Method Development by HPLC for Estimation of Rilpivirine in Unit Bulk and Matrix Form

Rilpivirine chemically 4-{[4-({4-[(E)-2-cyanoethenyl]-2,6dimethylphenyl} amino) pyrimidin-2-yl] amino}benzonitrile.  Rilpivirine is a  non-nucleoside  reverse  transcriptase inhibitor (NNRTI) of human immunodeficiency virus type 1 (HIV-1). It is a diarylpyrimidine, a class of molecules that resemble pyrimidine nucleotides found in DNA. Because of its flexible chemical  structure, resistance of rilpivirine is less likely  to develop than other NNRTI’s. FDA approved the Rilpivirine on May 20, 2011. Treatment of HIV-1 infections in treatment-naive patients with HIV-1 RNA ≤100,000 copies/mL in combination with at least 2 other antiretroviral agents. The matrix contains 25 mg of rilpivirine, which is equivalent to 25 mg  of  rilpivirine.  The  chemical  name  for  rilpivirine  hydrochloride  is  4- [[4- [[4- [(E)- 2- cyanoethenyl] -2,6dimethylphenyl] amino] 2-pyrimidinyl] amino] benzonitrile monohydrochloride. Its molecular formula is C22H18N6 [1].

Fig: 1. Structure of Rilpivirine

MATERIALS AND METHODS

Standard Drug

Rilpivirine was obtained as gift sample from Spectrum Labs India Pvt. Ltd. (Bangalore, India).

Chemicals and Reagents:

Methanol (HPLC Grade), Acetonitrile ((HPLC Grade) were obtained from Merck specialties Pvt. Ltd. (Mumbai, India).

Instruments and Equipment’s:

Table: 1 List of instruments used

Preparation of solutions:

a. Preparation of standard stock solution

Accurately weighed 10 mg Rilpivirine was transferred into 10 ml volumetric flask and shaken well till it gets dissolved. After that made up the volume up to the mark of 10 ml with Methanol and mixed to make 1000 µg/ml stock solution (Solution A).

b. Preparation of working standard solution

1 ml of stock solution (Solution A) was diluted with methanol to get 100 µg/ml as a working solution (Solution B). 0.5 ml of Rilpivirine standard solution (100 μg/ml) was pipetted into a volumetric flask and methanol was added up to 10 ml to obtain Rilpivirine solution (5 μg/ml). Dilutions were made up to 25 μg/ml using this procedure.

Preparation of sample solution:

For this, 250 mg of matrix was weighed accurately. A quantity of tablet powder equivalent to 10 mg of Rilpivirine was weighed and transferred to 100 mL volumetric flask containing about 60 mL of methanol and ultrasonicated for 15 min and volume was made up to the mark with the solvent. The solution was filtered through Whatman paper No. 41. One mL of this solution was transferred to 10 mL calibrated volumetric flask and volume was made up to the mark with the acetonitrile to get solution of concentration 10 µg mL-1. After setting the chromatographic conditions, the matrix sample solution was injected, chromatogram was obtained, and the peak areas were recorded. The injections were repeated six times and the amount of drug present per tablet was estimated from the respective calibration curve. The % assay was found to be 99.154 ± 0.48 (mean ± S.D.).

Mobile phase selection:

Mobile phase selection was done by various trials which was given in Table 3. In this, only one column was tried and confirmed.

Selection of Analytical wavelength:

A working solution containing Rilpivirine (5 μg/ml) was prepared using methanol as solvent and scanned over 200- 400 nm in UV –Spectrophotometer. It showed maximum absorbance at 310 nm. The UV Spectrum of Rilpivirine is given below.

Fig: 2 UV spectrum of Rilpivirine

Optimized chromatographic conditions:

Chromatographic conditions like column selection, mobile phase, wavelength, flow rate and run time were optimized.

Table 2 optimized chromatographic condition.

Fig: 3 Representative chromatogram of Rilpivirine (5 μg /ml, RT= 5.5 minutes)

Method Validation:

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

System suitability:

As per the test method the standard solution where prepaid and injected into HPLC from which the evaluated system suitability parameter are found to be within the limits.

Precision:

The degree of closeness of the agreement among individual test results when the method is applied to multiply samplings of a homogeneous sample .it is measure of either the degree of reproducibility (agreement under different conditions) or repeat ability (agreement under the same condition) of the method.

Accuracy:

Closeness of results was obtained by a method to the true value .it is a measure of the exactness of the method.

% Bias = measure value - true value x 100

True value

Linearity:

The ability of the method to produce results those are directly or indirectly proportional to the concentration of the analyt in sample within a given range.

Limit of detection (LOD):

Limit of detection is the lowest concentration in a sample that can be detected, but not necessarily quantified under the stated experimental condition.

LOD=3sa/b

Limit of quantification (LOQ):

Limit of quantification is the lowest concentration of analyte that can be determined with acceptable precision and accuracy.

LOQ=10sa/b

Where, Sa-the estimate represents peaks variation.

The related calibration curves slope is given by b.

Ruggedness:

Ruggedness of an analytical method is the degree of reproducibility of test results obtained by analysis of the same samples under a variety of conditions, such as different laboratories, different analyst, different instrument, different lots of reagents, different assay temperature and different days etc.

Robustness:

A methods robustness is defined by minor changes in parameters like the mobile phases PH, temperature, the strength of the organic solvents, the concentration of the buffer, etc, are made but there was no recognised change in the result and are within the range as per ICH guidelines. To assess a methods robustness, experimental conditions were purposefully changed, and chromatographic characteristics were assessed.

Results and Discussion:

Table: 3 Trail run for Rilpivirine HPLC.

Column used	Observation
Column used. Thermo scientific BDS Hypersil TmC8 column (250× 4.6 mm) Mobile phase Methanol: water (containing 0.1 % Ortho phosphoric acid) (80: 20) Wavelength: 310nm	Sharp peak with optimum asymmetry was obtained

The method for Rilpivirine was validated as per the ICH guidelines ICH Q2(R1) in terms of specificity, linearity, range, accuracy, precision, limit of detection, limit of quantitation, and robustness.

Specificity:

Specificity was analyzed by checking different solutions like blank, standard and sample. It was then proved that there were no peaks other than drug at retention time of drug 5.08 minutes.

The method was found specific.

Fig: 4 Representative chromatogram of rilpivirine blank

Fig: 5 Representative chromatogram of Rilpivirine standard (10μg/ml).

LINEARITY:

A standard solution of Rilpivirine (100 ug/ml) was injected in the HPLC UV system of the volume 5,10,15,20 and 25 ppm, thus leading to injected amounts in the range of 5-25 ppm. This procedure was repeated for 5 times. The correlation coefficient was found 0.9973with equation of y = 169912.052x + 37988The chromatogram was shown in the Fig. 6 for linearity. The calibration curve was obtained by plotting amount of drug injected (ppm/injection) Vs peak area shown in the. The results and details of the linearity was shown respectively.

Fig: 6 The representative chromatogram for linearity.

Fig: 7 The representative chromatogram for calibration curve.

ASSAY:

In a motor pestle, 25 mg of Rilpivirine was geometrically mixed with 169 mg microcrystalline cellulose and 56 mg lactose as excipients to make a synthetic mixture. From this mixture,100 mg of blend equal to 10 mg of medication) was properly weighed and diluted to 10 ml with methanol to produce a solution (1000 µg/ml). The solution was filtered and sonicated. 6 replicates of the sample solution (10 µg/ml) were made by diluting 1 ml of the filtrate above solution to 10 ml with methanol to get solution (10 µg/ml). Each sample solution was injected, and chromatogram was developed with mobile phase and scanned at 310 nm, with the peak area recorded. Assay of prepared blend was performed, and the % drug content was found to be 99.154% ± 0.487 (SD). The chromatogram was shown in the below fig 8

Fig: 8 Representative chromatogram for Assay.

ACCURACY:

Recovery study was carried out by performing standard addition method at 50%, 100% and 150% level. The standard drug Rilpivirine was added to pre analyzed sample solution at three levels. The basic concentration of was sample chosen was 10 µg/ml of Rilpivirine. The % mean recovery was found to be 99.828% for Rilpivirine. At 50%, 100% and 150% levels % recovery was found to be 99.506%, 100.209% and 99.769% respectively the peak area were calculated after peak was obtained and the result are shown in the below table chromatogram was shown in the below fig 9 for Accuracy.

Fig: 9 The Representative chromatogram for Accuracy (50.100 and 150%).

Table: 4 Recovery studies

The precision method was done by repeatability and intermediate precision studies. In repeatability precision, injection of three-three replicates of standard solution (10, 15 & 20ug/ml) was done on the same day after some time interval. In intermediate precision, injection of six replicates of standard solution (10, 15 & 20 ug/ml three of each) was injected in the HPLC system on two consecutive days.

The % RSD was calculated, and values were found less than 2%.

The precision result table is given in the Table. The chromatogram was shown in the Fig. 10

Fig: 10 The Representative chromatogram for precision (10ppm).

Table: 5 precision studies for Rilpivirine

1. Interday precision:

2. Intraday precision: