INTRODUCTION:

Rilpivirine, chemically known as4-{[4-({4-[(E2-cyanovinyl]-2,6-dimethylphenyl}amino)pyrimidin-2yl]amino} benzonitriles. It is a second generation non-nucleoside reverse transcriptase inhibitor (NNRTI) with higher potency and longer half-life. It has activity against NNRTI resistant viral strains due to the flexibility of interactions with the HIV RT. [1,2,3]

Literature review indicates that few UV spectrophotometric method [4-9] HPLC method [10-14], UPLC method [15,16] LC-MS[17] method, HPTLC method [18]. The above reported chromatographic methods employed sophisticated and expensive instrumentation that are generally not available in most of the quality control laboratories of underdeveloped and developing countries. As a result, the applications of these methods [4-18] for the quantification of Rilpivirine in biological samples, bulk and pharmaceutical formulations are limited.

In most of the developing countries, UV-Visible spectroscopy is the technique of preference for the precise, accurate and cost-effective determination of pharmaceutical substances. To the best of our knowledge, no method has been reported in the literature about the analysis of Rilpivirine in bulk and tablet formulations using a visible spectrophotometric method.

In the present work, simple and sensitive visible spectrophotometric method was developed and validated for the analysis of Rilpivirine with broad linearity, good precision and accuracy. This method could be applied for the quantitative determination of the Rilpivirine in their tablet formulations.

MATERIALS AND METHODS:

Instrumentation:

· A double beam UV spectrophotometer: LAB INDIA-3000 with UV WIN software and 1cm quartz cell.

· Weighing balance: Sartorius

Preparation of reagents and solutions

Standard stock solution of Rilpivirine:

Rilpivirine working standard was procured from HiQ Pharma Labs Pvt Ltd., Hyderabad, India. Standard stock solution of Rilpivirine was prepared by dissolving accurately weighed 10mg of drug in 20ml of 0.1 M NaOH in 50ml volumetric flask and diluted up to the mark with distilled water.

Standard solution of Sodium nitroprusside: 0.4% w/v of sodium nitroprusside was prepared by dissolving 0.400g in distilled water in 100ml volumetric flask and diluted up to the mark with water.

Standard solution of Hydroxyl amine HCl: 0.4% w/v of Hydroxyl amine HCl was prepared by dissolving 0.400g in distilled water in 100ml volumetric flask and diluted up to the mark with water.

Standard solution of Sodium Carbonate: 0.4% w/v of Sodium Carbonate was prepared by dissolving 0.400g in distilled water in 100ml volumetric flask and diluted up to the mark with water.

Preparation of sample solution:

General procedure: Aliquots (0.2-1.0ml) of Rilpivirine standard solution were transferred in to 10ml volumetric flask. To each flask 1ml of Sodium nitroprusside, 0.8ml of Hydroxyl amine HCl, and 0.8ml of Sodium Carbonate solutions were added. The volume was then made up to the mark with water and absorbance of each solution was measred at 445nm.

Analysis of Rilpivirine in tablet formulation: The tablet formulation of Rilpivirine labeled to contain 25mg was purchased. Twenty tablets were accurately weighed and finely powdered in a mortar. A portion of tablet powder equivalent to 25mg was weighed and transferred into 50ml volumetric flask. 20ml of 0.1 M NaOH was added and the mixture was sonicated for 15mins. The mixture was filtered through Whatman No. 1 filter paper. The solution was made up to the mark with distilled and contents were analysed by the proposed method.

RESULTS AND DISCUSSION:

Method Development:

The reaction of Sodium nitroprusside with amino group has been reported these reactions demonstrate that Sodium nitroprusside is a valuable reagent in the development of simple spectrophotometric methods for the quantification of many pharmaceutical amines [19-21]. In the present investigation, Rilpivirine drug contains diaryl pyrimidine nucleus. It acts as a donor due to the presence of aromatic tertiary nitrogen in the side chain of pyrimidine nucleus. Sodium nitroprusside in the presence of hydroxyl amine and alkali exists as aquo ferrocyanide [Fe(CN)₅H₂O]^3-. In a general way it may be expected that the electron transfer depends upon the extent of delocalization of the donor and acceptor metal orbitals of the intervening ligands (Figure 1).

Figure 1: Reaction Pathway between Sodium Nitroprusside and Rilpivirine

The reaction was studied under various conditions of Sodium nitroprusside, Hydroxyl amine HCl, Sodium carbonate concentration and reaction time was studied to determine the optimum conditions for the method.

The effect of the volume of 0.4% w/v Hydroxyl amine HCl on the absorbance of the yellow colored complex was studied in the range of 0.2-2.0ml. The absorbance increases with the increase in the volume of Hydroxyl amine HCl up to 0.8ml. Further addition of Hydroxyl amine HCl showed decrease in the absorbance. Therefore, 0.8 ml of 0.4% w/v Hydroxyl amine HCl was chosen as an optimum value (Figure 2).

Figure 2: Effect of volume of Hydroxyl amine HCl

The effect of volume of 0.4% w/v Sodium carbonate on the absorbance of yellow colored complex was studied in the range 0.2-2.0ml. The absorbance increases with increase in the volume of Sodium carbonate and becomes constant at 0.8ml. Further addition of Sodium carbonate does not show any change in the absorbance. Hence 0.8ml of 0.4% w/v Sodium carbonate was selected as an optimum value (Figure 3).

Figure 3: Effect of volume of Sodium carbonate

The effect of volume of 0.4% w/v Sodium nitroprusside on the absorbance of yellow colored complex was studied in the range 0.2-2.0ml.The absorbance increases with increase in the volume of Sodium nitroprusside up to 1ml. Further addition of Sodium nitroprusside showed decrease in the absorbance. Hence 1ml of 0.4% w/v Sodium nitroprusside was selected as an optimum value (Figure 4).

Figure 4: Effect of volume of Sodium nitroprusside

The effect of time on the formation of the yellow colored complex was also optimized. At room temperature, the color intensity of the product increased by the elapse if time, maximum absorbance was obtained at 2 minutes and remained constant for 20 minutes. The results indicated that the yellow colored product obtained was stable for 10 hrs.

In order to choose proper solvent for dilution, different solvents (methanol, dichloromethane, chloroform, isopropanol and Acetonitrile) were tested. The highest absorbance values were obtained when methanol was used as diluting solvent. In methanol medium, the absorbance values were found to be stable for 10hrs.Hence methanol was chosen as dilution solvent.

Association constant and the free energy changes of the complexes

The association constant of complex was determined by employing the Benesi–Hildebrand method [22], besides the association constant was calculated by using the following equation:

[Ao]/Aλ=1/ε+ (1/Kc.ε).1/[Do]

Where,

[Do] = Concentration of the drug,

[Ao] = Concentration of the reagent,

Aλ = Absorbance of the complex at 445nm,

ε = Molar absorptivity of the complex at 445nm,

Kc = Association constant of the complex.

The ΔG° (the standard free energy of complexation) and the association constant Kc are related by the following equation [20]

ΔG°=−2.303RTlogKc

Where,

ΔG° = Free energy change of the complex,

R = Gas constant (1.987 cal mol−1 degree−1),

T = Temperature in Kelvin,

K = Association constant (L mol−1) of the drug-reagent complex.

The results are summarized in the (Table 1). The negative values of the standard free energy indicated that the complexes are stable and started to form spontaneously.

Stoichiometry of ion pair complex:

Jobs method of continuous variation was employed to determine the composition of the ion pair complex [23]. For this, solutions of identical concentrations (1.1 x 10^-3M) of the drug and reagent were prepared and mixed in varying volume ratios such that total volume of each solution was constant. The absorbance of the resulting complex was measured at 445nm.The absorbance values were plotted against mole fraction of drugs. The stoichiometry ratio observed for drug dye ion pair complex was 1:1 as evident from the results in (Fig. 5)

Figure 5: Mole fraction of drug

Method Validation

Sensitivity:

According to the ICH guidelines [24], the sensitivity parameters like molar absorptivity, Sandell’s sensitivity, Limit of Detection and Limit of Quantification were calculated and summarized in (Table 1).

Table 1: Thermodynamic studies, linearity and Sensitivity of the proposed methods

Parameter	Result
Beer’s law limit (μg/mL)	4-20
Regression Equation(y=mx+c)	y = 0.0441x + 0.0105
Slope (m)	0.04335
Intercept (x)	0.022
Molar Absorptivity	1.6 x 10³
Regression coefficient (r2)	0.999
Sandell’s sensitivity (µg cm-2/0.001 Absorbance unit)	0.023068
LOD (μg/ml)	0.10
LOQ (μg/ml)	0.34
Association constant (L mole-1)	5.1 x 10⁴
Free energy change	-7.07 x 10⁴

Linearity:

The relation between the absorbance and final concentration of Rilpivirine was found to be linear over the concentration range of 4-20μg/ml. Results are shown in (Figure 6).

Figure 6: Linearity graph of Rilpivirine

Precision:

The repeatability (intra-day precision) of the proposed method was determined by replicate analysis (n=5) of standard solutions at three concentration levels (2μg/ml, 6μg/ml and 10μg/ml).

The intermediate precision (inter-day precision) was conducted by repeating the analysis over a period of three consecutive days.

The precision of the methods was expressed as standard deviation (SD) and percentage relative standard deviation (%RSD). The results are summarized in (Table 2).The SD and % RSD obtained by both methods are found to be in the acceptable range. Therefore, it can be considered to be satisfactory.

Table 2: Accuracy and Precision of the proposed methods

Type of Assay	Concentration((μg/mL) Taken	Found	SD	%RSD	%Recovery	%Error
Intra- day	4	4.003	0.016	0.408	99.9	0.1
	12	11.951	0.029	0.243	99.59	0.4
	20	19.95	0.03	0.13	99.73	0.3
Inter- day	4	3.95	0.03	0.75	98.87	1.1
	12	11.90	0.05	0.39	99.19	0.8
	20	19.95	0.02	0.11	99.77	0.2

Accuracy:

The accuracy of the proposed method was established by performing intra-day and inter-day assays by determining at different levels of drug concentrations [lower concentration (50%), intermediate concentration (100%) and higher concentration (150%)] within 1 day and 3 consecutive days, respectively. The accuracy of the methods is expressed as percentage recoveries and percentage error. The results obtained by both the methods are found to be in the acceptable range. Therefore, we can say it can be considered as satisfactory.

In addition, accuracy and validity of the proposed methods were determined by standard addition technique. The pre analyzed samples were spiked with additional 50,100 and 150% were once again analyzed by the proposed methods. The accuracy of the methods was evaluated by percentage recovery of the Rilpivirine. The average recovery and percentage standard deviation values (Table 3) of the methods lying in the acceptable range show that the methods are accurate.

Table 3: Results of standard addition technique of proposed method

S.No	Tablet Concentration(mg)	Spiked	Found	SD	%RSD	%Recovery
1	25	10	9.990	0.041	0.408	99.9
2	25	20	19.84	0.08	0.39	99.19
3	25	30	29.66	0.22	0.75	98.87

Robustness:

The robustness of the proposed methods was checked for each operational parameter and investigated. The operational parameters were:

Volume of Sodium Carbonate: 0.8 ± 0.1 mL

Volume of 0.4% SNP: 1 ± 0.1 mL

Volume of 0.4% HA HCL: 0.8 ± 0.1 mLThe robustness of the methods was assessed by analyzing the Rilpivirine at two different concentration levels (4 and 20 μg/mL).The percent recovery and % RSD of the method (Table 4) was found to be satisfactory, indicating that the method is robust

Table 4: Robustness of proposed method

S.NO

Experimental Parameter

Volume

(ml)

TEN Taken(4 μg/ml)

Absorbance

%Recovery

%RSD

TEN Taken

(20 μg/ml)

Absorbance

%Recovery

%RSD

Hydroxyl Amine HCL

0.7

0.8

0.9

0.205

0.202

0.207

99.52

97.58

100

1.29

0.75

0.39

0.881

0.887

0.884

99.55

100.23

99.89

1.09

0.45

0.79

Sodium Nitroprusside

0.9

1.0

1.1

0.206

0.203

0.205

99.52

98.07

99.03

0.37

0.40

0.71

0.883

0.885

0.884

99.77

100

99.89

1.21

0.51

0.62

Sodium Carbonate

0.7

0.8

0.9

0.204

0.203

0.206

98.55

98.07

99.52

0.28

0.42

0.68

0.882

0.886

0.881

99.65

100.11

99.59

0.45

0.15

0.49

Application of the proposed methods to analysis of Rilpivirine in tablet formulations.

It is obvious from the above mentioned results that the proposed methods gave satisfactory results with Rilpivirine in bulk. Therefore, Rilpivirine tablet formulations were subjected to the analysis of their Rilpivirine contents by the proposed methods. The percent recovery and %RSD (Table 5) clearly showed no interference of any excipients of formulation, thus proving accuracy and precision in the quantification of Rilpivirine.

Table 5: Results of analysis in tablet formulation

Formulation	Labeled claim(mg)	Found ± SD (n=5)	RSD(%)	Recovery(%)
EDURANT	25	24.96 ± 0.098	0.968	99.60

CONCLUSION:

The proposed methods don’t require any expensive sophisticated apparatus. The methods are simple, rapid and robust and have high precision and accuracy. The SNP and HA HCL are inexpensive reagents and are available in any analytical laboratory. Hence, these methods are valuable for its routine application in quality control laboratories for the analysis of Rilpivirine.

ACKNOWLEDGEMENT:

I express my sincere thanks to Dr. M. Bhagavan Raju, principal for his support and encouragement throughout my research work. I am also thankful to Sri Venkateshwara College of Pharmacy for providing chemicals and instruments and HIQ pharma laboratories limited Hyderabad for providing drug samples for research.

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Received on 19.09.2017 Modified on 28.12.2017

Research J. Science and Tech. 2018; 10(1):19-27

DOI: 110.5958/2349-2988.2018.00004.9