INTRODUCTION:

Rilipivirine, a diarylpyrimidine non nucleoside- reverse transcriptase inhibitor. Non-nucleoside reverse-transcriptase inhibitors ^1-2 are antiretroviral drugs used in the treatment of human immunodeficiency virus (HIV). HIV-1 non-nucleoside reverse transcriptase inhibitors ³ (NNRTIs) nowadays represent most promising anti-AIDS drugs that specifically inhibit HIV-1 reverse transcriptase. Rilpivirine ^4-6 is recently approved Anti-AIDS drug which blocks the chemical step of DNA synthesis by binding to reverse transcriptase , an enzyme that controls the replication of the genetic material of HIV. It is a used in the treatment of HIV infections and activity is mediated by non competitive inhibition of HIV-1 reverse transcriptase. Chemically ⁷ it is 4-{[4-({4-[(E)-2-cyanovinyl]-2, 6-Dimethyl phenyl} amino) pyrimidin-2- yl] amino} benzonitrile.

Figure: 1 Chemical Structure of Rilpivirine

MATERIAL AND METHODS:

Reagents and Chemicals:

Rilpivirine was obtained as gift sample from Hetero Drugs in Hyderabad and marketed product Edurant 25 mg was purchased from local market. Acetonitrile, Water, were obtained from Merck. Mumbai and Potassium dihydrogen ortho phosphate, Ortho Phosphoric Acid obtained from RANKEM Mumbai. All solvents used in this work are HPLC grade.

Standard solutions and Chromatographic conditions

Preparation of Buffer pH 6:

Transfer 6.8gm of Potassium dihydrogen ortho phosphate in to a 1000ml beaker add about 800ml of milli-Q water and sonicate and make up to the final volume.

Preparation of working standard stock solution:

Accurately Weighed and transferred 10 mg of Rilpivirine hydrochloride working Standard into a 10 ml clean dry volumetric flask, and 7 ml of (HPLC grade Water and ACN(50:50)) diluent added, sonicated for 5 minutes and make up to the final volume with diluent to get a concentration of 1gm/ml(standard stock).

Sample preparation of Rilpivirine in tablet dosage form:

5 tablets of rilpivirine (EDURANT- 25mg) were weighed and crushed into powder and transferred into a 100 ml of volumetric flask. 70mL of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered. From the filtered solution 0.8ml was pipeted out into a 10ml volumetric flask and make up the volume with diluents.

HPLC INSTRUMENTATION AND CONDITIONS:

Instrument:

The chromatographic separations were performed with Waters 2695 seperation module, an inbuilt auto sampler and waters 2996 Photodiode Array Detector employed for detection of the analytes. The column used was Kromosil C18, 150 x 4.6 mm, 5m. A digisum DI 707 digital pH meter used to measure the pH of the buffer. The Empower 2 software was employed for LC peak integration along with data acquisition and data processing. Ultrasonic bath was used for sonication and degassing of mobile phase.

Optimized Chromatographic conditions:

Chromatography was performed by using Phosphate Buffer: Methanol: Acetonitrile in the ration of 45:5:55 (v/v/v) as mobile phase at 1.2 ml/min flow rate. The mobile phase was prepared freshly and it was filtered through membrane filter (0.45μm) and degassed with ultra sonicator. The pump pressure was maintained at 1500-2000 psi at an ambient temperature (30^oC). Samples were analyzed at 280 nm at an injection volume of 10 μL and separation was carried by using Kromosil C-18, (150 x 4.6 mm, 5m) and retention time for Rilpivirine was found to be 3.31 min.

The standard chromatogram of rilpivirine is shown in figure 2 and optimized chromatographic conditions are given in Table 1

Assay of Rilpivirine in Tablet:

Assay of marketed product was carried out by applying the developed method. Sample solutions were prepared and injected into HPLC system. The sample solution was scanned at 280 nm. The % drug estimated was found to be 99.86. A single peak was observed with retention time 4.69. The chromatogram of rilpivirine in formulation is shown in figure 3.

Table 1: Optimized chromatographic conditions

S.NO	PARAMETER	OPTIMIZED CONDITION
1	Instrument	Waters 2695 Separation module RP-HPLC
2	Column	Kromosil C-18, 150 x 4.6 mm, 5m.
3	Mobile phase	Phosphate Buffer: Methanol: Acetonitrile in the ratio of 45:5:55 (v/v/v)
4	Flow Rate	1.2 ml/ min
5	Detector	PDA Detector at 280 nm
6	Injection volume	10µl
7	Temperature	30 ⁰C
8	Retention Time	4.74 min

Figure 2: RP-HPLC Chromatogram of Rilpivirine

Figure 3: Rilpivirine chromatogram in Tablet dosage form

Table 2: % Recovery data of Rilpivirine

S.No	Concentration	% Recovery	Amount added (µg/ml)	Amount found (µg/ml)	Mean %Recovery ± S.D (n=3)	%RSD
1	50%	100.02	50	50.01	99.93±0.35	0.356
		99.55	50	49.77
		100.24	50	50.12
2	100%	99.28	100	99.28	98.98±0.30	0.302
		98.99	100	98.99
		98.68	100	98.68
3	150%	98.77	150	148.15	98.95±0.16	0.164
		99.02	150	148.53
		99.08	150	148.62

RESULTS AND DISCUSSIONS:

Method Validation:

Accuracy:

The assay performed by preparing three standard drug solution concentration levels 50%, 100%, and 150 % and injected. It was repeated over three consecutive days to obtain intermediate precision data. Good recovery of the spiked drug was obtained at each added concentration, and the mean percentage recovery of Rilpivirine was found to be 99 % which suggest that method development is very accurate. The acceptance limit of % recovery should be between 98.0-105.0%. The % recovery data of rilpivirine is shown in table 2.

Precision:

A known standard solution of rilpivirine was injected five times and corresponding peak areas were recorded. The % relative standard deviation of peak area was determined. Based on the results obtained from the data it was suggested that developed method is precise and the precision data is given in Table 3.

Table 3: Precision method of proposed RP-HPLC method

Injection	Area	Retention Time (Rt)
1	3441406	4.69
2	3498538	4.70
3	3433249	4.72
4	3530573	4.70
5	3488537	4.68
6	3458866	4.73
Average	3475195	4.70
SD	37302.62	0.018
% RSD	1.07	0.38

Linearity:

The linearity of measurement was evaluated by analyzing different concentrations of standard solutions of Rilpivirine. After analysis, the areas of the peaks were recorded. Calibration curve was constructed by plotting average peak area versus and concentrations. The regression equation is y = 34443 x- 8259.4 and regression coefficient r ²is 0.9997. The linearity graph is shown in Figure 2 and data is given in table 4.

Figure 2: Linearity curve

Table 4: Linearity data

S.No	Concentration (µg/ml)	Area
1	25	850163
2	50	1668331
3	75	2587435
4	100	3481893
5	125	4314059
6	150	5122945
8	Correlation coefficient	0.9997

Robustness:

The robustness was evaluated by changing the original conditions of flow rate and temperature. Flow rate as per the method is 1ml and column temperature is 30^oC. The flow rate was changed to 0.8ml and 1.2ml. The effect of column temperature was studied at 35 and 25°C. It was found that there was no drastic changes occur on chromatogram when flow rate and column temperature changes. The robustness data is given in table 5.

System Suitability:

System suitability was performed for Rilpivirine standard solution by injecting five replicates. A standard solution was prepared and was injected five times in to the HPLC. The % RSD of the retention time and area of the peak was calculated from the chromatograms obtained. It was found that the number of theoretical plates for Rilpivirine is more than 2000 and tailing factor is below 2.0. The results of system suitability and system suitable parameters were given in Table 6 and 7.

Table 5: Robustness Data

S.No	Parameters			Peak Area	Retention Time	Plate Count	Tailing Factor
S.No		Optimized	Used	Peak Area	Retention Time	Plate Count	Tailing Factor
1	Flow rate ( ±0.2)	1.2ml	1ml	4158171	5.89	2950	1.29
			1.2	3502649	4.69	2395	1.26
			1.4ml	4138445	5.83	2930	1.29
2	Temperature(±5)	30 ⁰C	25⁰C	3488306	4.32	2952	1.23
			30	3502649	4.69	2395	1.26
			35⁰C	3481688	4.38	2995	1.27

SD- Standard Deviation, RT- Retention Time % RSD- Relative Standard Deviation

Table 6: System suitability for RP HPLC Method

STANDARD NO	AREA	RETENTION TIME	THEORETICAL PLATES	TAILING
1	3452528	4.68	2330	1.26
2	3458741	4.66	2419	1.26
3	3501450	4.73	2352	1.27
4	3499067	4.69	2351	1.29
5	3458741	4.66	2419	1.26
AVRG	3474105	4.68
SD	24023.54	0.028
%RSD	0.69	0.59

AVRG- Average, SD- Standard Deviation, % RSD- Relative Standard Deviation

Table 7: System Suitability parameters of proposed RP-HPLC method

S. No	Parameters	Values
1	Retention time (min)	4.74
2	Theoretical plates	2395
3	Tailing factor	1.26
4	Wavelength (λmax)	280 nm
5	Regression equation	y = 34443 x- 8259.4
6	Correlation coefficient(r²)	0.9997

CONCLUSION:

In the present study the attempt has been undertaken to develop most simple, economic, sensitive and accurate RP-HPLC method for the estimation of Rilpivirine. The method was validated successfully using parameters like accuracy, precision, linearity, robustness and found to be simple, sensitive, accurate and precise. The %RSD for all parameters was found within the limits, which indicates the validity of the method and therefore the proposed method can be used for routine analysis for Rilpivirine in bulk and pharmaceutical formulation.

ACKNOWLEDGEMENT:

The authors are wish to thanks hetero drugs laboratory for providing gift sample.

REFERENCES:

1. http://www.drugs.com/monograph/rilpivirine-hydrochloride.html

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3. Chen X, Zhan P, Li D. Recent advances in DAPYs and related analogues as HIV-1 NNRTIs. Current Medicinal Chemistry. 18; 2011:359-76.

4. Johnson BC, Pauly GT, Rai G, Patel D, Bauman JD, Baker HL, Das K, Schneider JP, Maloney DJ, Arnold E, Thomas CJ, Hughes SH . A comparison of the ability of rilpivirine (TMC278) and selected analogues to inhibit clinically relevant HIV-1 reverse transcriptase mutants. Retrovirology. 5(9);2012:99

5. Miller CD, Crain J, Tran B. Rilpivirine: a new addition to the anti-HIV-1 armamentarium. Drugs Today. 47; 2011:5-15.

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7. Somsubhra G, Mithiles K, Satyabrata J, David B, Subhadip Roy. Method development and validation of Rilpivirine in bulk and pharmaceutical dosage form by using UV-Visible Spectroscopic method. Asian Journal of Research in Chemistry, 5(12); 2012:1472-1475

Received on 14.02.2013 Modified on 06.03.2013

Asian J. Research Chem. 6(4): April 2013; Page 319-322

Formulation	Dose	Concentration(µg/ml)	Amount Added ( mg)	Amount found (mg)	% Drug Estimated
Edurant Tablets	25 mg	100	25	24.96	99.86