Stability Indicating RP-UPLC-PDA Method Development, Validation of Multi Drug Combination of Emtricitabine, Tenofovir Alafenamide and Rilpivirine in Bulk Drug and its Tablet Formulation

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^1-3 .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 patience ,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.

Materials and Methods

Details of Chemicals

Emtricitabine, Rilpivirine, and Tenofovir Alafenamide Standards (Aurabindo Pharma Pvt Ltd), KH₂PO₄ (FINE chemical LTD), Water and Methanol for UPLC (LICHROSOLV (MERCK), Acetonitrile (Batch: IA51F65129, Merck)

Instruments used

UPLC: WATERS, Aquity Empower, 2695 separation module, PDA detector

UV/VIS spectrophotometer, LABINDIA UV3000⁺

 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

300ppm of Emtricitabine, 450ppm of Tenofovir Alafenamide and 37.5ppm 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 200mg of Emtricitabine, 300mg of Tenofovir Alafenamide and 25mg of Rilpivirine powder sample in a 100mL 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 300ppm of Emtricitabine, 450ppm of Tenofovir Alafenamide and 37.5ppm of Rilpivirine

Procedure

Inject 2 mL of the standard, sample (each six replicates) into the UPLC system and calculate the Percentage RSD and calculate the %Assay by using the formulae.

Method Validation

Precision

Procedure

The standard solution was injected for six times and measures 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 measured 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.

Calculation of S/N Ratio

Average Baseline Noise obtained from Blank:    66 µV

Signal Obtained from LOD solution:    198 µV

S/N =198/66 =   3.00

Limit of Quantification of Tenofovir Alafenamide

45 µg/ml, 0.61 µg/ml solutions were Prepared by taking suitable aliquots from Stock by using mobile Phase as diluents,degassed,filtered through membrane filter .

Calculation of S/N Ratio

Average Baseline Noise obtained from Blank:    66 µV

Signal Obtained from LOQ solution:    659µV

S/N = 659/66 = 9.98

Acceptance Criteria

S/N Ratio value shall be 10 for LOQ solution.

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

100ppm of Emtricitabine, 150ppm of Tenofovir Alafenamide and 12.5ppm of Rilpivirine

Sample – II

200ppm of Emtricitabine, 300ppm of Tenofovir Alafenamide and 25ppm of Rilpivirine

Sample – III

300ppm of Emtricitabine, 450ppm of Tenofovir Alafenamide and 37.5ppm of Rilpivirine

Sample – IV

 400ppm of Emtricitabine, 600ppm of Tenofovir Alafenamide and 50ppm of Rilpivirine

Sample – V

500ppm of Emtricitabine, 750ppm of Tenofovir Alafenamide and 62.5ppm 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

Optimized Chromatographic Conditions

Temperature:        Ambient

Column:        Thermosil Octa Decyl Column (4.6 x 50mm, 1.7 mm)

Buffer:        0.1M Tri Ethyl Amine   of   P^H 3.0

Mobile phase:        35% buffer 65% Acetonitrile

Rate of flow into UPLC:        0.3 mille liters per minute

Wavelength:        260 mm

Injection volume:        2 ml

Sample Run time:        3.0min

The %RSD for the area of six replicate samples was found to be within the specified limits for Precision, Intermediate Precision

Table 1: Percentage Degradation

Degradation aid	Emtricitabine	Tenofovir Alafenamide	Rilpivirine
Standard	0.8	1.2	1.3
Acid	3.15	3.01	8.08
Base	5.77	4.27	3.44
Peroxide	4.15	4.97	8.37
Thermal	8.07	6.47	8.74
Photo	5.31	4.37	6.08

 

Table 2:  Accuracy results for Tenofovir Alafenamide

%Concentration (at specification Level)	Amount Added (mg)	Amount Found (mg)	% Recovery	Mean Recovery
50%(n=6)	150	151.87	101.25	100.48
100%(n=6)	300	301.88	100.63
150%(n=6)	450	448.04	99.57

 

Figure 1: Typical chromatogram of Standard   Click here to View Figure

 

Figure 2: Typical chromatogram of Sample Click here to View Figure

 

Figure 3: Typical chromatogram of base  degradation   Click here to View Figure

 

Figure 4: Typical chromatogram of acid degradation Click here to View Figure

 

Figure 5: Structure of degradation Product at 1.975 Rt Click here to View Figure

 

Discussion

The Method Validation is done  for all  parameters  and the results were within the acceptance criteria, Structure of  acid, base ,peroxide ,photolytic degradation product  at the retention time of 1.975 is collected by using preparative UPLC , degradation 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.

---

This work is licensed under a Creative Commons Attribution 4.0 International License.