INTRODUCTION:

The combination of Dolutegravir and Rilpivirine (Figure 1) was approved for the treatment of HIV infection in which Dolutegravir acts as an integrase strand transfer inhibitor by blocking the strand transfer step of the integration of the viral genome into the host cell and Rilpivirine acts as a non-nucleoside reverse transcriptase inhibitor¹.

This is marketed in the name of Juluca.

Dolutegravir is chemically (4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido [1',2':4,5] pyrazino[2,1-b] [1,3] oxazine-9-carboxamide with molecular formula C₂₀H₁₉F₂N₃O₅and molecular weight 419.385 g/mole. Rilpivirine is chemically 4-[[4-[[4-[(E)-2-cyanoethenyl]-2, 6-dimethylphenyl] amino]-2-pyrimidinyl] amino] benzonitrile with molecular formula C₂₂H₁₈N₆and molecular weight 364.417 g/mole.

Figure 1A: Doultegravir (DLT)

Figure 1B: Rilpivirine (RLP)

There are many analytical methods developed in the literature for the estimation of Dolutegravir and Rilpivirine such as LC-MS², LC-MS/MS³, HPLC for the assay of pharmaceutical dosage forms as well as biological fluids.

Grégoire developed a liquid chromatography-tandem mass spectrometry assay² for the quantification of Rilpivirine and Dolutegravir in human plasma using phenyl-hexyl column in presence of two internal standards, Rilpivirine-d6 and Dolutegravir-13C d5 with Analyst 1.4.2 software package (Applied Biosystems, Foster City, CA, USA) and linearity was shown as 25-2000 ng/ml.

Zheng et al., HPLC-MS/MS method³ for the simultaneous quantification of Dolutegravir, Rilpivirine, Ritonavir, Raltegravir and Raltegravir-β-d-glucuronide in human plasma using Kinetex phehyl-hexyl column and mobile phase consisting 0.05 % formic acid and Methanol (0.05 % formic acid) (55: 45) with flow rate 0.5 ml/min and the linearity was reported as 60-15000 ng/ml for Darunavir, 20-5000 ng/ml for Dolutegravir and Elvitegravir, 10-2500 ng/ml for Ritonavir, Raltegravir and Raltegravir-β-d-glucuronide, Ritonavir and Rilpivirine.

Wahab et al., developed a stability indicating RP-HPLC method⁴ for the simultaneous estimation of Rilpivirine and Dolutegravir in bulk and tablet dosage forms using Box-Behnken design. A mixture of 0.1% ortho phosphoric acid buffer and acetonitrile (60:40, v/v) was used as mobile phase with flow rate 1.0 ml/min (Kromasil C 18 column) (UV detection at 260 nm) where Rilpivirine and Dolutegravir were eluted at 2.167 min and 2.716 min respectively. Linearity was found to be was 6.25-37.5 µg/ml and 12.5-75 µg/ml for Rilpivirine and Dolutegravir respectively.

Niranjan Babu and Chandrasekar developed a stability indicating RP-HPLC method⁵ for the simultaneous estimation of Dolutegravir and Rilpivirine using Inertsil ODS C18 column. A mixture of 0.1% ortho phosphoric acid buffer and acetonitrile (60:40, v/v) was used as mobile phase with flow rate 1.0 ml/min (UV detection at 230 nm) where Rilpivirine and Dolutegravir were eluted at 2.167 min 4.3 and 3.4 min respectively. Linearity was found to be was 25-125 µg/ml and 50-250 µg/ml for Rilpivirine and Dolutegravir respectively.

Veeraswami and Naveen developed a RP-HPLC method⁶ for the simultaneous estimation of Dolutegravir and Rilpivirine rat plasma using Phenomenex C18 column. A mixture of 0.1% ortho phosphoric acid buffer and acetonitrile (60:40, v/v) was used as mobile phase with flow rate 1.0 ml/min (UV detection at 262 nm) where Rilpivirine and Dolutegravir were eluted at 7.73 min and 4.35 min respectively. Linearity was found to be was 5-75 µg/ml and 10-150 µg/ml for Rilpivirine and Dolutegravir respectively.

Sivagami et al., developed a stability indicating RP-HPLC method⁷ (Isocratic mode) for the simultaneous estimation of Rilpivirine and Dolutegravir in bulk and pharmaceutical dosage forms by using INERTSIL ODS column. A mixture of Phosphate buffer (pH 6.8): Acetonitrile (35: 65) was used as mobile phase with flow rate 1.0 ml/min (UV detection at 259 nm) where Rilpivirine and Dolutegravir were eluted at 3.285 min and 4.635 min respectively. Linearity was found to be was 50-250 µg/ml for Dolutegravir and 30-150 µg/ml for Rilpivirine.

Gopinath et al., developed a stability indicating RP-HPLC method⁸ (Isocratic mode) for the simultaneous estimation of Rilpivirine and Dolutegravir in bulk and pharmaceutical dosage forms by using a mixture of Acetonitrile: 0.1% aq. Tri ethyl amine (pH 2.5 adjusted with 0.1% ortho phosphoric acid) (40:60) with flow rate 1.0 ml/min (UV detection at 230 nm). Linearity was found to be was 10-150 µg/ml for Dolutegravir and 5-75 µg/ml for Rilpivirine.

Reddy et al., developed a stability indicating RP-HPLC method⁹ for the simultaneous estimation of Rilpivirine and Dolutegravir in bulk form using Hypersil ODS column. A mixture of methanol and water (80:20, v/v) was used as mobile phase with flow rate 1.0 ml/min (UV detection at 282 nm) where Rilpivirine and Dolutegravir were eluted at 5.14 min and 6.72 min respectively. Linearity was found to be was 10-100 µg/ml for Rilpivirine and Dolutegravir.

In the present study the authors have proposed a new stability indicating RP-HPLC method for the determination of Doultegravir, Rilpivirine, Rilpivirine Z Isomer and Doultegravir hydroxy impurity and the method was validated as per ICH guide lines.

MATERIALS AND METHODS:

Doultegravir, Rilpivirine, Rilpivirine Z Isomer and Doultegravir hydroxy impurity were obtained from Mylan Laboratories Ltd (India). HPLC grade Acetonitrile, Potassium dihydrogen ortho phosphate, Triethylamine and Ortho-phosphoric acid were obtained from Rankem Chemicals (India) (AR grade) and HPLC grade Milli Q water was used for the entire chromatographic study.

Weighing balance (Denver), pH meter (BVK enterprises, India), Ultra sonicator (Fast clean), UV-VIS spectrophotometer (Labindia TG 1800) and Water HPLC System (PDA detector) and auto sampler integrated with Empower 2 Software were used for the present study.

PROCEDURE

Preparation of phosphate buffer (0.01N) solution (pH 4.8)

1.36 gm of Potassium dihydrogen ortho phosphate was accurately weighed and transferred in to a 1000 ml volumetric flask and about 900 ml of milli-Q water was added, sonicated and finally the volume was made up with water with the addition of 1 ml of Triethylamine and the pH was adjusted to 4.8 with dil. ortho phosphoric acid solution. A mixture of water and acetonitrile (50:50) was used as the diluent.

Preparation of impurity stock solution

0.5 mg of Rilpivirine Z Isomer (B-158) and Dolutegravir Hydroxy impurity (D-166) are accurately weighed and transferred to a 10 ml volumetric flask and were makeup with the diluent gives 50 µg/ml solution of impurity.

Preparation of Standard stock solution

0.5 mg of Dolutegravir and 0.5 mg of Rilpivirine were weighed accurately and dissolved in a 10 ml volumetric flask with the diluent. The solution was sonicated for 5 min and the solution was make up with diluent to 10 ml which gives 50 µg/ml solution.

Preparation of Standard solution

0.2 ml of each of the above standard stock solutions of Dolutegravir & Rilpivirine and impurity stock were transferred to a 25 ml volumetric flask, sonicated for 5 min and the volume was made up to volume with the diluent.

Optimized chromatographic conditions

Water HPLC System (PDA detector) and auto sampler integrated with Empower 2 Software with Inertsil (250 × 4.6 mm, 5 μ) C18 column (PDA detector) was used for the present study. A mixture of 0.01N phosphate buffer and acetonitrile (50: 50, v/v) (pH adjusted to 4.8 with TEA and ortho phosphoric acid) was used as mobile phase for the chromatographic study (Flow rate: 1.0 ml/min; Injection volume: 10 μl; Detection wavelength: 257 nm) with run time 12 mins.

Method validation^10-12

Linearity

Linearity study was performed by preparing a series of solutions (0.25-1.5 µg/ml) consisting of Dolutegravir, Rilpivirine, Rilpivirine Z Isomer (B-158) and Dolutegravir hydroxy impurity (D-166) and there by injecting 10 µl of these solutions in to HPLC system and the mean peak area (n=3) of the chromatogram was noted. Calibration curves were drawn by plotting the concentration of Dolutegravir, Rilpivirine, Rilpivirine Z Isomer (B-158) and Dolutegravir hydroxy impurity (D-166) solutions on the x-axis and the corresponding mean peak area on the y-axis.

Precision study

Method precision were evaluated by spiking the Dolutegravir and Rilpivirine bulk drug formulation extracted solution with Rilpivirine Z Isomer (B-158) and Dolutegravir hydroxy impurity (D-166) and finally the % RSD was calculated.

0.5 mg of Impurity D-166, B-158 and Rilpivirine (A-153) were accurately weighed and transferred to a 10 ml volumetric flask and the volume was made up to volume with the diluent (50 µg/ml). Bulk drug formulation equivalent to 10 mg of Dolutegravir was weighed and transferred to a 10 ml volumetric flask and labelled as precision spiked. 0.2 ml of solution from the impurity stock solution was transferred to a 10 ml volumetric flask and labelled as precision spiked and made up to volume with diluent.

Accuracy study

0.5 mg of Impurity D-166, B-158 and Rilpivirine (A-153) were accurately weighed and transferred to a 10 ml volumetric flask and were made up to volume with the diluent which gives 50 µg/ml solution of impurity (Impurity stock solution).

Bulk drug formulation equivalent to 10 mg of Dolutegravir was weighed and transferred to three different 10 ml volumetric flasks and labelled as 50%, 100% and 150% spiked and 0.1, 0.2 and 0.3 ml of solution from impurity stock solution was transferred to the volumetric flask labelled and 50%, 100% and 150% spiked and the volume was made up to volume with the diluent.

Stress degradation studies

Stress degradation studies were carried out to study the stability of Dolutegravir, Rilpivirine, Rilpivirine Z Isomer (B-158) and Dolutegravir hydroxy impurity (D-166) and also to identify the degradation products.

Acidic degradation was performed by treating the mixture of Dolutegravir, Rilpivirine, Rilpivirine Z Isomer (B-158) and Dolutegravir hydroxy impurity (D-166) with 1N HCl for 2 hrs and then neutralized with NaOH solution and finally the volume was made up to volume with the diluent. Alkaline degradation was performed by treating the mixture of Dolutegravir, Rilpivirine, Rilpivirine Z Isomer (B-158) and Dolutegravir hydroxy impurity (D-166) with 1N NaOH solution for 2 hrs and then neutralized with HCl and finally the volume was made up to volume with the diluent. Oxidative degradation was performed by treating the mixture of Dolutegravir, Rilpivirine, Rilpivirine Z Isomer (B-158) and Dolutegravir hydroxy impurity (D-166) with 30% H₂O₂ for 2 hrs and finally the volume was made up to volume with the diluent. Thermal degradation was performed by heating the mixture of Dolutegravir, Rilpivirine, Rilpivirine Z Isomer (B-158) and Dolutegravir hydroxy impurity (D-166) to 50°C in a thermostat for 2 hrs and finally the volume was made up to volume with the diluent. Photolytic degradation was performed by treating the mixture of Dolutegravir, Rilpivirine, Rilpivirine Z Isomer (B-158) and Dolutegravir hydroxy impurity (D-166) to UV light (254 nm) for 2 hrs and finally the volume was made up to volume with the diluent. Hydrolysis degradation was performed by heating the mixture of Dolutegravir, Rilpivirine, Rilpivirine Z Isomer (B-158) and Dolutegravir hydroxy impurity (D-166) to 60°C in a thermostat for 2 hrs and finally the volume was made up to volume with the diluent. After degradation treatments the samples were cooled to room temperature, diluted with the diluent and injected in to the HPLC system for the chromatographic analysis.

RESULTS AND DISCUSSION:

The authors have proposed a new validated stability indicating RP-HPLC method for the quantification of Doultegravir and Rilpivirine related impurities Rilpivirine Z Isomer and Doultegravir hydroxy impurity using Water HPLC System (PDA detector) and auto sampler integrated with Empower 2 Software with Inertsil (250 × 4.6 mm, 5 μ) C18 column (PDA detector) was used for the present study. A mixture of 0.01N phosphate buffer and acetonitrile (50: 50, v/v) (pH adjusted to 4.8 with TEA and ortho phosphoric acid) was used as mobile phase for the chromatographic study (Flow rate: 1.0 ml/min; Injection volume: 10 μl; Detection wavelength: 257 nm) with run time 12 mins. The analytical techniques so developed till today for the simultaneous estimation of Doultegravir and Rilpivirine and its impurities were summarised in Table 1.

Method validation

Linearity, Precision, Accuracy and Robustness

Linearity study was performed by preparing a series of solutions (0.25-1.5 µg/ml) of Dolutegravir, Rilpivirine, Rilpivirine Z Isomer (B-158) and Dolutegravir hydroxy impurity (D-166) and there by injecting 10 µl of these solutions in to HPLC system and the mean peak area (n=3) of the chromatograms was noted (Table 2). The representative chromatograms of blank, placebo, Dolutegravir, Rilpivirine, Rilpivirine Z Isomer (B-158) and Dolutegravir hydroxy impurity (D-166) were shown in Figure 3. The LOD and LOQ (Figure 3) obtained for Dolutegravir, Rilpivirine, Rilpivirine Z-isomer (B-158) and Dolutegravir hydroxy impurity (D-166) were shown in Table 3.

Table 1: Literature survey

Mobile phase (v/v) / Detection wavelength (nm)	Method / Column	Linearity (µg/ml)	Ref
0.1% ortho phosphoric acid buffer: Acetonitrile (60:40) / 260	RP-HPLC Kromasil C 18	6.25-37.5 (RLP) 12.5-75 (DLT)	4
0.1% ortho phosphoric acid buffer: Acetonitrile (60:40) / 230	RP-HPLC Inertsil ODS C18	25-125 (RLP) 50-250 (DLT)	5
0.1% ortho phosphoric acid buffer: Acetonitrile (60:40) / 262	RP-HPLC Phenomenex C18	5-75 (RLP) 10-150 (DLT)	6
Phosphate buffer (pH 6.8): Acetonitrile (35: 65) / 259	RP-HPLC Inertsil ODS C18	30-150 (RLP) 50-250 (DLT)	7
Acetonitrile: 0.1% aq. Tri ethyl amine (pH 2.5 adjusted with 0.1% ortho phosphoric acid) (40:60) / 230	RP-HPLC Inertsil ODS C18	5-75 (RLP) 10-150 (DLT)	8
Methanol: Water (80:20) / 282	RP-HPLC Hypersil ODS C18	10-100 (RLP) 10-100 (DLT)	9
Phosphate buffer: Acetonitrile (50: 50) (pH adjusted to 4.8 with TEA and ortho phosphoric acid)	RP-HPLC Inersil C18	0.25-1.50 (Impurities)	Present method

Table 2: Linearity

Conc. (µg/ml)	*Average peak area
Conc. (µg/ml)	Dolutegravir	Rilpivirine	B 158	D 166
0.25	231502	323328	352587	253807
0.5	438092	541240	589126	448220
0.75	634509	786979	830781	633836
1	817579	994860	1056498	807674
1.25	1008140	1260766	1319014	1010421
1.5	1178868	1503297	1565092	1201497

*Mean of three replicates

A) Blank

B) Placebo

C) Rilpivirine (Rt: 4.107 min); Rilpivirine Z Isomer (B-158) (Rt: 3.254 min); Doultegravir (Rt: 5.699 min) and Dolutegravir hydroxy impurity (D-166) (Rt: 2.691 min)

Figure 2: Representative chromatograms of A) Blank, B) Placebo C) Rilpivirine, Rilpivirine Z Isomer, Doultegravir and Dolutegravir hydroxy impurity

Figure 3A: Representative chromatogram for LOD solution

Figure 3B: Representative chromatogram for LOQ solution

Table 3: LOD and LOQ

Conc. (µg/ml)

Rilpivirine

Dolutegravir

Dolutegravir hydroxy impurity

(D 166)

Rilpivirine Z Isomer (B 158)

LOD

0.01

0.03

LOQ

0.04

0.05

0.06

0.12

Calibration curves were drawn by taking the concentration of Dolutegravir, Rilpivirine, Rilpivirine Z Isomer (B-158) and Dolutegravir hydroxy impurity (D-166) on the x axis and the corresponding average peak area on the y axis (Figure 4). The precision results of Dolutegravir, Rilpivirine, Rilpivirine Z Isomer (B-158) and Dolutegravir hydroxy impurity (D-166) were shown in Table 4 and that of the accuracy results were shown in Table 5.

Doultegravir

Rilpivirine

Rilpivirine Z Isomer (B 158)

Dolutegravir hydroxy impurity (D 166)

Figure 4: Calibration curves

Table 4: Precision study

S. No	Rilpivirine	Dolutegravir	D 166	B 153
1	10087602	8289263	043915	816264
2	10106403	8218869	1064937	811897
3	10182792	8304543	1052683	811525
4	10114569	854580	1039296	818826
5	10122678	8419863	1062721	816369
6	10103806	8292045	1064134	806079
Mean	1011964	8345064	1054614	813493
Std. Dev.	3309.3	117798.9	11097.4	4599.5
% RSD	0.3	1.4	1.1	0.6

Table 5: Accuracy study

Spike level (%)	Rilpivirine Z Isomer (B 158)				Dolutegravir hydroxy impurity (D 166)
	Conc. (µg/ml)		Recovery (%)	Mean (%)	Conc. (µg/mL)		Recovery (%)	Mean (%)
	Added	Recovered	Recovery (%)	Mean (%)	Added	Recovered	Recovery (%)	Mean (%)
50	48	49	99.38	98.61	49	49	99.51	100.68
	48	48	98.41		49	50	101.48
	48	48	98.04		49	50	101.05
100	97	97	99.26	99.04	98	98	99.88	99.67
	97	97	98.79		98	98	99.36
	97	97	99.08		98	98	99.79
150	146	147	100.20	100.26	147	148	100.38	100.03
	146	146	99.63		147	147	99.56
	146	148	100.96		147	148	100.15

Table 6: Robustness study

Rilpivirine
Flow rate (± 0.1 ml/min)	USP plate count	USP Tailing	Organic Phase (± 5%)	USP Plate Count	USP Tailing	Column oven temp (°C)	USP plate count	USP Tailing
0.9	10277	1.43	45	9486	1.45	28	10644	1.43
1.1	9826	1.48	55	9953	1.44	32	9659	1.44
Dolutegravir
0.9	19752	1.32	45	19277	1.33	28	19277	1.33
1.1	20941	1.33	55	19752	1.37	32	20524	1.33
Rilpivirine Z Isomer (B 158)
0.9	9509	1.40	45	8912	1.37	28	10869	1.45
1.1	9689	1.49	55	10905	1.43	32	10985	1.46
Dolutegravir hydroxy impurity (D 166)
0.9	7051	1.44	45	7.52	1.47	28	7690	1.46
1.1	7246	1.52	55	6985	1.52	32	7853	1.44

Stress degradation studies

The stability studies were performed for Dolutegravir, Rilpivirine, Rilpivirine Z Isomer (B 158) and Dolutegravir hydroxy impurity (D 166) and the corresponding chromatograms were represented in Figure 5.

During the acidic degradation Rilpivirine and Dolutegravir were eluted at 4.021 min and 5.693 min respectively with an extra peak at 2.513 min. The theoretical plates were found to be 10321 and 20409 for Rilpivirine and Dolutegravir (>2000) respectively with resolution values 12.6 and 9.6 (>2) which are within the acceptable criteria. During the alkaline degradation Rilpivirine and Dolutegravir were eluted at 4.037 min and 5.682 min respectively with an extra peak at 2.480 min. The theoretical plates were found to be 10245 and 22410 for Rilpivirine and Dolutegravir (>2000) respectively with resolution values 12.3 and 9.8 (>2) which are within the acceptable criteria. During the oxidative degradation Rilpivirine and Dolutegravir were eluted at 4.042 min and 5.634 min respectively with an extra peak at 2.635 min and 3.067 min. The theoretical plates were found to be 10459 and 21693 for Rilpivirine and Dolutegravir (>2000) respectively with resolution values 2.9, 8.3 and 10.1 (>2) which are within the acceptable criteria. During the thermal degradation Rilpivirine and Dolutegravir were eluted at 4.053 min and 5.660 min respectively. The theoretical plates were found to be 10382 and 20326 for Rilpivirine and Dolutegravir (>2000) respectively which are within the acceptable criteria. During the UV degradation Rilpivirine and Dolutegravir were eluted at 4.048 min and 5.652 min respectively. The theoretical plates were found to be 10515 and 18593 for Rilpivirine and Dolutegravir (>2000) respectively with tailing factor 1.3 (<2.0) which are within the acceptable criteria. During the hydrolysis degradation Rilpivirine and Dolutegravir were eluted at 4.059 min and 5.640 min respectively. The theoretical plates were found to be 10552 and 18547 for Rilpivirine and Dolutegravir (>2000) respectively with tailing factor 1.3 (<2.0) which are within the acceptable criteria.

Acidic degradation

Basic degradation

Oxidative degradation

Thermal degradation

UV degradation

Hydrolysis degradation

Figure 5: Representative chromatograms of Rilpivirine and Doultegravir during stress degradation studies

Table 7: Stress degradation studies

Degradation condition	% Content (% degradation)		Peak purity for Rilpivirine		Peak purity for Dolutegravir
Degradation condition	Rilpivirine	Dolutegravir	Purity angle	Purity threshold	Purity angle	Purity threshold
Control	100.0	100.0	0.537	0.736	0.460	0.493
Acidic degradation (1N HCl / 2 hrs)	94.91 (5.09)	95.49	0.432	0.783	0.361	0.370
Basic degradation (1N NaOH / 2 hrs)	95.54 (4.46)	95.96	0.419	0.749	0.333	0.366
Oxidative degradation (30% H₂O₂ / 2 hrs)	94.71 (5.29)	94.74	0.498	0.681	0.460	0.493
Thermal degradation (50°C / 2 hrs)	97.39 (2.61)	96.96	0.532	0.863	0.388	0.439
UV degradation (254 nm / 2 hrs)	98.29	98.02	0.539	0.881	0.441	0.483
Hydrolysis degradation (60°C / 2 hrs)	99.54	99.15	0.537	0.736	0.460	0.493

CONCLUSION:

The current RP-HPLC method is specific, precise, accurate, linear, stable and robust for the quantification of Dolutegravir and Rilpivirine related impurities Rilpivirine Z Isomer (B 158) and Dolutegravir hydroxy impurity (D 166) in Doultegravir and Rilpivirine bulk drug formulations. The developed method can be applied successfully to quality control and for other analytical purposes.

REFERENCES:

1. Blair HA. Dolutegravir/Rilpivirine: A review in HIV-1 infection. Drugs. 2018;78(16): 1741-1750.

2. Grégoire M, Deslandes G, Renaud C, Bouquié R, Allavena C, Raffi F, Jolliet P and E. Dailly. A liquid chromatography-tandem mass spectrometry assay for quantification of Rilpivirine and Dolutegravir in human plasma. Journal of Chromatography B. 2014; 971: 1-9.

3. Zheng Y, Aboura R, Boujaafar S, Lui G, Hirt D, Bouazza N, Foissac F, Treluyer JM, Benaboud S and Gana I. HPLC-MS/MS method for the simultaneous quantification of Dolutegravir, Elvitegravir, Rilpivirine, Darunavir, Ritonavir, Raltegravir and Raltegravir-β-d-glucuronide in human plasma. J Pharm Biomed Anal. 2020; 182: 113119.

4. Wahab S, Khalid M, Ahmad S and Sweilam SH. Rilpivirine and Dolutegravir simultaneously measured via RP-HPLC-PDA with Box-Behnken design application: A study of forced degradation under various conditions. Separations. 2023; 10(3):185.

5. Niranjan Babu M and Chandrasekar R. Development and validation of stability indicating RP-HPLC method for the simultaneous estimation of Dolutegravir and Rilpivirine by forced degradation studies. International Journal of Pharmaceutical Sciences and Research. 2021; 12(9): 4954-4963.

6. Veeraswami B and Naveen VMK. Development and validation of RP-HPLC method for the estimation of Dolutegravir and Rilpivirine in bulk and pharmaceutical dosage form and its application to rat plasma. Asian J Pharm Clin Res. 2019; 12(2): 267-271.

7. Sivagami B, Sharmil Kumar LM, Chandrasekar. R, Niranjan Babu. M. Development and validation for the simultaneous estimation of Rilpivirine and Dolutegravir in bulk and pharmaceutical dosage forms by RP-HPLC Method. Research Journal of Pharmacy and Technology. 2022; 15(11): 5302-5306.

8. Gopinath K, Padmavathi K V, Murali Krishna N, & Subbarao M. Validated stability indicating RP-HPLC method for the determination of Dolutegravir and Rilpivirine in bulk and pharmaceutical dosage forms. International Journal of Research in Pharmaceutical Sciences. 2021; 12(3): 1961-1966.

9. Reddy KS, Shirisha SS and Kumar KP. Validated stability indicating RP-HPLC method for the simultaneous estimation of Rilpivirine and Dolutegravir in bulk form. International Journal of Pharmaceutical Sciences and Research. 2020; 11(10): 4991-4997.

10. ICH Validation of analytical procedures: Text and methodology Q2 (R1), International Conference on Harmonization (2005).

11. ICH Stability testing of new drug substances and products Q1A (R2), International Conference on Harmonization (2003).

12. ICH Q3A (R2) Impurities of new drug substances (2006).

Received on 29.10.2023 Modified on 30.11.2023

Research J. Pharm. and Tech 2024; 17(2):595-602.

DOI: 10.52711/0974-360X.2024.00093