Development and Validation of Quality by Design based RP-HPLC Method for determination of Tenofovir Alafenamide Fumarate from Bulk drug and Pharmaceutical dosage form and its application to Forced Degradation Studies

Aakanksha J. Wankhade*, Purnima D. Hamrapurkar

Department of Pharmaceutical Analysis, Prin. K. M. Kundnani College of Pharmacy,

Plot No. 23, Jote Joy Building, Rambhau Salgaonkar Road, Cuffe Parade, Mumbai, Maharashtra, India.

*Corresponding Author E-mail: aakankshaw1996@gmail.com

ABSTRACT:

The present research work entails the development and validation of a simple, robust and sensitive stability indicating reverse phase high performance liquid chromatographic method using Quality by Design methodology for estimation of Tenofovir alafenamide fumarate in bulk drug and its marketed dosage form. Chromatographic separation was performed in isocratic mode using Inertsil C₁₈ column, mobile phase composed of Acetonitrile: ammonium formate buffer pH 5(40: 60) at a flow rate of 1mL/min with Photo Diode Array (PDA) detection at 261nm. A Box-Behnken statistical design with 3 factors and 3 levels was selected for the optimization study. Interaction effects of Critical Quality Parameters (Buffer pH, Organic Phase-% acetonitrile, flow rate) were evaluated for critical quality attributes (Retention time, NTP and symmetry factor). The optimized method was validated according to the current International Conference Harmonization (ICH) Q2 R1 guidelines. The method was found to be linear, covering the range from 1.5 μg/mL – 4.5 μg/mL with correlation coefficient r² of 0.9996 and precision was found to be less than 2%. Limit of Detection and Limit of Quantification was found to be 1µg/mL and 1.2µg/mL respectively. Accuracy was between 99% and 102%. Forced degradation study was conducted under degradation conditions like light, oxidation, dry heat, acidic, basic, hydrolysis in order to establish the intrinsic stability of molecule. The method was successfully applied for the determination of the drug in commercial dosage form. This holistic stability indicating chromatographic method focused on QbD approach ensured reproducibility and was found to be useful for routine analysis of Tenofovir alafenamide fumarate.

KEYWORDS: RP-HPLC-PDA, Quality by Design, Box Behken design, forced degradation, validation, stability indicating method.

1. INTRODUCTION:

Tenofovir alafenamide fumarate (TAF) belongs to the class of selective reverse transcriptase nucleotide inhibitor (NRTI). It is novel ester (phosphonamidate) prodrug of antiretroviral tenofovir¹. Tenofovir suppresses viral replication by inhibiting HBV reverse transcriptase. TAF is indicated for the treatment of chronic hepatitis B in adults with compensated liver disease³.

Chemically, it is (E)-but-2-enedioic acid; propan-2-yl (2S)-2-[[[(2R)-1-(6-aminopurin-9-yl) propan-2-yl] oxymethyl-phenoxyphosphoryl]amino] propanoate with molecular formula of C₂₃H₃₁N₆O₇P and a molecular weight- 476.47 g/mole ².

Figure 1 Structure of Tenofovir alafenamide fumarate ³

It is revealed from the literature search that fFew analytical methods are reported for the determination of Tenofovir alafenamide Fumarate in bulk drug, plasma and pharmaceutical dosage forms along with the degradation study^4,8. However, there are no methods reported for routine analysis of the drug by using mobile phase which is MS compatible^4,7. Hence, an attempt was made to develop and validate a simple, precise and robust stability indicating reverse phase HPLC method focused on the Quality by Design methodology. Implementation of analytical Quality by Design (AQbD) was required for better understanding of influence of critical chromatographic factors on method performance ^5,6. The anticipated method has distinct advantages over previously stated methods with its cost effectiveness by using acetonitrile and ammonium formate buffer, short run time and is devoid of any interference of formulation excipients.

2. EXPERIMENTAL:

2.1 Chemicals and reagents:

The Tenofovir alafenamide fumarate standard was procured from Lupin Pvt. Ltd. as a gift sample along with its certificate of analysis. HPLC grade Acetonitrile and ammonium formate was used for performing the study. Purified water was obtained from Milli‐Q (Millipore) water purification system. All the other solvents and chemicals used were of analytical grade. Emtaf-B, a commercial dosage form of the drug was purchased from local market for conducting the assay.

2.2 Instrumentation:

The drug was estimated on Waters HPLC (2695 separation module), equipped with photo diode array detector (Waters 2996) and a quaternary pump on a reverse phase Inertsil C₁₈ column (250mm x 4.6mm x 5μm). The data was integrated using Empower software. Instruments such as pH meter (Labindia), sonicator (Spectralab), autopipettes (Eppendorf) and analytical balance (Citizon) were used for sample preparation.

2.3 Chromatographic conditions:

Chromatographic separation was achieved on Inertsil C₁₈ (250mm x 4.6mm x 5μm) in an isocratic mode at a flow rate of 1mL/min. Mobile phase composed of Acetonitrile and ammonium formate buffer (adjusted to pH 5) in ratio 40:60 was selected after numerous trials based on pH, solubility and retention⁸. The column oven temperature was maintained at 25^◦C. Sample injection volume was 10 µL. Prior to the injection of drug solution, the column was equilibrated with the mobile phase¹⁵. The sample was eluted at 261nm with run time of 20 minutes. Peak purity was obtained directly from analysis report produced on Empower software. The forced degradation samples were analysed in scan mode covering the wavelength range of 200–400nm.

2.4 Preparation of standard stock solution:

50mg of working standard of Tenofovir alafenamide fumarate was accurately weighed and transferred to 50 mL of volumetric flask. To it, about 15mL of Acetonitrile was added. It was sonicated and volume was made up with Acetonitrile to give stock solution of 1000 µg/mL (solution A). 5mL of this solution was further diluted to 50mL with Acetonitrile to give a concentration of 100µg/mL (solution B). 5mL of this solution B was further diluted to 50mL with Acetonitrile to give a concentration of 10µg/mL (solution C).

2.5 Preparation of mobile phase:

The optimized mobile phase consisted of Acetonitrile and ammonium formate buffer in a ratio of 40:60. Buffer was prepared by dissolving accurately weighed 315.3mg of ammonium formate in 500mL of Milli-Q water and pH of the solution was adjusted to 5 with formic acid. The mobile phase was prepared daily, filtered through a 0.2μ membrane filter and degassed using sonicator (for 15min) prior to use.

2.6 Initial method development:

a) Choice of analytical wavelength - 10mg of TAF was accurately weighed and dissolved in 10mL of Acetonitrile to give concentration of 1000μg/mL (solution A). 1mL of this solution A was further diluted to 10mL with Acetonitrile to give concentration of 100 μg/mL (solution B). 1mL of this solution B was further diluted with 10mL Acetonitrile to give final concentration of 10μg/mL (solution C). UV spectrum of solution C was recorded by scanning it over a range of 400nm to 200nm using acetonitrile as blank. The drug showed maximum absorbance at 261nm, hence considered as λ_max for the experimental work.

b) Choice of mobile phase - Different solvents such as water, methanol and acetonitrile were checked for optimum solubility and sharp peak as tabulated below. Use of buffer was required to control the ionization state of analyte¹⁵. The mobile phase was selected on the basis of MS compatibility. Tenofovir alafenamide fumarate has pka in the range 3.19. Hence pH within the range of 5-6 would be suitable for anticipated ionization of analyte. Ammonium acetate: ACN mixture was examined. It showed a broad peak. Ammonium acetate interacted with analyte to produce polar ionized species more than the unionized ones leading to inconsistent retention of analyte on the column. The next trial was conducted with ammonium formate. It provided a sharp peak with desired ionization of analyte. Eventually, mobile phase composition of ACN: ammonium formate buffer (40:60) at pH 5 was finalized using QbD trials.

Table 1 Trials for selection of mobile phase

Mobile phase composition	Observation	Inference
Water: Methanol	Interference of methanol	Use of ACN required
Water: ACN	Less precision in retention time. Peak tailing	Use of buffer required
ACN: ammonium acetate	Broad peak	Use of ammonium formate required
ACN: ammonium formate	sharp peak. Peak area improved	Satisfactory

2.7 Analytical method development using Quality by Design approach:

Quality by design methodology was implemented into the experimental work.

a) Method design based on risk assessment – A Box-Behnken design with response surface methodology was executed out for optimization of the chromatographic conditions¹⁴. Buffer pH (A), % Acetonitrile (B) and flow rate (C) were selected as Critical Method Parameters for attaining the desired analytical target profile. The influence and interactions between critical method parameters were studied using a Design of Experiments (DOE) approach. A 3 factor 3 level Box Behnken Design constructed 13 experimental trials. The factors such as retention time, theoretical plates and symmetry factor were found to be critical, hence considered as critical quality attributes⁶. Statistical analysis was performed using the Design Expert 12 software.

Table 2 Box Behnken experimental design

Critical method Parameters (CMP)	Levels Used
Critical method Parameters (CMP)	Low (-)	Center (0)	High (+)
Buffer pH	4.5	5	5.5
%ACN	35	40	45
Flow rate	0.5	1	1.5

2.8.1. Validation of optimized analytical method:

The method was validated⁹ for system suitability, linearity, precision, accuracy, specificity, limit of detection (LOD), limit of quantification (LOQ) and robustness to confirm the suitability of the method for its intended purpose.

2.8.2. Forced degradation study for validated RP-HPLC method:

Force degradation studies were conducted^10,12,21 to determine the stability indicating ability of the optimized method. 10mg of working standard of Tenofovir alafenamide fumarate was accurately weighed and dissolved in 10mL of diluent to give a solution of 1000 µg/mL. 2mL of this solution was further diluted to 10 mL to give a solution of 200µg/mL. These standard samples were exposed to different stress conditions²⁰. All samples were then diluted accordingly to give a final concentration of 200µg/mL. The samples were neutralized if required and filtered prior to injection. The degraded samples are then analyzed using the method to determine if there are interferences with the active. Acid hydrolysis-Solutions for acid degradation were prepared using 0.01 and 0.1 N HCl, protected from light, and stored at room temperature. Samples were withdrawn at 0, 1, 2, 3 hrs time points and diluted as described above. Base hydrolysis - Solutions were prepared using and 0.01N and 0.1 N NaOH, protected from light and stored at room temperature. Samples were withdrawn at 0, 1, 2, and 3 hrs time points and diluted as described above. Oxidative degradation - Solutions for oxidation studies were prepared using 6% H₂O₂, protected from light, and stored at room temperature. Samples were withdrawn at 0, 3 and 6 hrs time points and diluted as described above. Thermal degradation - Standard solution was kept in water bath at 80°C for 3 and 6 hours and diluted as described above. Photolytic degradation - The solution was kept in sunlight for 3 and 6 hours and diluted as described above.

2.9 Application of validated method on the marketed formulation:

10 tablets containing Tenofovir alafenamide fumarate (25mg) were accurately weighed and the average weight of a tablet was recorded. Then 5 tablets were finely powdered and powder equivalent to 50mg of Tenofovir alafenamide fumarate was taken and transferred into a 50 mL volumetric flask. To it, 25mL of diluent was added. It was sonicated with occasional shaking for few minutes. The volume was made up to 50mL with diluent to give a concentration of 1000µg/mL (solution A) and was sonicated for a while. 1mL of this solution was diluted to 10mL with diluent to give a concentration of 100µg/mL (solution B). 1mL of this solution was further diluted to 10mL with diluent to give a concentration of 10µg/mL (solution C). 3mL of solution C was diluted to 10mL with diluent to give a working level concentration of 3µg/mL (solution D). This solution D was injected into the HPLC system. The average area of 3 such injections was taken for calculation^5,11,13.

3. RESULTS AND DISCUSSION:

3.1 HPLC method optimization by Quality by design methodology:

Impact of factors such as Buffer pH (A), % Acetonitrile (B) and flow rate (C) on response variables retention time (Y1), theoretical plates (Y2) and symmetry factor (Y3) was studied by applying Box Behnken multivariate analysis. Evaluation of all of the above critical method parameters with a Box-Behnken lead to 13 experimental trials due to permutation and combination of these parameters²³. These trials and their responses are tabulated as follows-

Table 3 Factor screening by Box Behnken design

Run	Coding X1,X2,X3	Critical Method Parameters			Critical Quality Attributes (Response)
Run	Coding X1,X2,X3	pH	%ACN	Flow rate mL/min	Retention time (min)	NTP	Asymmetry factor
1	--0	4.5	35	1	8.075	4481	1.11
2	-+0	4.5	45	1	4.389	4750	1.22
3	+-0	5.5	35	1	4.9	3800	1.04
4	++0	5.5	45	1	5. 8	5281	1.08
5	-0-	4.5	40	0.5	11.11	4558	1.06
6	-0+	4.5	40	1.5	3.773	4521	1.16
7	+0-	5.5	40	0.5	6.4	5227	1.01
8	+0+	5.5	40	1.5	4.3	4800	1.05
9	0--	5	35	0.5	15.49	3423	0. 815
10	0-+	5	35	1.5	5.3	3587	1.07
11	0+-	5	45	0.5	8.531	4620	1.11
12	0++	5	45	1.5	2. 86	2227	0.93
13	000	5	40	1	5.463	3631	1.15

The response variables were statistically evaluated using the following contour plots and subsequent ANOVA results.

Retention time

Analysis of variance (ANOVA) was used to assess the design data and the importance of the regression coefficients that were estimated by consistent p-values (i.e. p value is consistent at alpha = 0.05).

Table 4 ANOVA summary statistics

Response	R1(Rt)	R2(NTP)	R3(Symmetry factor)
Standard deviation	1.51	330.68	0.017
Mean	6.79	4369	1.06
C.V.%	22.23	7.57	1.65
R- squared	0.9659	0.97	0.9927
Adjusted R squared	0.8638	0.91	0.97

The model F value for retention time, NTP and symmetry factor was found to be 9.46, 15.90, 45.30 respectively. There is only a 4.5%, 2.19%, 0.48% chance (for respective responses) that an F-value this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model terms are significant. The optimum chromatographic conditions were established to be percentage of acetonitrile (40), flow rate (1.0 mL/min) at pH 5 gave the maximum peak area for the Tenofovir alafenamide fumarate.

3.2 Validation of an optimized RP-HPLC method:

3.2.1. System suitability:

System suitability was performed to verify resolution and reproducibility of chromatographic method^16,18. It was carried out by injecting 6 replicates of 3μg/mL standard solution to evaluate parameters like retention factor, theoretical plates and tailing⁹. The System Suitability parameters were found to be within the limits for Tenofovir alafenamide fumarate as listed in table 5

Table 5 System suitability data

Validation parameters	Observation
System suitability	NTP = 4877 %RSD = 0.67 Tailing factor = 1.03 Retention time = 5.495
Specificity	No interference found at the retention time of analyte
Linearity	R²= 0.9998 (Linear) with equation y = 18894x – 80.58
Accuracy	101.2%
Intraday precision	0.71%
Interday precision	0.68%
LOD	1 µg/mL
LOQ	1.2 µg/mL

Figure 2 Chromatogram for system suitability

3.2.2 Specificity:

It was determined by recording the chromatogram of standard stock solution of Tenofovir alafenamide fumarate (10 μg/mL) and blank chromatogram (only diluent). The method was quite specific for the drug as there was no interfering peak around retention time of the drug (RT 5.495). Also, the standard peak of Tenofovir alafenamide fumarate was found to be pure.

Figure 3 Chromatogram of blank (Acetonitrile)

3.2.3. Linearity:

The linearity was tested over the working range of 1.5μg/ mL – 4.5 μg/mL for Tenofovir alafenamide fumarate. Each level was injected six times into HPLC. Chromatograms and their respective peak areas were recorded. Calibration curve plotted with the concentration (in μg/mL) on the x-axis and the response (area) on the y-axis. The correlation coefficient (R2) was found to be 0.998 and the equation of the line was y = 18894x+ 80.58 as evident from the below calibration curve. Thus, the data shows that the response is found to be linear.

Figure 4 Calibration curve for Tenofovir alafenamide fumarate at 261 nm

Table 6 Linearity data

Conc.	Lin1	Lin2	Lin3	Lin4	Lin5	Lin6	Mean	SD	%RSD
1.5	28450	29653	29486	29251	28169	28571	33930	335.26	0.998
2.25	41577	41472	42543	41573	41530	42013	41785	382.21	0.8347
3	56472	56079	55951	56019	56292	56604	56236.2	267.93	0.472
3.75	72944	72201	72262	71996	72319	72506	72371.3	297.39	0.4549
4.5	84653	84542	84858	84141	84113	84113	84627	417.28	0.524

Table 7 Interday precision data

Conc.	Lin1	Lin2	Lin3	Lin4	Lin5	Lin6	Mean	SD	%RSD
1.5	29267	28905	29312	28883	28761	28409	28922.8	335.26	0.9883
3	57164	56740	56923	56617	56519	56454	56736.17	267.93	0.4722
4.5	84056	84842	85045	84923	84143	84679	84614.67	417.28	0.5241

Table 8 Intraday precision data

Conc.	Lin1	Lin2	Lin3	Lin4	Lin5	Lin6	Mean	SD	%RSD
1.5	28421	28905	28983	29064	28737	28583	28822	247.79	0.7334
3	56788	56305	57025	56441	56053	56774	56564.3	360.59	0.6374
4.5	84553	84610	85072	85047	84755	85906	84823.8	618.05	0.7742

3.2.6. Limit of quantification (LOQ) and limit of detection (LOD):

The LOD and LOQ of the developed method were determined by injecting gradually low concentrations of the standard solution of Tenofovir alafenamide fumarate ²². This was done until a signal to noise ratio of NLT 3:1 and NLT 10:1 is maintained for LOD and LOQ respectively. The LOD and LOQ of Tenofovir alafenamide fumarate was found to be 1μg/mL and 1.2 μg/mL. These values indicate that the method developed is sensitive.

3.2.7. Robustness:

Robustness of the method was evaluated by analysing the system suitability parameters after alteration in pH (±0.2), flow rate (±0.2) and % mobile phase (±2.0). A working standard of 3μg/mL was used. %RSD was calculated. The method was found to be robust against small changes in chromatographic conditions.

3.2.8. Stock solution stability:

The stability studies were evaluated for three different concentrations i.e. 1.5μg/mL, 3μg/mL and 4.5μg/mL which were stored for 3 days. Stability was calculated by comparing the results of six replicate injections of stored solutions with fresh samples. The stability test results indicated that the drug solutions are stable up to 48 hours.

3.3 Results for forced degradation study:

Forced degradation studies were performed under various stress conditions. % total degradation ^[18,20] is presented in Table 10. The drug was found to be highly susceptible to hydrolytic conditions, while less susceptible to the oxidative stress condition. The drug was found to be stable under thermal and photolytic conditions.

Figure 5 Chromatogram of acid hydrolysis

Figure. 6 Chromatogram of base hydrolysis

Figure 7 Chromatogram of oxidation

Figure 8 Chromatogram of photolytic degradation

Figure 9 Chromatogram of thermal degradation

Table No. 10 Forced degradation data

Sr. No.	Parameter	Stressed condition	Retention time for degradation products	% degradation
1	Acid hydrolysis	0.01N HCl, 2min	Degradation pdt 1 RT- 2.119 Degradation pdt 2 RT- 7.144	8.169
2	Base hydrolysis	0.01N NaOH, 2min	Degradation pdt 1 RT- 2.115 Degradation pdt 2 RT- 7.156	7.485
3	Oxidative degradation	6%H2O2, 3 hrs	RT- 2.041	1.9805
4	Photolytic degradation	Sunlight, 6 hrs	-	0.0
5	Thermal degradation	Dry heat at 80^ₒC, 6 hrs	-	0.0
	Total degradation			17.63

3.4 Estimation of drug in marketed formulation:

The validated method was successfully applied to estimate^4,17,19 Tenofovir alafenamide fumarate from marketed formulation (EMTAF Tablets 25mg). The representative chromatogram is shown below.

Figure 10 Chromatogram for Tenofovir alafenamide fumarate in the formulation

4. CONCLUSION:

A simple, rapid, cost effective and sensitive stability-indicating analytical method has been developed employing the systematic Quality by Design methodology for determination of Tenofovir alafenamide fumarate in bulk drug and its marketed pharmaceutical formulation. Validation of the method corroborated excellent linearity, precision, accuracy, specificity, system suitability and robustness. Statistical analysis demonstrates the suitability of method for estimation of the drug in pharmaceutical formulation with no interference from the excipients. The method can successfully be extended to routine quality control analysis and bioequivalence studies.

5. ACKNOWLEDGEMENT:

The authors appreciate the technical assistance received from Ultrapure Analytics Pvt. Ltd and are grateful to Lupin Pharma Pvt. Ltd. for providing Tenofovir alafenamide fumarate standard.

6. CONFLICT OF INTEREST:

Authors declare no conflict of interest.

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Received on 05.10.2020 Modified on 11.03.2021

Research J. Pharm. and Tech. 2022; 15(5):2127-2134.

DOI: 10.52711/0974-360X.2022.00353

Conc. (µg/mL)	Area			Mean area	Recovered conc.	Accuracy (%)
Conc. (µg/mL)	Inj. 1	Inj.2	Inj.3	Mean area	Recovered conc.	Accuracy (%)
1.5	28653	28486	28223	28787.3	1.52	99.51
3	56229	56219	56604	56350.6	3.02	100.03
4.5	84600	84972	84858	84810	4.54	100.37

Drug	Amount Labelled	Retention time (min)	Area	NTP	Symmetry Factor	% Assay
Tenofovir alafenamide fumarate	25mg	5.458	56714	3336	1.098	99.75