INTRODUCTION:

Rilpivirine hydrochloride is a di-amino pyrimidine derivative. Chemically, it is 4-[[4-[[4-[(E)-2-cyanoethenyl]- 2,6-dimethylphenyl] amino]-2-pyrimidinyl] amino] benzonitrile monohydrochloride structure are shown in Figure 1.

N⁴-{4-[(E)-2-isocyanoethenyl]-2,6-dimethylphenyl}-N²-(4-isocyanophenyl)pyrimidine-2,4-diamine

Figure1. Chemical Structure of Rilpivirine

Rilpivirine hydrochloride is a human immunodeficiency virus type 1 (HIV-1) specific nonnucleoside reverse transcriptase inhibitors (NNRTI) indicated for used in single and combination of other drugs like tenofovir, emcitribine and effarveniz. Rilpivirine (TMC278) is NNRTI, which was approved by the FDA in May 2011.^1-3 It is a basic, white, amorphous powder which is readily soluble in methanol, dichloromethane and insoluble in water. Rilpivirine hydrochloride is not yet official in I.P and B.P. A thorough literature survey has revealed that UV spectroscopy,^4-6 HPLC^7-9 method for rilpivirine hydrochloride with combination of other drugs, UPLC,¹⁰ LC-MS,¹¹ GC-MS for its estimation in bulk, pharmaceutical dosage forms and biological samples

The objective of present investigation deals with development of simple, accurate, precise and more economical UV spectroscopic method using derivative methods for estimation of rilpivirine in bulk and formulation and their validation as per ICH guidelines.¹²

MATERIALS AND METHODS:

Materials:

Rilpivirine hydrochloride was gift sample from Hetero Laboratories Ltd. (Hyderabad, A.P, INDIA). Methanol was purchased from Merck Chemical Company (India). The commercially tablets of are not available in Indian market, hence we have manufactured immediate release tablet containing 25 mg, acacia, micro crystalline cellouse, lactose, magnesium sterate and talc from S D Fine Chem. Ltd. (Mumbai, India).

Instruments:

The derivative technique was performed in double beam UV-VIS spectrophotometer (UV-1800, Shimadzu, Japan) connected to computer loaded with spectra manager software UV Probe was employed with spectral bandwidth of 1nm and wavelength accuracy of ± 0.3 nm with a pair of 10 mm matched quartz cells and sonicator.

Preparation of Standard stock solution of rilpivirine hydrochloride:

Standard stock solution of rilpivirine hydrochloride (1 mg/mL) was prepared by transferring 10 mg of rilpivirine hydrochloride into a 10 ml volumetric flask containing 4mL of (8:2) methanol and water for zero order and first order derivative spectrophotometric analysis. It was then sonicated for 15 minutes and solution was diluted up to the volume by methanol and water. From these, further dilutions were made using (8:2) methanol and water to produce solution of rilpivirine hydrochloride (100µg/ml).

Selection of wavelength for analysis of rilpivirine hydrochloride:

0.1 ml of standard stock solution of rilpivirine hydrochloride was transferred into a 10 ml volumetric flask and diluted to a mark with methanol : water (8:2) to give concentration of 1 μg/ml. The resulting solution was scanned in the UV range (200–400 nm) for zero order and first order derivative spectrophotometric analysis.¹³

Preparation of sample solution:

Twenty tablets are analysed for their drug content by UV spectrophotometric methods. The tablet contents were crushed into a fine powder and suitably diluted in methanol: water (8:2) to yield a concentration of 1.0 mg/ml for rilpivirine hydrochloride. The mixture was sonicated to dissolve, make up the volume with methanol: water (8:2). The above solutions were filtered through Whatman filter paper and the solution was transferred into volumetric flask and was made up to the mark with methanol: water (8:2) to obtain a final concentration of 1.5 µg/ml. The spectrum was recorded at 305 nm and 271 for both zero order and first order derivative spectrophotometric method, against blank solution of methanol : water (8:2).

VALIDATION OF PROPOSED METHOD:

The method was validated according to ICH guidelines in order to determine the linearity, precision, accuracy and ruggedness of the method.

Linearity:

Linearity was evaluated by seven point standard curve in concentration range of 0.5- 3.5 µg/ml (0.5, 1, 1.5, 2, 2.5, 3 and 3.5 µg/ml) of rilpivirine hydrochloride. The calibration curve was obtained by plotting absorbance against concentration (μg/ml) for both zero order and first order derivative spectrophotometric method.¹⁴ Each set was analyzed to plot a calibration curve. Standard deviation (SD), slope, intercept, and correlation coefficient of determination (r²) of the calibration curves were calculated to ascertain the linearity of the method.

Method precision (repeatability):

The precision of the instrument was checked by repeated scanning and measurement of the absorbance of solution (n=6) for rilpivirine hydrochloride (1.5µg/ml) without changing the parameter of the proposed UV method for both zero order and first order derivative spectrophotometric method. The %RSD was calculated.

Intermediate Precision (reproducibility):

The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses on the same day and next day for three different concentration of standard solution of rilpivirine hydrochloride (0.5, 1.5 and 2.5µg/ml)for both zero order and first order derivative spectrophotometric method. The result was reported in terms of relative standard deviation (%RSD).

Accuracy:

Accuracy of the proposed method was determined using recovery studies by standard addition method. The recovery studies were carried out by adding different amounts (50, 100 and 150%) of the pure drug to the pre-analysed formulation. The solutions were prepared in triplicates and the % recovery was calculated for both zero order and first order derivative spectrophotometric method.

Limit of Detection and Limit of Quantitation:

The parameters LOD and LOQ were determined on the basis of response and slope of the regression equation. The limit of detection (LOD) and the limit of quantitation (LOQ) of the drug were derived by calculating the signal-to-noise ratio (S/N, i.e., 3.3 for LOD and 10 for LOQ) using the following equations designated by International Conference on Harmonization (ICH) guidelines.

LOD = 3.3 × σ/S, LOQ = 10 × σ/S

Where σ = the standard deviation of the response and S = slope of the calibration curve

Ruggedness Studies:

Ruggedness studies were performed by preparing three replicates of 1.5µg/ml, analysing by two different analyst and on two different instruments and the results are reported as %RSD for both zero order and first order derivative spectrophotometric method.

RESULTS AND DISCUSSION:

The zero order and first order derivative spectra for rilpivirine hydrochloride were recorded at the wavelength of 305 nm and 271 nm was shown in Figure 2.

Figure2. Zero and first order derivative spectrum of rilpivirine hydrochloride

Linearity:

Standard solutions of rilpivirine hydrochloride in the concentration range of 0.5 to 3.5 µg/ml were observed in UV spectroscopy at 305nm and 271nm, the graph obtained for zero order and first order derivative spectra showed linear relationship was shown in Table no.1. The regression equations of calibration curves were y = 0.272x + 0.018 (r2 = 0.999) at 305 nm for zero order derivative spectrophotometry and y = 0.162x + 0.007 (r2 = 0.999) at 271 for first order derivative spectrophotometry was shown in Figure 3. The range was found to be 0.5-3.5 μg.mL-1 for both zero order and first order derivative spectrophotometric methods. A graph of absorbance (on Y-axis) versus concentration (on X-axis) was plotted (overlay of absorbance) was shown in Figure 4

Table No.1 zero order and first order calibration values of rilpivirine hydrochloride

Sample no.	Concentration (µg/ml)	Absorbance at 305nm	Absorbance at 271 nm
1	0.5	0.163	0.082
2	1.0	0.286	0.147
3	1.5	0.451	0.238
4	2.0	0.580	0.317
5	2.5	0.725	0.394
6	3.0	0.891	0.485
7	3.5	1.030	0.564

Figure 3.Zero order and first order calibration curve of rilpivirine hydrochloride

Method precision (repeatability):

Repeatability was determined by analyzing 1.5 μg/ml concentration of rilpivirine hydrochloride for six times and % RSD was found to be < 2 which shown in Table no 2 for both zero order and first order derivative spectrophotometric method.

Figure 4.Zero order and first order overlay spectrum of rilpivirine hydrochloride

The precision of the developed method was expressed in terms of percent relative standard deviation (% RSD). These results show reproducibility of the assay. The % RSD values was found to be less than 2 that indicate this method precise for the determination of the pure form. The interday and intraday precision results were mentioned in Table no.3a and 3b for both zero order and first order derivative spectrophotometric method.

Accuracy:

Accuracy is determined by performing recovery studies at 3 levels in which known amount of analyte shall be added and recovery shall be carried out in three replicates of each concentration level and the % recovery was calculated. The mean recovery was found between 100-101 % and %RSD between 0.3-0.7. The accuracy results are shown in Table No. 4 for both zero order and first order derivative spectrophotometric method.

Limit of Detection and Limit of Quantitation:

The parameters LOD and LOQ were determined on the basis of response and slope of the regression equation. LOD and LOQ values are 0.358 and 1.086 for both zero order and first order derivative spectrophotometric method.

Ruggedness Studies:

This study was performed by analyzing 1.5µg/ml of by two different analysts and on two instruments, results of the study were given in Table no. 5 and % RSD obtained was less than 2 which is within the acceptance limits for both zero order and first order derivative spectrophotometric method.

Table No. 2 Zero order and first order repeatability studies of rilpivirine hydrochloride at 305 and 271 nm Intermediate Precision (reproducibility)

Concentration (µg/ml)	Absorbance at 305 nm	Mean=0.461 S.D=0.008 %RSD=1.71	Concentration (µg/ml)	Absorbance at 271 nm	Mean=0.238 S.D=0.0038 %RSD=1.61
1.5	0.473		1.5	0.236
1.5	0.449		1.5	0.239
1.5	0.458		1.5	0.234
1.5	0.464		1.5	0.241
1.5	0.459		1.5	0.238
1.5	0.462		1.5	0.245

Table No.3a Intraday precision of rilpivirine hydrochloride at 305 nm and 271 nm

Concentration (µg/ml)	Absorbance at 305nm	%RSD	Concentration (µg/ml)	Absorbance at 271nm	%RSD
0.5	0.162± 0.003	1.88	0.5	0.088± 0.0017	1.96
1.5	0.456± 0.009	1.98	1.5	0.240± 0.0041	1.72
2.5	0.727± 0.014	1.91	2.5	0.386± 0.0072	1.86

Table No.3b Interday precision of rilpivirine hydrochloride at 305 nm and 271 nm

Concentration (µg/ml)	Absorbance At 305nm	%RSD	Concentration (µg/ml)	Absorbance at 271nm	%RSD
0.5	0.165± 0.002	1.21	0.5	0.089± 0.001	1.12
1.5	0.457± 0.005	1.91	1.5	0.241± 0.0045	1.86
2.5	0.726± 0.009	1.57	2.5	0.397± 0.0072	1.56

Application of the Proposed Method for Pharmaceutical Formulation:

The proposed method was able to remove the interferences of the other excipients present in the pharmaceutical formulations (tablets) are assessed with a high percent of recovery. The percentage recovery for tablet formulation was found to be 99 -101 % enlisted in table no.6. The results for assay are within acceptable limits for both zero order and first order derivative spectrophotometric method.

Table No.6 Assay results for the determination of rilpivirine hydrochloride in pharmaceutical dosage form

Parameter

Labelled amount (mg)

Amount taken

(ug/ml)

Amount found

(ug/ml)

% Recovery

Zero order derivative

1.015

101.5

First order derivative

1.004

100.4

CONCLUSION:

The above proposed UV method is very simple, precise, accurate, rapid and cost effective for the quantification of rilpivirine hydrochloride from its pharmaceutical dosage forms for both zero order and first order derivative spectrophotometric method¹⁵. The solvent is also very cheaper when compared the other methods. Hence it can be utilized for routine analysis in bulk and pharmaceutical dosage forms.

ACKNOWLEDGEMENTS:

The authors are grateful to, Malla Reddy College of Pharmacy for providing necessary research facilities to carry out the research work and to hetero drugs, India for providing the gift sample of the drug.

REFERENCES:

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[4] Masthanamma SK, Alekya Gottumukkala. Development and validation of uv spectrophotometric methods for estimation of rilpivirine in bulk and pharmaceutical formulation. International Journal of Pharmaceutical Sciences and Research. 5(2): 2014: 483-489.

[5] Girija B, Bhavar, Sanjay S, Pekamwar, Kiran B, Aher, Sanjay R, Chaudhari. Development and validation of UV spectrophotometric method for estimation of rilpivirine hydrochlorine in bulk and pharmaceutical formulations. American Journal of PharmTech Research.3(1):2013: 450- 458.

[6] GhoshSomsubhra, Kumar Mithlies, Jena Satyabrata, Banji David, Roy Subhadip. Method development and validation of rilpivirine hydrochloride in bulk and pharmaceutical tablet dosage form by using UV–visible spectrophotometric method. Asian Journal of Research in Chemistry. 5(12):2012:1472-1475.

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Received on 26.07.2014 Modified on 02.08.2014

Asian J. Research Chem. 7(8): August 2014; Page 717-721

Actual concentration	Observed concentration	%Recovery	Mean recovery	%RSD
Zero order derivative spectrophotometric method
1.5	1.51	100.5	100.2	0.336
2.0	2.01	100.1		0.603
2.5	2.49	100.1		0.651
First order derivative spectrophotometric method
1.5	1.50	100.1	100.4	0.420
2.0	2.01	100.6		0.626
2.5	2.51	100.5		0.525

DIFFERENT INSTRUMENT				DIFFERENT ANALYST
Concentration (µg/ml)	Absorbance (nm)		Mean=0.461,0.237 S.D=0.008,0.001 %RSD=0.178,0.64	Concentration (µg/ml)	Absorbance (nm)		Mean=0.463,0.235 S.D=0.008,0.0005 %RSD=0.165,0.24
	305	271			305	271
1.5	0.469	0.239		1.5	0.471	0.236
	0.453	0.236			0.456	0.235
	0.458	0.238			0.461	0.237