INTRODUCTION:

Rilpivirine, chemically designated as 4-{[4-({4-[(E)-2-cyanovinyl]-2,6-dimethylphenyl}amino)pyrimidin-2-yl]amino}benzonitrile (Figure - 1), belong to the category of Anti-HIV drug ⁽¹⁾. There are very few analytical methods have been developed for its determination in pharmaceutical formulations and in bio-fluids either alone or in combination with other drugs, which includes liquid chromatography-tandem mass spectrometry⁽²⁾, HPLC⁽³⁾ and a Spectrophotometric analysis⁽⁴⁾.

Figure No 1: Chemical structures of Rilpivirine

According to the information collected from literature there are very few method^(5-9) reported for the determination of Rilpivirine out of these method only one method is in HPLC.

In the present work, we have therefore focused to achieve the optimum chromatographic conditions for the determination of Rilpivirine in tablet doses form. We describe a simple, sensitive and validated HPLC method with total run time less than 6 minutes for the determination of Rilpivirine. The developed method can be applied successfully to quality control and for other analytical purposes.

MATERIALS AND METHODS:

Standard drugs:

Rilpivirine of purity 99% w/w procured from Hetero Drugs Ltd as a gift sample.

Chemicals and reagents:

Acetonitrile -HPLC grade (Merck), Water for HPLC-milli-Q grade (Merck), Potassium dihydrogen phosphate (Merck), Orthophosphoric acid (Merck).

Apparatus:

Single pan balance (Mettler), pH meter (Thermo), Sonicator (Shimadzu),

HPLC - (Waters, UV – 2489). Column -XTerra– RP C8 (250 x 4.6mm, 5μmm)

Membrane filter (0.45μ) Centrifugation Apparatus, Volumetric flask borosilicate,

Pipettes (borosilicate)

Preparation of standard solutions:

Accurately weigh and transfer 10mg of Rilpivirine Working standard into a 10 mL volumetric flask add about 7 mL of Diluent and sonicate to dissolve it completely and made volume up to the mark with the same solvent (Stock solution). Further pipette 0.3 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluent to get concentration 30 µg/ mL. Mix well and filter through 0.45µm filter.

Sample Solution Preparation:

Weigh 20 Rilpivirine Tablets and calculate the average weight. Accurately weigh and transfer the sample equivalent to 10 mg of Rilpivirine into a 10 mL volumetric flask. Add about 7 mL of diluent and sonicate to dissolve it completely and make volume up to the mark with diluent. Mix well and filter through 0.45µm filter. Further pipette0.3 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45µm filter.

Optimized chromatographic conditions:

Equipment: High performance liquid chromatography equipped with Auto Sampler Waters Empower- 2, UV detector 2489

Column: Waters C8 (4.6 x 150mm, 5mm, Make: Waters)

Mobile phase: Acetonitrile : Phosphate buffer (60:40)

Flow rate: 1 ml per min

Wavelength: 282 nm

Injection volume: 10 ml

Column oven: Ambient

Run time: 6min

Technique: Isocratic.

Preparation of Potassium dihydrogen Ortho phosphate (pH= 3.5):

Accurately weighed 2.72 grams of KH₂PO₄ was taken in a 1000ml volumetric flask, dissolved and diluted to 1000ml with HPLC water and the volume was adjusted to pH 3.5 with Orthophosphoric acid.

Method Validation:

The suggested analytical method was validated according to international guidelines with respect to following parameters^(10,11) such as, precision, accuracy, linearity, robustness, ruggedness, LOD and LOQ.

Precision:

Method precision was determined both in terms of repeatability (injection and analysis) and intermediate precision (intra-day and inter-days reproducibility). In order to determine injection repeatability, samples spiked with 30 µg/ml of Rilpivirine were injected 5 times into HPLC system and repeatability of the retention time and peak area was determined and expressed as mean and %RSD calculated from the data obtained.

Accuracy:

Accuracy was determined in terms of percent recovery. Sample solution spiked with the analytes at three different concentration levels 15, 30, 45 µg/ml of Rilpivirine. Another set of standard mixtures at the same concentration levels was also prepared with the diluents. Sample and standard solutions are injected onto the HPLC system in triplicate. Percentage recoveries of Rilpivirine was calculated.

Linearity:

The linearity of the method was established by spiking a series of standard mixtures of Rilpivirine (10-50μg/ml). Above solutions are injected onto the HPLC system. Construct the Calibration curves for standard solutions by plotting their response (peak area of the analytes) against their respective concentrations. Linear regression was applied and slope (a), intercept (b), correlation coefficient (r) and standard error (Es) were determined.

Limit of detection & Limit of quantification:

Detection and quantification limits were determined through dilution method using S/N approach by injecting a 10μl sample. LOD was considered as the minimum concentration with a signal to noise ratio of at least three (S/N˜3), while LOQ was taken as a minimum concentration with a signal to noise ratio of at least ten (S/N˜10).

Robustness:

The robustness of the developed method was investigated by evaluating the influence of small deliberate variations in procedure variables like flow rate (±5%) and change in organic composition.

Ruggedness: The ruggedness of the method was investigated by evaluating the influence of different analyst, different time intervals.

Stability:

The stability studies of Rilpivirine sample was carried out over a period of 48 h at 25 0C (room temperature under laboratory light), 2–80C (refrigerator) and standard solutions for one month at 2–80C.

RESULTS AND DISCUSSION:

Sample preparation:

Initially the mobile phase tried was acetonitrile: water with various combinations as well as varying proportions and phosphate buffer: acetonitrile with various combinations of pH as well as varying proportions. Finally, the mobile phase was optimized to potassium dihydrogen phosphate with buffer (pH 3.5), acetonitrile in proportion 40: 60 v/v respectively (fig.-2).

Figure – 2: Chromatogram for Rilpivirine in pure form

Name

Retention time(min)

Area

(µV sec)

Height (µV)

USP tailing

USP plate count

Rilpivirine

2.75

1540351

195505

1.7

3291.9

Method validation:

Precision:

Precision data representing both repeatability (injection and analysis) and intermediate precision (different analysist) are summarized in Table No: 1 and Table No: 2, respectively. The %RSD values for both Precession & ID Precision were less than 2.0%, which indicates that the proposed method is precise.

Table No: 1. Precision results for Rilpivirine

S.NO	Area of Rilpivirine
1.	1536490
2.	1536351
3.	1539021
4.	1539344
5.	1540984
Mean	1538438
S.D	1987.0
R.S.D	0.13

Table No: 2. ID-Precision results for Rilpivirine (different analysist)

Analysts	Area of Rilpivirine
Analyst 1	1532019
Analyst 2	1532127
S.D	76.367
% R.S.D	0.004

Accuracy:

Average recoveries of Rilpivirine are 100.03%, 99.8%, 99.3%, at 50%,100% & 150% concentrations level respectively. The percentage recoveries of the drug is within the limits 99-101%. So the method is accurate, accuracy data for Rilpivirine are presented in Table No: 3.

Table No: 3. Accuracy of Rilpivirine

% Concentration (at specification Level)	Area	Amount Added (mg)	Amount Found (mg)	% Recovery	Mean Recovery
50%	1546245	5.0	5.0	100.3%	99.8%
100%	3077037	10.0	9.98	99.8%
150%	4591078	15.0	14.9	99.3%

Linearity:

The response was found linear over a concentration range of 10-50 µg/mL of Rilpivirine. The correlation co-efficient were found to be 0.998 for Rilpivirine. So the method is linear, data is presented in Table No: 4. Linearity curve of Rilpivirine is given in Figure: 3.

Table No: 4. Linearity results for Rilpivirine

Concentration (µg/ml)	Rilpivirine area
10	506172
20	1061027
30	1542964
40	2083016
50	2539881
Correlation Coefficient	0.999

Figure – 3: Calibration graph of Rilpivirine

Limit of detection:

The LOD for Rilpivirine standard solutions were found to be 0.005µg/ml given in Figure No: 4.

Figure – 4: Chromatogram of Limit of Detection

Limit of quantification:

The LOQ Rilpivirine standard solution was found to be 0.17µg/ml given in Figure - 5.

Robustness:

Minor deliberate changes in different experimental parameters such as flow rate (±5%) and wavelength (±5units) did not significantly affect the retention time & peak area of Rilpivirine indicating that the proposed method is robust which is mentioned in Table – 5 & 6.

Figure – 5: Chromatogram for Limit of Quantification

Table No: 5. Study of Robustness results for Rilpivirine (Flow Rate)

Flow rate	Retention time
1ml/min	2.744
1.5ml/min	2.742

Table No: 6. Study of Robustness results for Rilpivirine (Wavelength)

Wavelength	Area
275nm	1536916
280nm	1538575

Ruggedness:

The method is rugged by different analyst, different time intervals and the method did not significantly affect the recoveries, peak area and retention time of all the above drugs indicating that the proposed method is rugged.

Stability:

Results from the stability studies of samples and standard solutions indicated that samples were stable for 48 h when stored at room temperature (25⁰C), refrigerator (2–8⁰C) and while the standard solutions demonstrated stability for one month at 2–8 ⁰C. A validation summary is given in Table No: 7 which includes all parameters at a glens.

Table No: 7. Validation summary of Rilpivirine

S. NO	Validation parameters	Acceptance criteria	Observation
1.	Linearity range (µg/ml)	-	10-50
2.	Correlation coefficient	NLT 0.999	0.999
3.	Number of Theoretical Plates	NLT 2000	3291.9
4.	Method precision	% RSD (NMT 2%)	0.13
5.	System precision	% RSD (NMT 2%)	0.19
6.	Ruggedness	% RSD (NMT 2%)	0.004
7.	% Recovery	98% to 102%	99.8
8	Limit of Detection (µg)	NMT 3	2.98
9.	Limit of Quantification (µg)	NMT 10	9.98

CONCLUSION:

A novel, simple, rapid and cost effective RP-HPLC method was successfully developed for determination of Rilpivirine. The proposed method was optimized and validated for the various experimental parameters. Influence of pH of the mobile phase, column oven temperature and various particulate columns on the analysis of Rilpivirine was evaluated. All the analytes were well resolved and separated in less than 5 min. Our developed method will be applied for assessing the pharmacokinetics and drug–drug interaction studies of this drug with other commonly prescribed drugs. This method could also be used for the analysis of this drug in pharmaceutical preparations and routine laboratory analysis with slight modification in the extraction procedure. Overall, the proposed method provides high throughput for determination of Rilpivirine with excellent accuracy, precision, selectivity and reproducibility.

AKNOWLEDGEMENT:

We are very thankful to authorities of Nalanda College of Pharmacy for providing the facilities to complete this research work.

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Received on 12.12.2012 Modified on 30.12.2012

Research J. Pharm. and Tech. 6(3): March 2013; Page 240-243