INTRODUCTION:

Dolutegravir¹ sodium (CAS No. 1051375-19-9) is chemically 2H-Pyrido [1',2':4, 5] pyrazino [2,1-b][1,3]oxazine-9-carboxamide,N-[(2,4-difluorophenyl)methyl]-3,4,6,8,12,12a-hexahydro-7-hydroxy-4-methyl-6,8-dioxo-, sodium salt (1:1), (4R,12aS) with molecular formula, C₂₀H₁₈F₂N₃NaO₅and molecular weight 441.367g/mol. Dolutegravir is a HIV Integrase Inhibitor. Dolutegravir is an orally bioavailable integrase strand-transfer inhibitor with activity against HIV-Type 1 infection.

Lamivudine^2-3 (CAS No. 134678-17-4) is chemically 4-amino-1-[(2R, 5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one with molecular formula, C₈H₁₁N₃O₃S and molecular weight 229.256g/mol. Lamivudine is a synthetic nucleoside analogue and is phosphorylated intracellularly to its active 5'-triphosphate metabolite, lamivudine triphosphate (L-TP). This nucleoside analogue is incorporated into viral DNA by HIV reverse transcriptase and HBV polymerase, resulting in DNA chain termination.

Tenofovir disoproxil fumarate⁴ (CAS No. 202138-50-9) is chemically bis (1-methylethyl) 5-{[(1R-2-(6-amino-9H-purin-9-yl)-ethylethoxy] methyl}-5-oxo-2, 4, 6, 8-tetraoxa-5-λ5-phosphanonanedioate (ester) hydrogen (2E)-but-2-enedioate with molecular formula, C₁₉H₃₀N₅O₁₀P. C₄H₄O₄ and molecular weight 635.52 g/mol. It is an antiretroviral agent acts as a reverse Transcriptase inhibitor.

The chemical structures of Dolutegravir sodium, Lamivudine and Tenofovir disoproxil fumarate (Tenofovir DF) were shown in Figure 1. Only three liquid chromatographic methods^5-8 were developed for the simultaneous estimation of Tenofovir disoproxil fumarate, Lamivudine and Dolutegravir in tablets and their potential impurities.

Kalpana⁵ et al., developed a RP-HPLC method for the simultaneous estimation of Lamivudine, Tenofovir disproxil and Dolutegravir on gradient program using a mobile phase mixture of 0.1% v/v trifluoro acetic acid in water and methanol and a diluent consisting of water and acetonitrile in 50: 50 ratio with flow rate 1.0ml/min using Luna C8 column (Detection wavelength 260nm). Lamivudine, Tenofovir and Dolutegravir were eluted at 2.023min, 5.330min and 7.673 and the linearity was found to be 75-225, 75-225 and 12.5-37.5µg/ml for Lamivudine, Tenofovir and Dolutegravir respectively.

Mallikarjuna⁶ Rao et al., developed a RP-HPLC method for the simultaneous estimation of Lamivudine, Tenofovir and Dolutegravir on gradient program using a mixture of acetonitrile and 0.05 M phosphate buffer pH 6.2±0.05 adjusted with dilute potassium hydroxide solution and acetonitrile as mobile phase with flow rate 1.0ml/min and Inertsil ODS-3V C18 column (Detection wavelength 260nm). Lamivudine, Tenofovir and Dolutegravir were eluted at 2.8min, 5.2min and 11.5 and the linearity was found to be 27-162, 27-162 and 4.5-28 µg/ml for Lamivudine, Tenofovir and Dolutegravir respectively.

Varaprasad⁷et al., developed a stability-indicating HPLC method for the determination of potential impurities in the combined dosage forms of Dolutegravir, Lamivudine and Tenofovir disoproxil fumarate tablets on gradient program with run time of 140 mins. Buffer solution consisting of 10mM sodium dihydrogen phosphate monohydrate with 0.5g/L of 1- octane sulfonic sodium salt monohydrate (pH adjusted to 2.5 with ortho phosphoric acid) was used as an aqueous phase i. e. Mobile phase A. A mixture of Buffer: Acetonitrile: Methanol (20: 60: 20) was used as Mobile phase B. Core-shell Bi-phenyl polar stationary phase of Phenomenex Kinetex Biphenyl column (Detection wavelength 260nm). Lamivudine, Tenofovir disoproxil and Dolutegravir were eluted at 51.70min, 80.98 min and 92.16 and the linearity was found to be 1.5-4500, 1.5-4500 and 0.25-750µg/ml respectively.

Saravanan⁸ et al., developed a stability-indicating HPLC method for the assay method of analysis for Dolutegravir/Lamivudine/Tenofovir Disoproxil fumarate tablets on gradient mode using a mobile phase mixture of 0.1% (v/v) trifluoroacetic acid buffer and methanol with flow rate 1.0 ml/min using Inertsil ODS C18 column (Detection wavelength 260nm). Lamivudine, Tenofovir disoproxil and Dolutegravir shows linearity was found to be 6-90, 6-90 and 1-15 µg/ml respectively.

In the present study, the authors have proposed a new stability-indicating UHPLC method for the simultaneous estimation of Dolutegravir sodium, Lamivudine and Tenofovir DF in tablet dosage forms and the method was validated as per ICH guidelines.

Tenofovir disoproxil fumarate

Lamivudine

Dolutegravir sodium

Figure 1: Chemical structures of anti-viral drugs

MATERIALS AND METHODS:

Lamivudine (%purity 99.91), Tenofovir DF (%purity 100.01) and Dolutegravir sodium (%purity 99.89) were procured and used as it is without further purification. HPLC grade acetonitrile, AR grade HCl, NaOH, H₂O₂, Triethylamine, Trifluoro acetic acid, dibasic potassium hydrogen phosphate (K₂HPO₄) and o-phosphoric acid were procured from Merck and used without further purification. Mobile phase A was prepared by using 0.01 % aqueous Tri ethyl amine (pH adjusted to 4.6±0.05 using ortho phosphoric acid) and mobile phase B was prepared by using 0.01% Tri fluoro acetic acid in acetonitrile. A mixture of phosphate buffer (adjusted the pH 3.0±0.05 with ortho phosphoric acid) and acetonitrile in 72: 28 ratio was used for the chromatographic study. Phosphate buffer (pH 3.0) was prepared by dissolving 4.40g of anhydrous dibasic potassium hydrogen phosphate (K₂HPO₄) in water in a 1000ml volumetric flask and the pH was adjusted to 3.0±0.05 with ortho phosphoric acid.

Instrumentation and Chromatographic conditions:

Shimadzu NexeraX2 Model UPLC system with PDA detector and Zorbax SB-C18 column (100mm × 4.60 mm, 3.5μ) was employed for the study on gradient mode (Table 2) with 1.0mL/min flow rate (Detection wavelength 260nm) and column temperature 50°C (Injection volume 1µL) within a run time of 5 mins.

Phosphate buffer (pH 3.0) was prepared by dissolving 4.4grams of anhydrous Di basic potassium hydrogen phosphate (K₂HPO₄) in a 1000mL volumetric flask in water by adjusting the pH to 3.00±0.05 with the help of ortho phosphoric acid. A mixture of phosphate buffer (pH 3.0) and acetonitrile (62:38, v/v) was used as a diluent for the study.

An aqueous solution of 0.1% Triethylamine, adjusted to pH 4.60±0.05 with ortho phosphoric acid was used as mobile phase A and a 0.1% TFA solution prepared in acetonitrile was used as mobile phase B and a diluent consisting of a mixture of phosphate buffer (pH 3.0) and acetonitrile (62: 38) was used for the study.

Preparation of stock and working standard solutions of Tenofovir Disoproxil Fumarate, Lamivudine and Dolutegravir sodium

25mg of Lamivudine, 25mg of Tenofovir disoproxil fumarate and 25mg of Dolutegravir sodium were weighed accurately and dissolved in acetonitrile in three different 25ml volumetric flasks and from this stock solutions a mixture of these three drugs were prepared on dilution with the diluent as per the requirement for the validation as well as stress degradation studies.

Method validation⁹

Linearity, Precision and Accuracy

A series of Lamivudine (5-900µg/mL), Tenofovir DF (2-700µg/mL) and Dolutegravir sodium (2-150µg/mL) solutions were prepared from their stock solutions(1000 µg/mL) and diluted with the diluent and 1.0µL each of these solutions were injected into the UPLC system three times and the average peak area was calculated from the respective chromatograms. A calibration graph was drawn by plotting the concentration of the drug solutions on the x-axis and the corresponding mean peak area on the y-axis. The intraday precision studies were conducted on the same day at different equal time intervals and the interday precision studies were conducted on three successive days (Day 1, Day 2 and Day 3) and the data was analysed. Accuracy studies were performed by spiking the formulation solution with 80, 100 and 120% of API solution and % recovery was calculated. The percentage relative standard deviation was calculated in all the validation parameters.

Assay of combined dosage forms of Lamivudine, Tenofovir disoproxil fumarate and Dolutegravir (Tablets):

20 Tablets of two different brands of tablet dosage forms consisting of Lamivudine (300mg), Tenofovir DF (300 mg) and Dolutegravir (50mg) available with brand name, Viropil from Emcure Pharmaceuticals Ltd (India) and Acriptega (Mylan Laboratories Ltd (India) were weighed, powdered and tablet powder equivalent toLamivudine (300mg), Tenofovir disoproxil fumarate (300mg) and Dolutegravir (50mg) was transferred in to a 100ml volumetric flask and acetonitrile was added. The mixture was sonicated for 30minutes filtered through 0.45µ Nylon filter. Then the required concentrations were prepared on dilution with the diluent and 1.0μLof these solutions were injected in to the UHPLC system and the chromatograms were recorded. The percentage purity was determined from the peak area of the chromatogram obtained.

Stress degradation studies¹⁰

Stress degradation studies of Lamivudine, Tenofovir disoproxil fumarate and Dolutegravir were performed to identify the specificity of the method.

Acid hydrolysis was carried out by treating the mixture of Tenofovir DF, Lamivudine and Dolutegravir drug solution with 1ml of 0.2N HCl and allowed to stand at room temperature for 30 mins and then neutralized with 0.2N NaOH in a volumetric flask and volume was made up to volume with the diluent. 1.0µl of this solution was injected in to the UPLC system after filtering through 0.45µm nylon filter and the peak area of each of Tenofovir Disoproxil Fumarate, Lamivudine and Dolutegravir was noted from the respective chromatogram.

Alkaline hydrolysis was carried out by treating the mixture of Tenofovir DF, Lamivudine and Dolutegravir drug solution with 1ml of 0.1N NaOH and allowed to stand at room temperature for 30 mins and then neutralized with 0.1N HClin a volumetric flask and volume was made up to volume with the diluent. 1.0µl of this solution was injected in to the UPLC system after filtering through 0.45µm nylon filter and the peak area of each of Tenofovir DF, Lamivudine and Dolutegravir was noted from the respective chromatogram.

Oxidative degradation was carried out by treating the mixture of Tenofovir DF, Lamivudine and Dolutegravir drug solution with 1ml of 3% H₂O₂and allowed to stand at room temperature for 30 mins in a volumetricflask and volume was made up to volume with the diluent. 1.0 µl of this solution was injected in to the UPLC system after filtering through 0.45µm nylon filter and the peak area of each of Tenofovir DF, Lamivudine and Dolutegravir was noted from the respective chromatogram.

Thermal degradation was carried out by treating the mixture of Tenofovir DF, Lamivudine and Dolutegravir drug solution in hot air oven for 4 hours at 65°C and cooled to room temperature in a volumetric flask and volume was made up to volume with the diluent. 1.0µl of this solution was injected in to the UPLC system after filtering through 0.45µm nylon filter and the peak area of each of Tenofovir DF, Lamivudine and Dolutegravir was noted from the respective chromatogram.

Neutral degradation was carried out by treating the mixture of Tenofovir DF, Lamivudine and Dolutegravir drug solution with diluent for 60 mins at room temperature in a volumetric flask and volume was made up to volume with the diluent. 1.0µl of this solution was injected in to the UPLC system after filtering through 0.45µm nylon filter and the peak area of each of Tenofovir DF, Lamivudine and Dolutegravir was noted from the respective chromatogram.

RESULTS AND DISCUSSION:

A new stability indicating RP-UPLC method for the simultaneous determination of Lamivudine, Tenofovir DF and Dolutegravir sodium in tablets. The previously published liquid chromatographic methods in the literature were thoroughly reviewed and some of the parameters were compared with the present proposed method in Table 1.

The representative chromatogram obtained for the placebo and API were shown in Figure 2A and Figure 2B with system suitability parameters within the acceptance criteria.

Table 1: Literature survey

Mobile phase (v/v)	λ (nm)	Linearity (µg/mL)	Comment	Ref
0.1% v/v Trifluoro acetic acid in water and methanol Diluent: Water: Acetonitrile (50: 50)	260	75-225 (LM) 75-225 (TDF) 12.5-37.5 (DT)	RP-HPLC (Gradient mode) (Stability indicating)	5
Mobile phase A: Acetonitrile and 0.05 M phosphate buffer pH 6.2 ± 0.05 adjusted with dilute potassium hydroxide solution Mobile phase B: Acetonitrile	260	27-162 (LM) 27-162 TDF) 4.5-28 (DT)	RP-HPLC (Gradient mode) (Stability indicating)	6
Buffer: Solution consisting of 10 mM sodium dihydrogen phosphate monohydrate with 0.5 g/L of 1- octane sulfonic sodium salt monohydrate (pH adjusted to 2.5 with ortho phosphoric acid) Mobile phase A: Buffer Mobile phase B : Buffer: Acetonitrile: Methanol (20: 60: 20)	260	1.5-4500 (LM) 1.5-4500 (TDF) 0.25-750 (DT)	RP-HPLC (Gradient mode) (Stability indicating) Potential impurities	7
0.1% Trifluoroacetic acid buffer and methanol	260	6-90 (LM) 6-90 (TDF) 1-15 (DT)	RP-HPLC (Gradient mode) (Stability indicating)	8
Mobile phase A: 0.1% aqueous Triethylamine adjusted to pH 4.60 ± 0.05 with ortho phosphoric acid Mobile phase B: 0.1% TFA solution prepared in acetonitrile	260	5-900 (LM) 2-700 (TDF) 2-150 (DT)	UHPLC (Stability indicating)	Present method

Table 2: Gradient program

Time (min)	Mobile phase A	Mobile phase B
0.0	97.0	3.0
2.34	50.0	50.0
4.0	50.0	50.0
4.5	97.0	3.0
5.0	97.0	3.0

Linearity, Precision and accuracy:

Lamivudine, Tenofovir DF and Dolutegravir sodium obey Beer-Lambert’s law over the concentration range 5-900, 2-700 and 2-150µg/mL (Table 3) with linear regression equations y = 1724.x-11307 (R² = 0.999)(Figure 3A), y = 1737.x-1009 (R² = 0.999)(Figure 3B) and y = 3538.x+587.5 (R² = 0.999) (Figure 3C) for Lamivudine, Tenofovir DF and Dolutegravir sodium and the method was validated as per ICH guidelines. The LOQ values were found to be 4.4926, 1.8342 and 1.7963 µg/mL and that of LOD values as 1.4811. 0.6024 and 0.5903µg/mL for D Lamivudine, Tenofovir DF and Dolutegravir sodium respectively.

A) Placebo

B) API:Lamivudine (1.939 min), Tenofovir DF (3.422 min) and Dolutegravir sodium (4.034 min)

C) Tablet dosage form

Figure 2: Representative chromatograms of the combination of Lamivudine, Tenofovir DF and Dolutegravir

Table 3: Linearity

Conc. (µg/mL)	*Mean peak area
Conc. (µg/mL)	LM	TDF	DT
0	0	0	0
2	-	3584	7081
5	7584	8541	18524
10	15061	17216	35013
20	30132	34879	71259
40	60325	68532	142425
50	74469	85214	181647
60	90294	102596	209526
80	120611	139562	284191
100	147122	170921	352102
120	206119	196537	429957
150	235684	265478	528892
200	329154	351012	-
300	492659	519847	-
400	668265	702154	-
500	832628	856247	-
600	1031124	1032654	-
700	1211265	1226587	-
800	1376782	-
900	1538276	-	-

*Mean of three replicates

Precision study was performed by injecting the mixture of LM, TDF and DT six times into the UHPLC system and the chromatographs were recorded. The mean peak area, standard deviation and the relative standard deviation were calculated from the respective linear regression equations.

The %RSD in intraday precision was found to be 0.4277, 0.0097 and 0.0213 whereas for interday precision it was found to be 0.0669, 0.0699 and 0.0416 for Lamivudine, Tenofovir DF and Dolutegravir sodium respectively (Table 4) which was found to be less than 2.0% showing that the method is precise.

In the accuracy study the %RSD was found to be 0.44-0.81 for Lamivudine (%Recovery: 99.73 - 99.91), 0.39-0.82 for Tenofovir DF (%Recovery: 99.54 - 99.77) and 0.36 - 0.91 for Dolutegravir sodium (%Recovery: 99.61-99.94) respectively which is less than 2.0 showing that the method is accurate (Table 5).

The %RSD in robustness study was also found to be less than 2% (Lamivudine: 0.19 - 0.61; Tenofovir DF: 0.21-0.71; Dolutegravir sodium: 0.22 - 0.87) showing that the method is robust (Table 6).

Figure 3A: Calibration curve of Lamivudine (LM)

Figure 3B: Calibration curve of Tenofovir disoproxil fumarate (TDF)

Figure 3C: Calibration curve of Dolutegravir sodium (DT)

Table 4: Precision study

Intraday precision study
S. No.	R_t (min)			Peak area
S. No.	LM	TDF	DT	LM	TDF	DT
1	1.875	3.425	4.021	492659	519847	181647
2	1.874	3.421	4.015	494247	519768	181712
3	1.883	3.427	4.028	498321	519719	181629
4	1.871	3.416	4.015	493318	519811	181731
5	1.872	3.422	4.018	496133	519722	181684
6	1.865	3.418	4.020	493725	519789	181693
Mean	-	-	-	494733.83	519776	181682.67
SD	-	-	-	2116.12	50.33	38.62
% RSD	-	-	-	0.4277	0.0097	0.0213
Interday precision study
Day				Day 1	Day 2	Day 3
LM				492659	493984	494452
TDF				519847	519264	519181
DT				181647	181754	181793
Mean				494218.00	519430.67	181731.33
SD				330.93	362.94	75.59
% RSD				0.0669	0.0699	0.0416

Table 5: Accuracy study

Level %	Spiked Conc. (µg/mL)			Formulation (µg/mL)			Total Conc. (µg/mL)			*% Recovery (% RSD)**
Level %	LM	TDF	DT	LM	TDF	DT	LM	TDF	DT	LM	TDF	DT
80	240	240	40	300	300	50	540	540	90	99.91 (0.49)	99.71 (0.62)	99.79 (0.36)
100	300	300	50	300	300	50	600	600	100	99.84 (0.81)	99.77 (0.39)	99.61 (0.57)
120	360	360	60	300	300	50	660	660	110	99.73 (0.44)	99.54 (0.82)	99.94 (0.91)

*Mean of three replicates

Table 6: Robustness study

LAMIVUDINE
Conditions	% Assay	Theoretical plates	Tailing factor	% RSD
Controlled Condition	99.09	96460	1.12	0.33
Increased Flow (1.1ml)	99.82	78834	1.15	0.19
Decreased Flow (0.9ml)	99.73	102207	1.23	0.19
Low column temperature (45°C)	99.81	86415	1.17	0.61
High column temperature (55°C)	99.98	97813	1.16	0.55
Low Buffer pH (4.4)	99.69	58452	1.21	0.42
High Buffer pH (4.8)	99.79	90782	1.33	0.29
TENOFOVIR DF
Controlled Condition	99.26	138628	1.23	0.31
Increased Flow (1.1ml)	99.37	127119	1.17	0.28
Decreased Flow (0.9ml)	99.28	137882	1.14	0.41
Low column temperature (45°C)	98.34	162552	1.34	0.52
High column temperature (55°C)	99.61	178639	1.21	0.21
Low Buffer pH (4.4)	99.87	97581	1.11	0.33
High Buffer pH (4.8)	99.78	132664	1.18	0.71
DOLUTEGRAVIR
Controlled Condition	99.98	79256	1.15	0.54
Increased Flow (1.1ml)	99.77	68331	1.23	0.26
Decreased Flow (0.9ml)	99.92	61756	1.17	0.22
Low column temperature (45°C)	98.64	78254	1.15	0.87
High column temperature (55°C)	99.65	88597	1.17	0.47
Low Buffer pH (4.4)	99.88	40769	1.19	0.81
High Buffer pH (4.8)	99.28	60594	1.21	0.72

Table 6: Stress degradation studies

	R_t (min)			*% Recovery (%Decomposition)**			Theoretical Plates (>2000) (Tailing factor)			Resolution (>2)
Drug	LM	TDF	DT	LM	TDF	DT	LM	TDF	DT	LM	TDF	DT
Acidic hydrolysis 0.2N HCl/ 30 mins	1.928	3.415	4.016	98.11 (1.89)	89.23 (10.77)	99.9 (0.1)	96434 (1.13)	139582 (1.22)	79981 (1.01)	-	29.14	10.21
Alkaline hydrolysis 0.1N NaOH/ 5 mins	1.945	3.433	4.035	93.12 (6.88)	83.4 (16.6)	97.9 (2.1)	96913 (0.81)	144341 (0.62)	81029 (0.25)	-	32.74	10.49
Oxidative degradation 3 % H₂O₂/ 30 mins	1.937	3.343	3.907	88.98 (11.02)	97.8 (2.2)	99.3 (0.7)	99851 (0.32)	137261 (0.54)	78697 (0.91)	-	28.93	10.32
Thermal degradation 65°C/4 Hrs	1.933	3.415	4.021	97.1 (2.9)	99.1 (0.09)	99.9 (0.1)	96785 (0.75)	139982 (0.38)	80265 (0.77)	-	30.14	10.91
Neutral degradation 60 mins	1.931	3.413	4.017	95.1 (4.9)	99.51 (0.49)	99.4 (0.6)	98927 (0.72)	143659 (0.42)	79745 (0.39)	-	31.23	10.81

*Mean of three replicates

Alkaline hydrolysis

Acidic hydrolysis

Oxidative degradation

Thermal degradation

Neutral degradation

Figure 4: Typical chromatograms of Lamivudine, Tenofovir disoproxil fumarate and Dolutegravirduring the stress degradation studies

CONCLUSION:

The proposed new stability indicating RP-UPLC method for the estimation of Lamivudine, Tenofovir disoproxil fumarate and Dolutegravir is simple, accurate, precise and robust. The method is useful for the routine analysis of Lamivudine, Tenofovir disoproxil fumarate and Dolutegravir tablets in pharmaceutical industries. The method is specific and there is no interference of excipients during the study.

ACKNOWLEDGEMENT:

The authors are grateful to Emcure Pharmaceuticals Ltd (India) for providing the gift samples of Lamivudine, Tenofovir disoproxil fumarate and Dolutegravir and there is no conflict of interest.

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Received on 19.07.2022 Modified on 21.08.2022

Research J. Pharm. and Tech 2022; 15(9):3823-3830.

DOI: 10.52711/0974-360X.2022.00641