 ICH Harmonised Tripartite Guideline. Validation of Analytical Procedures: Text And
Methodology Q2 (R1).Nov2005.
.

37.  Joshi M, Nikalje AP, Shahed M, HPTLC methodfor the simultaneous estimation of
emtricitabine andtenofovir in tablet dosage form. Indian J Pharm Sci.2009; 71:95–97.
 Venkateswara Rao B, Vidyadhara S, Nagaraju B, A Novel Stability Indicating RP-HPLC
Method DevelopmentAnd Validation For The Determination Of Tenofovir Disoproxil
Fumarate And Emtricitabine In Bulk And Pharmaceutical formulations. Int J of Pharm Sci
and Res. 2017; 8(5):2168–2176.
 Yenumula BRR, Singampalli MR, Challa BSR. Simultaneous Estimation of Emtricitabine
and Tenofovir DisoproxilFumarate in Tablet Dosage Form by Reverse Phase
Highperformance Liquid Chromatography. SOJ Chromatograph Sci. 2015; 1:6.
 Sharma R, Gupta P. A Validated RP - HPLC method for Simultaneous estimation of
Emtricitabine and TenofovirDisoproxil fumarate in a tablet dosage form. Eurasian J Anal
Chem. 2009; 4(3):276–284.
 Shelke A, Shinde M, Mogal R, Application of UV Spectrophotometric methods for
simultaneous estimation of Emtricitabine and Tenofovir alafenamidefumarate in bulk.
Asian J Pharm Techn. 2018;8: 2231–5713
 Mastanamma S, Venkata Reddy D, Saidulu P, Developmentand Validation of Stability
Indicating RP-HPLCMethod for the Simultaneous estimation of EmtricitabineTenofovir
Alafenamide bulk and their combined dosage form. J. Chem. Pharm. 2017; 9 (9):70–80