This document presents the development and validation of a new RP-UPLC method for the simultaneous estimation of lamivudine, tenofovir, and efavirenz in tablet dosage forms. The optimized method uses a C18 BEH column, a mobile phase of 35% phosphate buffer (pH 3.0) and 65% methanol, and detects the drugs at 260 nm. The method was validated per ICH guidelines and found to be specific, precise, accurate, linear, robust, and stability-indicating for the simultaneous analysis of the drugs without interference from excipients. The developed and validated RP-UPLC method provides a simple, rapid, and economical approach for the routine quality control analysis of fixed

1. Presented by
SK.MADEESH (10P21S007)
Institutional guide:
Mr. Y ISMAIL M.Pharm (Phd.,).
Assistant professor
Department of Pharmaceutical Analysis,
Rao’s College of Pharmacy.
A NEW VALIDATED RP-UPLC METHOD DEVELOPMENT AND VALIDATION FOR THE
SIMULTANEOUS ESTIMATION OF LAMIVUDINE ,TENOFOVIR AND EFAVIRENZ IN ITS
BULK AND PHARMACEUTICAL DOSAGE FORM
1
Industrial guide:
Mr. G. Chandrasekhar Reddy, M.Sc,
Director technical,
PharmaTrain ,
Hyderabad.

2. DRUG PROFILE
Drug name : LAMIVUDINE
Description of the drug : White Crystalline powder
Chemical name 2(1H)-Pyrimidinone,4-amino-1-[2-(hydroxymethyl)
-1,3-oxathiolan-5-yl]-,(2R-cis)-. (–)-1-[(2R,5S)-2-
(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine [134678-
17-4].
Empirical formula : C8H11N3O3S
Structure :
Molecular weight : 514.63gm/mol
Category : Anti Retro viral drug
Solubility : very soluble in water.
Brand name : Combivir, Epzicom, Trizivir.
2

3. Drug name : TENOFOVIR
Description of the drug : White Crystalline powder
Chemical name : ({[(2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)
phosphonic acid.
Empirical formula : C23H34N5O14P
Structure :
Molecular weight : 287.2123 [g/mol]
Category : Anti Retro viral drug.
Solubility : very soluble in alcohol
Official : IP,BP Pharmacopoeias
Brand name : Atripla, Truvada.
3

4. 4
Drug name : EFAVARENZ
Description of the drug : White Crystalline powder
Chemical name : (4S)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoro-methyl)
-2,4-dihydro-1H-3,1-benzoxazin-2-one.
Empirical formula : C14H9ClF3NO2
Structure :
Molecular weight : 315.675[g/mol]
Category : Anti Retro viral drug.
Solubility : very soluble in alcohol
Brand name : Atripla.

5. 5
D.H. Shewiyo, E. Kaale et
al
developed and validated a method for the simultaneous
analysis of lamivudine (LVD), stavudine (STV) and
nevirapine (NVP) using high-performance thin-layer
chromatography (HPTLC) with densitometric detection.
Sockalingam
Anbazhagan et al
developed simultaneous quantification of stavudine
(SV), lamivudine (LV) and nevirapine (NV) in tablets by UV
spectroscopy, reverse phase HPLC (RP-UPLC) and
HPTLC methods were developed.
Mirna El Barkil et al
A sensitive high-performance liquid chromatography
method coupled to UV and single mass spectrometry
(MS) detection was developed for the determination of
tenofovir in human plasma.
Eda Ross Montgomery
et al
A stability-indicating high performance liquid
chromatographic (HPLC) method was developed for the
assay of efavirenz, a non-nucleoside reverse
transcriptase inhibitor used in the treatment of AIDS
LITERATURE REVIEW
AUTHORS WORK

6. 6
Bregt S. Kappelhoff
et al
Efavirenz and nevirapine are non-nucleoside reverse
transcriptase inhibitors for the treatment of HIV-1-infected
individuals. A simple and rapid high-performance liquid
chromatographic method for the simultaneous quantification
of efavirenz and nevirapine in human plasma suitable for
therapeutic drug monitoring is described
Jayaseelan S et al
have reported the new analytical method development and
validation for the simultaneous estimation of Lamivudine and
Stavudine in tablet dosage form by RP-UPLC Method
Kumar M et al
have reported the method development and validation of RP-
UPLC method for simultaneous determination of Lamivudine
and Zidovudine using Altima
Rajesh S and Pooja G
et al
have reported the Validated RP-UPLC method for
simultaneous estimation of Emitricitabine and Tenofovir
Disoproxil Fumarate in a tablet dosage form
Jayaseelan S et al
have reported the Bio analytical method development and
validation of Lamivudine by RP-UPLC method

7. 7
Anna PN et al
have reported the stability indicating HPLC method for
the determination of Efavirenz in bulk drug and in
pharmaceutical dosage form using Nova-pak phenyl
column
Malipatil S M et al have reported the determination of Tenofovir
Disoproxil Fumarate by a sensitive simple isocratic
RP-UPLC method
Mandloi D K et al
have reported the method development and validation
of a RP-UPLC in the application of in-vitro dissolution
study of Lamivudine in bulk drug and tablet
formulation,

8. AIM AND OBJECTIVE
8
AIM
 The literature survey carried out and it revealed that several analytical methods have been reported
for estimation of these drugs as individual or in combination with other drugs.
 Present study have been aims to develop a specific, simple and rapid Stability indicating RP-UPLC
method for simultaneous estimation and validation of lamiudine, tenofovir and efavirenz in tablet
dosage form.
OBJECTIVE
 To develop new simple, sensitive, accurate and economical analytical
method for the simultaneous estimation of Lamivudine, Tenofovir and
Efavirenz.
 To validate the proposed method in accordance with USP and ICH
guidelines for the intended analytical application i.e., to apply the
proposed method for analysis of the Lamivudine ,Tenofovir and
Efavirenz in dosage form.

9. 9
 Preparation of Buffer
 Preparation of Mobile Phase
 Preparation of Standard Solution
 Preparation of Sample Solution
EXPERIMENTAL WORK

10. 10
TRIALS
Mobile phase : phosphate buffer (pH 3.0)
Methanol (50:50v/v)
Column : C18 BEH(4.6 × 50 mm, 1.7µm)
Flow rate : 0.4 ml/min
Run time : 10 min
Injection volume : 8µl
Wave length : 260 nm
Mobile phase : phosphate buffer (pH 3.0)
methanol (40:60v/v)
Column : C8 (4.6 × 50 mm, 1.7µm)
Flow rate : 0.3 ml/min
Run time : 10 min
Injection volume : 6 µl
Wave length : 260 nm

11. 11
Mobile phase : phosphate buffer (pH 3.0)
Methanol (60:40 v/v)
Column : C18 BEH (4.6 × 50 mm, 1.7µm)
Flow rate : 1 ml/min
Run time : 8 min
Injection volume : 6µl
Wave length : 260 nm

12. 12
 Column : C18 BEH (4.6 x 50mm,1.7µm,make: Waters)
 Buffer : phosphate buffer (3 pH)
 Mobile Phase : 35% Buffer,65% Methanol
 Flow rate : 0.3ml/min
 Wave length : 260 nm
 Injection volume : 6 µl
 Temperature : Ambient
 Run time : 5 min
OPTIMIZED CHROMATOGRAPHIC CONDITIONS

13. 13
Parameters Lami teno
Resolution 4.13
Retention time (min) 2.976 3.94
No. of Theoretical plates 2612.40 2643.90
Tailing factor 1.17 1.13
RESULTS AND DISCUSSION
Optimized chromatogram
System suitability Parameters
Chromatogram for blank

14. 14
10µg/ml of Lamivudine,Tenofovir and 20µg/ml of
Efavirenz
20µg/ml of Lamivudine,Tenofovir and 40µg/ml of Efavirenz
30µg/ml of Lamivudine,Tenofovir and 60µg/ml of
Efavirenz
40µg/ml of Lamivudine,Tenofovir and 80µg/ml of
Efavirenz
50µg/ml of Lamivudine,Tenofovir and 100µg/ml of Efavirenz
LINEARITY

15. 15
LINEARITY STUDIES OF LAMIUVDINE
There exists a linear relationship in the concentration range of 10 to 50µg/ml for Lamiuvdine. The data
are tabulated in the below table.
CONCENTRATION
(µg/ml)
PEAK AREA
10
20
30
40
50
839286
1067774
1246474
1439994
1639065

16. LINEARITY STUDIES OF TENOFAVIR
16
CONCENTRATION (µg/ml) PEAK AREA
10
20
30
40
50
626221
778750
931447
1070162
1196060
There exists a linear relationship in the concentration range of 5 to 25µg/ml for
Tenofavir. The data are tabulated in the below given table.

17. LINEARITY STUDIES OF EFAVIRENZ
17
CONCENTRATION (µg/ml) PEAK AREA
20
40
60
80
100
626221
753615
899796
1035191
1194356
There exists a linear relationship in the concentration range of 5 to 25µg/ml for
Efavirenz. The data are tabulated in the below given table.

18. ANALYTICAL PERFORMANCE PARAMETERS OF LAMIVUDINE,TENOFOVIR AND
EFAVERINZ
18
PARAMETERS Efavirenz
Linear Dynamic range
Correlation coefficient
Slope(m)
Intercept(c)
20-100µg/ml
0.999
53592
50245
PARAMETERS Lamivudine,Tenofovir
Linear Dynamic range
Correlation coefficient
Slope(m)
Intercept(c)
10-50µg/ml
0.999
66574
12529
ANALYTICAL PERFORMANCE PARAMETERS OF Efavirenz

19. PRECISION
METHOD PRECISION:
Chromatogram of sample
Chromatogram of standard
Precision was determined by injecting 5 injections of sample and
standard solutions.
19
Acceptance criteria:
• %RSD for sample
should be NMT 2
S.No Sample area
Lami teno Efav
1 1247256 935035 954854
2 1248579 929353 937615
3 1243273 930459 950694
4 1243262 932389 940252
5 1249574 922057 922057
Average 1246389 929858.6 945423.4
SD 2965.62 4865.16 7200.575
%RSD 0.23793 0.5232 0.761

20. 20
Chromatogram for sample concentration-50% Chromatogram for sample concentration-100%
Chromatogram for sample concentration-150%
Sample solutions at different concentrations (50%, 100%, and 150%) were prepared and the
% recovery was calculated.
ACCURACY

21. 21
RECOVERY STUDIES
Sample Concentrati
on
Amount
added
Amount
found
%
Recovery
% Mean
Recovery
Lamivudin
e,
Tenofovir
50% 5
4.9
101.2
100% 10
9.95
99.9
100.7
150% 15
14.99
101
Efavirenz
50% 10
10.2
100.4
100% 20
20.2
100.5
100.8
150% 30
29.6
101.4
Acceptance criteria:
The percentage recovery at each level should be between (98-102%).
The results obtained for recovery at 50%, 100%, 150% are within the limits. Hence the method is accurate.

22. 22
LIMITS OF MEASUREMENT
• There are two important categories within the level of measurement. They are Limit of
detection (LOD) and Limit of quantification (LOQ).
• LIMIT OF DETECTION FOR LAMIVUDINE, TENOFOVIR AND EFAVIRENZ
Sample Baseline noise(µV) Signal obtained
(µV)
S/N
ratio0
Lamivudine
Tenofovir
Efavirenz
47
47
47
139
142
138
2.95
3.02
2.93
Acceptance criteria:
Signal to noise ratio
should be 3 for LOD
solution.
Chromatogram of Lamivudine,Tenofovir and Efavirenz showing LOD

23. 23
LIMIT OF QUANTIFICATION FOR Lamivudine,Tenofovir AND Efavirenz
Chromatogram of Lamivudine,Tenofovir and Efavirenz showing LOQ
Sample Baseline noise(µV) Signal obtained
(µV)
S/N ratio
Lamivudine
Tenofovir
Efavirenz
47
47
47
469
417
468
9.97
10
9.95
Acceptance criteria:
Signal to noise ratio should
be 10 for LOQ solution.

24. 24
Less flow (0.2 ml/min) More flow (0.4 ml/min)
Less organic (55 %) More organic (65 %)
ROBUSTNESS

25. 25
 Effect of Variation in flow
S.No Less flow (0.2ml/min)Rt More flow (0.4 ml/min) Rt
Lamivudine, Tenofovir Efavirenz Lamivudine, Tenofovir Efavirenz
1
0.516 0.796 2.932 0.362 0.560 1.934
2
0.514 0.795 2.852 0.368 0.561 1.937
3
0.513 0.791 2.835 0.368 0.561 1.937
Mean 0.513 0.7912
2.8488
0.368 0.5624 1.9388
%RSD 0.4358
0.5465
1.7613 1.0167 0.4462 0.2107
Acceptance criteria:
• %RSD for sample should be NMT 2

26. Effect of variation in mobile phase composition
26
S.No Less organic(55 %)Rt More organic (65 %) Rt
Lamivudine Tenofovir Efavirenz Lamivudine Tenofovir Efavirenz
1 0.432 0.785 4.014 0.424 0.576 1.478
2 0.433 0.787 4.019 0.428 0.579 1.480
3 0.434 0.789 4.024 0.428 0.579 1.480
4 0.434 0.789 4.024 0.430 0.582 1.484
5 0.436 0.790 4.042 0.432 0.586 1.488
Mean 0.4338 0.788 4.024 0.4284 0.5804 1.482
%RSD
0.3419
0.02538
0.2627 0.6924
0.6515
0.2699
Acceptance criteria:
• %RSD for sample should be NMT 2

27. CONCLUSION
27
The proposed Stability indicating RP-UPLC method was found to be simple, specific,
precise, accurate, rapid and economical for simultaneous estimation of lamiudine, tenofovir
and efavirenz in combined tablet dosage form. These method was validated as per ICH
guidelines. The sample recoveries in all formulations were in good agreement with their
respective label claims and they suggested non –interference of formulation excipients in
the estimation. Hence, this method can be easily and conveniently adopted for routine
analysis of lamiudine, tenofovir and efavirenz in combined tablet dosage form.

28. BIBLIOGRAPHY
28
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30. 30