INTRODUCTION:

Rilpivirine (Figure 1) is a non-nucleoside reverse transcriptase inhibitor^2-3. It is chemically 4-[[4-[[4-[(E)-2-cyanoethenyl]-2, 6-dimethylphenyl] amino]-2-pyrimidinyl] amino] benzonitrile. Rilpivirine (C₂₂H₁₈N₆) (Mo. wt. 364.417 g/mol) is used to treat infections caused by human immuno deficiency virus¹. It acts by binding directly to reverse transcriptase enzyme non-competitively by which conformational changes occurs and the reverse transcriptase functions are altered.

Figure 1: Chemical structure Rilpivirine

Rilpivirine was earlier estimated using various analytical techniques such as LC-MS, LC-MS/MS, HPTLC, HPLC, and spectrophotometry in biological fluids as well as dosage forms.

Ajay Gupta² et al., developed LC-MS/MS assay for the estimation of Rilpivirine in human plasma using Gemini C18 column in presence of an internal standard, Rilpivirine-d6 and the linearity was 5-200 ng/ml.

Chilukuri³ et al., developed LC-MS method on gradient mode for the estimation of Rilpivirine and 6 impurities using Shim-pack XR-ODS-II column with a mixture of 0.1 M Ammonium acetate (pH adjusted to 4.0 with acetic acid): Acetonitrile as mobile phase in the ratio 50: 50, v/v with flow rate 0.5 ml/min (Detection wavelength 295 nm).

Shibata⁴ et al., developed LC-MS method on gradient mode for the estimation of Rilpivirine

in human plasma using Sunfire C18 column using a mixture of 0.1 mM EDTA in acetic acid: Acetonitrile: Methanol (Gradient mode) in presence of an internal standard, 6,7-dimethyl-2,3-dipyridyl) quinoxaline and the linearity was 18-715 ng/ml.

Raju and Appala Raju⁵ developed a LC-MS/MS method for the estimation of Rilpivirine in sprague dawley rat serum using Discovery C18 column in presence of an internal standard, Didanosine and the linearity was 2-1000 ng/ml. A mixture of Acetonitrile: Methanol: 0.1% Acetic acid in 5 mM Ammonium acetate was used as a mobile phase in the ratio 20:25:55, v/v with flow rate 0.6 ml/min.

Laxminarayana⁶et al., developed a LC-MS method for the estimation of Rilpivirine in human plasma using C18 column using a mixture of Acetonitrile and 0.1% formic acid buffer as a mobile phase in the ratio 80:20, v/v with flow rate 0.5 ml/min. and the linearity was 51-200 ng/ml.

Rilpivirine was also estimated by using UFLC⁷, HPLC^8-13 and HPTLC¹⁴ in biological fluids as well as dosage forms with different columns and mobile phases. In the present study the authors have proposed a new stability indicating RP-UPLC method for the determination of Rilpivirine and its N-Oxide impurity and the method was validated as per ICH guide lines.

MATERIALS AND METHODS:

Rilpivirine Hydrochloride and N-Oxide Impurity were obtained from Mylan Laboratories Ltd (India). HPLC grade Methanol, Acetonitrile, Potassium dihydrogen ortho phosphate (AR grade), Triethylamine (AR grade) were obtained from Rankem Chemicals (India). Ortho-phosphoric acid, sodium dihydrogen Ortho phosphate of AR grade were obtained from Avantor performance material India Pvt. Ltd and HPLC grade Milli Q water was used for the entire study.

Electronics Balance (Denver), Digital pH meter 7007 (Digisun Electronics, Hyderabad), Ultrasonicator (Labman), Vacuum pump (Crompton), Hot Air Oven (Servewell Instrument PVTLTD, Bangalore), UV-VIS spectrophotometer (PG Instruments T60) integrated with UV win 6 Software and WATERS UPLC Aqcuity SYSTEM with TUV detector integrated with Empower 2 Software were used for the study.

Procedure

Preparation of Rilpivirine stock & impurity N-Oxide solution

A mixture of water and Acetonitrile (50:50) was used as the diluent.10 mg of Rilpivirine drug is weighed accurately and transferred to 10 ml volumetric flask and dissolved in a small amount of diluent, sonicated for 5 min and the solution was made up with the diluent to 10 ml (1000 µg/ml) solution. 0.5 mg of N-Oxide Impurity was accurately weighed and transferred to a 10 ml volumetric flask and made up with the diluent that gives 500 µg/ml solution of impurity. 0.2 ml of each of the above solutions and transferred to another 10 ml volumetric flask and was made up to volume with the diluent to 10 ml to prepare standard solution.

Optimized chromatographic conditions

A mixture of 0.1% OPA: Methanol in the ratio of 50:50 (v/v) was used as the mobile phase for the chromatographic study (Flow rate: 1.0 ml/min; Detection wavelength: 279 nm). Injection volume: 10 µl). Waters UPLC Aqcuity SYSTEM with TUV detector integrated with Empower 2 Software and Kromasil C18 column (250 × 4.6 mm, 5μm) was used for the present study and the column oven temperature was 30 °C (Run time was 12 min).

Method validation¹⁵

Linearity

Linearity study was performed by preparing a series of solutions (0.25-1.5 µg/ml) of Rilpivirine and its N-Oxide impurity and there by injecting 10 µl of these solutions in to UPLC system and the mean peak area (n=3) was noted. Calibration curves were drawn by plotting the concentration of Rilpivirine and its N-Oxide impurity solutions on the x-axis and the corresponding mean peak area on the y-axis.

Precision study

Method precision and intermediate precision were evaluated by spiking the Rilpivirine tablet extracted solution with the N-Oxide impurity and thereby calculating the % RSD.

For precision study 0.5 mg of Impurity N-Oxide was accurately weighed and transferred to a 10 ml volumetric flask and made up with the diluent which gives 50 µg/ml solution of impurity (Impurity stock solution).

Tablet powder equivalent to 10 mg of Rilpivirine is weighed and transferred to a 10 ml volumetric flask and labelled as precision spiked. 0.2 ml of solution from impurity stock was transferred to the volumetric flask and labelled as precision spiked and made up to volume with the diluent (Spiking impurity).

Accuracy study

For accuracy study 0.5 mg of Impurity N-Oxide was accurately weighed and transferred to a 10 ml volumetric flask and made up with the diluent which gives 50 µg/ml solution of impurity. The accuracy of the method was proved by spiking the solutions with 50%, 100% and 150% level and the % recovery was calculated.

Tablet powder equivalent to 10 mg of Rilpivirine 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 the impurity stock was transferred to three volumetric flasks and labelled as 50%, 100% and 150% spiked and made up to 10 ml with the diluent.

Stress degradation studies^16-17

Stress degradation studies were carried out to study the stability of Rilpivirine and N-Oxide impurity and also to identify the degradation products. Acidic degradation was performed by treating the mixture of Rilpivirine and N-Oxide impurity 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 Rilpivirine and N-Oxide impurity 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 Rilpivirine and N-Oxide impurity 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 Rilpivirine and N-Oxide impurity 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 Rilpivirine and N-Oxide impurity to UV light (254 nm) for 2 hrs and finally the volume was made up to volume with the diluent. Water degradation was performed by heating the mixture of Rilpivirine and N-Oxide impurity 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 UPLC system for the chromatographic analysis.

RESULTS AND DISCUSSION:

The authors have developed a new validated stability indicating RP-UPLC method for the estimation of Rilpivirine and its N-Oxide impurity using WATERS UPLC Aqcuity SYSTEM with TUV detector integrated with Empower 2 Software equipped with Kromasil C18 column (250 × 4.6 mm, 5μm) (Column oven temperature: 30 °C) within a run time of 12 min (UV detection 279 nm). A mixture of 0.1% OPA: Methanol in the ratio of 50:50 (v/v) was used as the mobile phase for the chromatographic study with flow rate 1.0 ml/min. The analytical methods so far developed for the estimation of Rilp[ivirine were summarised in Table 1.

Method validation (Rilpivirine and N-Oxide impurity)

Linearity, Precision, Accuracy and Robustness

Linearity study was performed by preparing a series of solutions (0.25-1.5 µg/ml) of Rilpivirine and its N-Oxide impurity and there by injecting 10 µl of these solutions in to UPLC system and the mean peak area (n=3) was noted. The LOD and LOQ were found to be 9.130 µg/ml and 27.66 µg/ml for Rilpivirine and 0.27 µg/ml and 0.82 µg/ml for N-Oxide impurity respectively (Figure 2). The representative chromatograms of Rilpivirine and N-Oxide impurity were shown in Figure 3. A calibration graph was drawn by taking the concentration of Rilpivirine and N-Oxide impurity on the x axis and the corresponding average peak area on the y axis (Figure 4). The method precision and intermediate precision results of Rilpivirine and N-Oxide impurity were shown in Table 3 and that of the accuracy results were shown in Table 4.

Table 1: Literature review of liquid chromatographic methods

Mobile phase (v/v)	Method / Column	Linearity (µg/ml)	Ref
Rilpivirine-d6 (Internal standard)	LC-MS/MS / Gemini C18 (Human plasma)	(0.005-0.2)	2
0.1 M Ammonium acetate (pH adjusted to 4.0 with acetic acid): Acetonitrile (Gradient mode)	LC-MS (6 Impurities) Shim-pack XR-ODS-II	-	3
0.1 mM EDTA in acetic acid: Acetonitrile: Methanol (Gradient mode) 6,7-dimethyl-2,3-dipyridyl) quinoxaline (Internal standard)	LC-MS / Sunfire C18 (Human plasma)	(0.018-0.715)	4
Acetonitrile: Methanol: 0.1% Acetic acid in 5 mM Ammonium acetate (20:25:55) Didanosine (Internal standard)	LC-MS/MS / Discovery C18 (Sprague dawley rat serum)	(0.002-1.0)	5
Acetonitrile and 0.1% formic acid buffer (80:20)	LC-MS / C18 (Human plasma)	(0.051-0.2)	6
10 mM tetra butyl ammonium hydrogen sulphate: Acetonitrile (55: 45)	UFLC / Phenomenex C8	0.05-40	7
Acetonitrile: Phosphate buffer (pH 3.5) (60:40)	C8	HPLC 10-50	8
Acetonitrile: Acetate buffer (pH 4.0) (65 :35)	HPLC / Develosil ODS HG-5 RP C18	0-25	9
0.03M di potassium hydrogen phosphate (pH adjusted to 2.5 using dilute ortho- phosphoric acid) (Solvent A): Acetonitrile (Solvent B) (15: 85)	HPL Zorbax Eclipse XDB-C18	100-300	10
Acetonitrile: Potassium dihydrogen phosphate buffer (pH 2.8) with ortho phosphoric acid (40:60)	HPLC Develosil ODS HG-5 RP C18	0-30	11
0.1% Ortho phosphoric acid: Acetonitrile (65:35)	HPLC Develosil ODS HG-5 RP C18	20-70	12
Acetonitrile: 25 mM potassium dihydrogen phosphate (50:50)	HPLC / Gemini RP C18	0.025-2.0	13
Phosphate buffer (pH 6.8): Acetonitrile (60:40) Ethyl acetate: methanol: chloroform (80: 10: 10)	HPLC and HPTLC	1-10 (HPLC) 5-30 µg/spot (HPTLC)	14
0.1% OPA: Methanol (50:50)	RP-UPLC / Kromasil C18	0.25-1.25	Present method

Figure 2A: Chromatogram for LOD solution

Figure 2B: Chromatogram for LOQ solution

Table 4: Linearity of Rilpivirine and its N-Oxide impurity

Conc (µg/ml)	*Average peak area
Conc (µg/ml)	Rilpivirine	N-Oxide impurity
0.25	40700	20280
0.5	80549	40877
0.75	123099	61522
1	161596	80876
1.25	207872	104319
1.5	240913	121316

*Mean of three replicates

A-Blank

B-Placebo

C-Rilpivirine (Rt: 5.178 min) and N-Oxide impurity (Rt: 6.657 min)

Figure 3: Representative chromatograms of A) Blank, B) Placebo and Rilpivirine and its N-Oxide impurity

Calibration curve of Rilpivirine

Calibration curve of N-Oxide

Figure 4: Calibration curves of Rilpivirine and N-Oxide impurity

Table 3: Precision study

S. No	Rilpivirine			N-Oxide
	Method Precision	Intermediate Precision		Method Precision	Intermediate Precision
1	9715543	9715423		812435	813435
2	9708008	9708118		801973	801983
3	9622050	9622052		819608	819618
4	9723188	9723382		803864	803860
5	9647969	9647974		813135	813139
6	9636178	9636281		814583	814565
Statistical analysis: Mean peak area ± SD (% RSD)
	9675489 ± 44934.3 (0.5)		9675538 ± 44950.99 (0.46)	810933 ± 6722.1 (0.8)		811100 ± 6778.04 (0.83)
Plate Count	3746		4170	3728		3629
Tailing Factor	1.47		1.39	1.26		1.26
Resolution	--		--	3.7		3.8

Table 4: Accuracy study

Spike level	N-Oxide impurity
	Conc. drug (µg/ml)		Recovery (%)	Mean (%)
	Added	Recovered	Recovery (%)	Mean (%)
50 %	49	49	99.79	100.86
	49	50	101.45
	49	50	101.34
100 %	98	100	101.33	100.70
	98	99	100.85
	98	98	99.91
150 %	147	148	99.94	99.89
	148	144	97.42
	148	151	102.31

Stress degradation studies

The stability studies were performed for Rilpivirine and its N-Oxide impurity and the corresponding chromatograms were represented in Figure 5.

During the acidic degradation Rilpivirine was eluted at 5.966 min and N-Oxide impurity was eluted at 6.584 min with an extra degradant peak at 2.313 min. The theoretical plates were found to be 7220 and 4408 (>2000) for Rilpivirine and N-Oxide respectively with resolution values 15.3 and 4.2 (<2) which are within the acceptable criteria. During the alkaline degradation Rilpivirine was eluted at 5.525 min and N-Oxide impurity was eluted at 7.203 min. The theoretical plates were found to be 7320 and 4933 (>2000) for Rilpivirine and N-Oxide respectively with resolution 4.4 (<2) which are within the acceptable criteria. During the oxidative degradation Rilpivirine was eluted at 5.163 min and N-Oxide impurity was eluted at 6.758 min. The theoretical plates were found to be 3709 and 3148 (>2000) for Rilpivirine and N-Oxide respectively with resolution 3.5 (<2) which are within the acceptable criteria. During the thermal degradation Rilpivirine was eluted at 5.440 min and N-Oxide impurity was eluted at 7.104 min. The theoretical plates were found to be 3705 and 3139 (>2000) for Rilpivirine and N-Oxide respectively with resolution 3.6 (<2) which are within the acceptable criteria. During the UV degradation Rilpivirine was eluted at 5.364 min and N-Oxide impurity was eluted at 6.997 min. The theoretical plates were found to be 3699 and 2954 (>2000) for Rilpivirine and N-Oxide respectively with resolution 3.6 (<2) which are within the acceptable criteria. During the water degradation Rilpivirine was eluted at 5.265 min and N-Oxide impurity was eluted at 6.836 min. The theoretical plates were found to be 3408 and 2912 (>2000) for Rilpivirine and N-Oxide respectively with resolution 3.6 (<2) which are within the acceptable criteria.

Acidic degradation of Rilpivirine and N-Oxide

Basic degradation of Rilpivirine and N-Oxide

Oxidative degradation of Rilpivirine and N-Oxide

Thermal degradation of Rilpivirine and N-Oxide

UV degradation of Rilpivirine and N-Oxide

Water degradation of Rilpivirine and N-Oxide

Figure 5: Chromatograms of Rilpivirine and N-Oxide during stress degradation studies

Table 5: Robustness Study

Rilpivirine
Flow Rate (ml/min) (± 0.1)	USP Plate Count	USP Tailing	Organic phase (± 10%)	USP Plate Count	USP Tailing	Column oven temp (± 30°C)	USP Plate Count	USP Tailing
0.9	3668	1.74	40	5101	1.64	28°C	4642	1.83
1.1	5422	2.01	60	2743	1.98	32°C	4135	2.04
N-Oxide
0.9	3335	1.54	40	4645	1.17	28°C	4227	1.64
1.1	4324	1.89	60	3326	1.97	32°C	3587	1.95

Table 6: Stress degradation studies

Degradation condition	% Content			Peak purity Rilpivirine		Peak purity N-Oxide
Degradation condition	Rilpivirine	Rilpivirine % degradation	N-Oxide	Purity angle	Purity threshold	Purity angle	Purity threshold
Control	100.00	-	0.05	0.697	0.998	1.135	1.347
Acidic degradation (1N HCl / 2 hrs)	99.88	0.12	0.04	0.650	0.907	1.023	1.380
Basic degradation (1N NaOH / 2 hrs)	93.69	6.31	0.10	0.781	1.047	1.201	1.480
Oxidative degradation (30% H₂O₂ / 2 hrs)	98.21	1.79	0.14	0.659	0.914	1.084	1.416
Thermal degradation (50°C / 2 hrs)	99.34	0.66	0.17	0.693	0.920	1.110	1.377
UV degradation (254 nm / 2 hrs)	96.17	3.83	0.10	0.607	0.853	0.999	1.292
Water degradation (60°C / 2 hrs)	98.76	1.24	0.04	0.560	0.794	0.943	1.200

CONCLUSION:

A simple, precise, robust and accurate RP-UPLC method has been developed and validated as per ICH guidelines and the method can be applied for the Related Substances of any pharmaceutical formulations. The percentage decomposition in all the degradations was found to be less than 7% indicating that Rilpivirine is highly resistant towards all degradation conditions.

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Received on 22.08.2023 Modified on 26.11.2023

Research J. Pharm. and Tech 2023; 16(12):6110-6116.

DOI: 10.52711/0974-360X.2023.00992