Stability Indicating RP-UPLC-PDA Method Development , Validation of Multi drug Combination of Emtricitabine , Tenofovir Alafenamide and Rilpivirine In Bulk drug & its tablet formulation

Stability representing Ultra Performance LC method was developed for Assay of multi drug Combination of Rilpivirine, Emtricitabine and Tenofovir alafenamide in bulk active pharmaceutical ingredients & its tablet formulation, Validation was performed for all parameters .Retention times of Reference Standard Emtricitabine, Tenofovir Alafenamide and Rilpivirine was found to be 0.965, 1.528, 2.186 respectively, with the flow rate of 0.3 mille liters per minute by Injecting Volume of 2 micro liters by maintaining Run time of 3 minutes. Developed method was subjected to forced degradation studies under specified conditions, which meets the required criteria. Degradation product at 1.975 Rt was collected under various stress condition, cleavage of imidazole ring of tenafovir alafenamide confirmed with Proton NMR, ESI –MASS in + MODE.


INTRODUCTION
Emtricitabine, Rilpivirine, and Tenofovir Alafenamide combination is used in the treatment of HIV-1 as initial therapy as a part of anti retroviral therapy [1][2][3] .This combination was approved in March 2016 by US-FDA.Literature survey indicate no UPLC -PDA (stability indicating) method for this new fixed dose Alafenamide derivative combination 4-6.As the Tenofovir disproxil fumarate causing the release of formic acid with frequent administration in HIV-I patients, which ultimately cause GI disturbances, henceforth alternative analogue that is Tenafovir Alafenamide was proved alternative drug to that of previous analogue, The current work describes the stability indicating accurate, Precise Ultra Performance LC method for quantifying Emtricitabine, Rilpivirine, Tenofovir Alafenamide in bulk drug &its formulation by using PDA detector.
The ICH recommend stability testing for combined drug formulation for enhancing safety and to avoid the interference of degradation products during the routine analysis 7,8 .The Objective of current work was to develop the method for quantifying and to study the degradation process under various stress condition for assay of long term stability testing samples and in routine analysis.UV/vis spectrophotometer, LABINDIA UV 3000 3.

Details of Chemicals
Weighing Machine (afcoset ER-200A)) Preparation of Diluents Buffer 0.1M tri ethyl amine buffer was prepared , P H was adjusted to 3 with Ortho phosphoric acid and filtered through membrane filter of pore size 0.45 microns.

Mobile phase
To 35% of 0.1M tri ethyl amine buffer add 65% Acetonitrile HPLC Grade, degassed and filtered through membrane filter under vacuum condition.

Standard Solution Preparation
300 ppm of Emtricitabine, 450 ppm of Tenofovir Alafenamide and 37.5 ppm of Rilpivirine was prepared by using the mobile phase and injected into liquid chromatography (Standard Stock Solution).

Sample Solution Preparation
Accurately weigh 10 tablets, crush and take equivalent to 200 mg of Emtricitabine, 300 mg of Tenofovir Alafenamide and 25 mg of Rilpivirine powder sample in a 100 mL volumetric flask, transfer 65 mL of mobile phase and subject to sonication for 30 minutes make up the volume with diluent.Then it is filtered through 0.45 micron filter (Sample Stock solution) From the above take aliquots to prepare 300 ppm of Emtricitabine, 450 ppm of Tenofovir Alafenamide and 37.5 ppm of Rilpivirine.

Procedure
The standard solution was injected for six times and measure the area for all six replicate samples in UPLC.

INTERMEDIATE PRECISION/RUGGEDNESS Procedure
The standard solutions prepared in the precision were injected on the other day, for six times and measure the area for all six samples in UPLC.

Acceptance Criteria
The % RSD for the area of six standard injections results should not be more than 2%.

Limit of Detection of Tenofovir Alafenamide
450 µg/ml, 0.18 µg/ml solutions were Prepared by taking suitable aliquots from Stock by using mobile Phase as diluents.

LINEARITY
From the stock solution, suitable aliquots were taken to prepare the following samples of various drug concentrations by using mobile phase as diluent.

Sample-I
100 ppm of Emtricitabine, 150 ppm of Tenofovir Alafenamide and 12.5 ppm of Rilpivirine.

Procedure
2 micro liters of each level sample was injected into liquid chromatography, peak areas were measured, calculate correlation co efficient value by plotting the graph.

DEGRADATION STUDIES Hydrolytic degradation under acidic condition
Transfer 1.5 ml of standard stock to volumetric flask of 10ml and add 3 ml of 0.1N Hydrochloric acid, flask was subjected to temperature of 60ºC for a period of 6 hours and the sample is neutralized with 0.1 N Sodium hydroxide and make up the volume to 10ml with mobile phase diluents, filter ,inject into the system under optimized chromatographic condition .

H y d ro ly t i c d e g r a d a t i o n u n d e r a l k a l i n e condition
Transfer 1.5 ml of standard stock to the volumetric flask of 10ml and add 3 ml of 0.1 N Sodium hydroxide , flask was subjected to temperature of 60ºC for a period of 6 h and the sample is neutralized with 0.1N hydrochloric acid and make up the volume to 10ml with mobile phase diluents, filter ,inject into the system under optimized chromatographic condition.

Thermally induced degradation
Emtricitabine, Tenofovir Alafenamide and Rilpivirine sample was taken in Petri dish and kept in Hot air oven at 110 0 C for 24 hours.Then the sample was taken and diluted with diluents and injected into UPLC and analyzed.

Oxidative degradation
Transfer 1.5 ml of standard stock to the volumetric flask of 10ml and add 1 ml of 3% w/v of hydrogen peroxide, make up the volume to the mark and keep the flask at room temperature for a period of 15 minutes, filter, inject into the system under optimized chromatographic condition.The %RSD for the area of six replicate samples was found to be within the specified limits for Precision, Intermediate Precision.

Optimized Chromatographic Conditions
The Method Validation was done for all parameters and the results were within the Acceptance criteria.Acid, base, peroxide ,photolytic degradation product at the retention time of 1.975 was collected by using preparative UPLC , cleavage of imidazole ring of tenafovir alafenamide confirmed with Proton NMR, ESI -MASS in + MODE and developed method Proved to be effective even in the presence of degradation products.

CONCLUSION
Forced degradation studies were performed for the developed method as per US FDA, ICH guidelines, hence proved that developed method is stability indicating, can be very effective in bulk drug synthetic sites & even in formulation premises for quantifying the amount of drug in the tablet formulation .