Development, Validation and Forced Degradation Study of Emtricitabine and Tenofovir Alafenamide in its Pharmaceutical Dosage Form Using RP-HPLC

Aims: The present research was aimed to develop and validate a reverse phase high performance liquid chromatographic (RP-HPLC) method for the quantification of Emtricitabine (EMT) and Tenofovir Alafenamide (TEN) in combination. Methodology: Separation was achieved under optimized chromatographic condition on an Inertsil C18, 250 x 4.6 mm, 5μm column. Various composition of mobile phase was tried. Separation of EMT and TEN was started with Methanol: Buffer and Methanol finally using solvent system of Buffer (pH 3.5) and Methanol in ratio of (30:70) and flow rate adjust at 1.0 ml/min was used as solvent system, the detection was carried out at 262nm using Shimazdu UV-visible detector. The mobile phase run time for the developed analytical method was 10 minutes. Results: The standard curve was found linear in the concentration range of 20-60 μg/ ml (r0.9994) and 2.5-7.5 μg/ ml (r 2 -0.9992) for EMT and TEN respectively. The %RSD was found to be 0.80-0.95% and 0.63-1.09 for EMT and TEN respectively. Percentage (%) recoveries for EMT and Original Research Article TEN to be in range of 100%-100.6% and 99.32% the limit of quantification were found to be 4.80 μg/ ml and 14.7 μg/ ml respectively f 0.11 μg/ ml and 0.33μg/ ml respectively for TEN. Results of forced degradation study showed EMT degradation in acid and base medium while TEN was showed degradation in oxidative stress. The proposed developed RP-HPLC method was validated statist within the acceptable limits. Conclusion: In conclusion, the developed RP rugged for simultaneous estimation of EMT and TEN. Validation results of method was found within the acceptable limits. Hence it can be used for analysis of EMT and TEN.


INTRODUCTION
Human immunodeficiency virus (HIV) is a virus that damages the immune system. The immune system helps the body fight off infections. Untreated HIV infects and kills CD4 cells, which are a type of immune cell called also known as T cells. Over time, as HIV kills more CD4 cells, the body is more likely to get various types of infections and may lead to cancer. (EMT) is a nucleoside reverse transcriptase inhibitor (NRTI) for the treatment of HIV infection in adults. Emtricitabine (EMT) is an analogue of cytidine (Fig. 1). Tenofovir Alafenamide (TEN) is a nucleotide reverse transcriptase inhibitor (NRTI) and a novel ester prodrug of the antiretroviral tenofovir (Fig. 2).
The fixed dose of the EMT and TEN is approved by FDA. The formulation of EMT and TEN is commercially available in the Indian market for treatment of HIV [1,2]. So, as per FDA, valida is an essential requirement in any pharmaceutical industry to build and designed 38 100.6% and 99.32%-100.83% respectively. The limit of detection and the limit of quantification were found to be 4.80 μg/ ml and 14.7 μg/ ml respectively f 0.11 μg/ ml and 0.33μg/ ml respectively for TEN. Results of forced degradation study showed EMT degradation in acid and base medium while TEN was showed degradation in oxidative stress. The HPLC method was validated statistically and the values were found to be

Chemical Structure of EMT
In conclusion, the developed RP-HPLC method was found to be simple, specific, and rugged for simultaneous estimation of EMT and TEN. Validation results of method was found within the acceptable limits. Hence it can be used for analysis of EMT and TEN.

HPLC; EMT; TEN; validation.
Human immunodeficiency virus (HIV) is a virus that damages the immune system. The immune body fight off infections. Untreated HIV infects and kills CD4 cells, which are a type of immune cell called also known as T cells. Over time, as HIV kills more CD4 cells, the body is more likely to get various types of infections and may lead to cancer. Emtricitabine (EMT) is a nucleoside reverse transcriptase inhibitor (NRTI) for the treatment of HIV infection in adults. Emtricitabine (EMT) is an analogue of 1). Tenofovir Alafenamide (TEN) is a nucleotide reverse transcriptase inhibitor RTI) and a novel ester prodrug of the The fixed dose of the EMT and TEN is approved by FDA. The formulation of EMT and TEN is commercially available in the Indian market for treatment of HIV [1,2]. So, as per FDA, validation is an essential requirement in any pharmaceutical industry to build and designed quality, safety and efficiency of product. The literature survey reveals that these drugs have been analyzed along with many other Anti agents by analytical methods l spectrophotometric [3], HPTLC [4], RP Stability Indicating RP-HPLC [6], Bioanalytical LC-MS [7] etc. but no any RPhave been reported for the estimation of specifically EMT and TEN. Before commercial production, the company need t process validation of TEN and EMT dosage forms for assurance of the quality. Quantitative analysis of any drug is an important tool in industry, so it was thought of interest to develop and validate chromatographic method for TEN and EMT. The number of the drug and drug formulation introduced into the market has been increasing at alarming rate, so at the end of the project comparison of all the marketed TEN and EMT will be carried out. In the present research work, an attempt was made to Deve validate of chromatographic Method for Estimation of TEN and EMT in bulk and tablet dosage form. 100.83% respectively. The limit of detection and the limit of quantification were found to be 4.80 μg/ ml and 14.7 μg/ ml respectively for EMT and 0.11 μg/ ml and 0.33μg/ ml respectively for TEN. Results of forced degradation study showed EMT degradation in acid and base medium while TEN was showed degradation in oxidative stress. The ically and the values were found to be HPLC method was found to be simple, specific, and rugged for simultaneous estimation of EMT and TEN. Validation results of method was found within quality, safety and efficiency of product. The literature survey reveals that these drugs have been analyzed along with many other Anti-HIV agents by analytical methods like spectrophotometric [3], HPTLC [4], RP-HPLC [5], HPLC [6], Bioanalytical HPLC method have been reported for the estimation of specifically EMT and TEN. Before commercial production, the company need to perform the process validation of TEN and EMT dosage forms for assurance of the quality. Quantitative analysis of any drug is an important tool in industry, so it was thought of interest to develop and validate chromatographic method for TEN number of the drug and drug formulation introduced into the market has been increasing at alarming rate, so at the end of the project comparison of all the marketed TEN and EMT will be carried out. In the present research work, an attempt was made to Develop and validate of chromatographic Method for Estimation of TEN and EMT in bulk and tablet Chemical Structure of TEN

Chemicals and Reagents
Potassium Dihydrogen Phosphate was used in the method development was of HPLC grade. Other solvents like methanol and water was also of HPLC grade. Emtricitabine and Tenofovir Alafenamide was gifted by Emcure Pharmaceuticals Ltd, Ahmedabad. % purity of both EMT and TEN was 101.21% and 100.21% respectively, as per Certificate of Analysis (CoA) supplied by Emcure Pharmaceuticals Ltd, Ahmedabad.

Instrumentation
Shimadzu HPLC system containing LC-20AT pump and SPD-20AT UV-visible detector was used for the development of analytical method and forced degradation study. HPLC system was used an isocratic elution technique at a flow rate of 1ml/min on an Inertsil C18 (GL Sciences, Japan) 250 x 4.6 mm x 5μm column at ambient temperature. A Rheodyne injector (20 μl) was used for injecting the sample. Detection wavelength for EMT and Ten was 381nm and 272nm, respectively.

Preparation of Sample Solution
Standard stock solution of EMT (400 µg/ml) was prepared by adding accurately weighed quantity of EMT (40 mg) to 100 ml volumetric flask, dissolved and diluted up to the mark with Buffer (pH 3.5): Methanol (30:70) to give a stock solution of 400μg/ml. Standard stock solution of TEN (50 µg/ml) was prepared by adding accurately weighed quantity of TEN (5 mg) to 100 ml volumetric flask, dissolved and diluted up to the mark with Buffer (pH 3.5): Methanol (30:70) to give a stock solution of 50μg/ml. Transfer 1 ml of standard stock solution of EMT and TEN to 10 ml volumetric flask and dilute up to mark with Buffer (pH 3.5): Methanol (30:70). (Final Sample concentration 40μg/ml(EMT) & 5μg/ml (TEN). Each solution was scanned between 200-400 nm and the spectrum was recorded. The point at which drug shows absorbance was selected as wavelength for determination. Sensitivity of HPLC method that uses UV detection depends upon proper selection of detection wavelength. An ideal wavelength is the one that gives good response for the drugs that are to be detected.

System suitability test parameters
System suitability testing is an internal part of a liquid chromatographic method, and it is used to verify that the chromatographic method is able to produce good resolution between the peaks of interest with high reproducibility. The system suitability was determined by making six replicate injections from a freshly prepared standard solution of 5 µg/ml of TEN and 40 µg/ml of EMT and analyzing each solute for its retention time (Rt), Number of theoretical plates (N), resolution (RS) and tailing factor (T). The system suitability method acceptance criteria set in each validation run were-a %RSD<2% , Capacity factor > 2.0, tailing factor ≤ 2.0, and theoretical plates>2000 [8][9][10][11].

Selectivity
It is ability of the method to measure specifically the analyte of interest, in the presence of other components, such as impurities, degradation products, excipients that be expected to be present in the sample preparation.

Linearity and range (n=5)
Aliquots of working standard solution (0.5, 0.75, 1.0, 1.25, and 1.5 ml) of EMT (400 μg/ml) were transferred to a series of 10 ml volumetric flask. The volume was adjusted up to the mark with Diluent to obtain 20, 30, 40, 50, and 60 μg/ml of Emtricitabine. Aliquots of working standard solution (0.5, 0.75, 1.0, 1.25 and 1.5 ml) of TEN (50 μg/ml) were transferred to a series of 10 ml volumetric flask. The volume was adjusted up to the mark with Diluent to obtain 2.5, 3.75, 5, 6.25 and 7.5 μg/ml of TEN. An aliquot of 20µl of each solution was injected under operating chromatographic conditions. Plot the calibration curve of area versus respective concentration and find out correlation coefficient and regression line equation for EMT and. TEN Each response was an average of five determinations.

Precision
Intraday precision (n=3) was determined by analyzing of EMT and TEN standard solution in the range EMT (20, 40, and 60 µg/ml) & TEN (2.5, 5 and 7.5 µg/ml) were analyzed on three times on same day and % RSD was calculated. Interday precision (n=3) was determined by analyzing of EMT and TEN standard solution in the range EMT (20,40, and 60 µg/ml) & TEN (2.5, 5 and 7.5 µg/ml) were analyzed on three different successive and % RSD was calculated. Repeatability (n=6) was determined by analyzing EMT and TEN test solution having the concentration 40µg/ml & 5µg/ml of EMT and TEN Measure six times. Calculate %RSD for EMT and TEN [12,13,14].

Accuracy (n=3)
The accuracy of the method was determined at 50%, 100% and 150% by calculating recoveries of EMT and TEN by the standard addition method. Known amount of standard solutions of EMT and TEN were added to pre-quantified sample solution of EMT and TEN. Each solution was injected in triplicated and the percentage recovery was calculated by measuring the peak areas and fitting these values into the regression equation of the respective calibration curves [13].

Limit of detection (LOD) and limit of quantitation (LOQ)
LOD and LOQ of the drug were calculated using following equations according to ICH guideline. LOD = 3.3 σ/s and LOQ = 10 σ/s Where σ is the SD of the response and S is the slope of the calibration curve.

Robustness
The robustness study was performed to evaluate the influence of small but deliberate variation in the chromatographic condition. The robustness was checked by changing three small changes.

Forced Degradation Study
Stress degradation study was carried out under the acid catalysis and base catalysis. Study was also performed under oxidative stress, thermal degradation and photolytic degradations [15,16,17]. Standard stock solutions of EMT (400µg/ml) and TEN (50 µg/ml) was prepared by dissolving accurately weighed 40mg of EMT Reference Standard and 5mg of TEN Reference Standard to 100ml volumetric flask respectively. The final volume was made up with the Diluent.

Preparation of sample for acid degradation
1 ml of EMT Standard Stock solution (400 µg/ml) and TEN Standard Stock solutions (50 µg/ml) was transferred to 10ml volumetric flask, respectively. 2 ml of HCl of different concentration was added. Keep the solution for particular time period [18].

Preparation of sample for base degradation
1 ml of EMT Standard Stock solution (400 µg/ml) and TEN Standard Stock solutions (50 µg/ml) was transferred to 10ml volumetric flask, respectively. 2 ml of NaOH of different concentration was added. Keep the solution for particular time period [19,20].

Preparation of sample for oxidation degradation
1 ml of EMT Standard Stock solution (400 µg/ml) and TEN Standard Stock solutions (50 µg/ml) was transferred to 10ml Volumetric flask and 2ml different concentration of H 2 O 2 of was added, respectively. Keep for particular time period.

Sample preparation for photo degradation
Accurately weighed 40mg of EMT (40 mg) and TEN (5mg) Reference Standard was transferred to 100ml volumetric flask, respectively. The solutions were kept it into UV chamber for time period specified as per ICH. After specific time period the volume was made up with mobile phase.

Sample preparation for thermal degradation
Accurately weighed 40mg of EMT (40mg) and TEN (5mg) Reference Standard was transferred to 100ml volumetric flask, respectively. The solution was kept it into Oven at 80 0 C temperature for time period specified as per ICH, after time period the volume was made up with mobile phase.

Method Development of RP-HPLC Method for EMT and TEN
For development of method development various chromatographic condition trail was carried out in preliminary experimental work. Chromatographic condition of the HPLC system was consisted of Inertsil C18 column (250 x 4.6 mm x 5μm) as stationary phase. The mobile phase composition was set as Buffer (pH 3.5): Methanol (30:70). Flow rate of mobile phase was set to 1ml/min. volume of the HPLC injection was set to 20µL. The wavelength for detection was set to 262nm.
The final chromatographic condition for the method development was depicted in Table 1. Chromatogram of the mobile phase run was showed in Fig. 1.

Linearity
A method was found linear in a range of 20-60 mcg/ml & 2.5-7.5mcg/ml of EMT & TEN of standard concentration respectively were found linear as shown in Fig. 2. A correlation coefficient for EMT and TEN was 0.9994 & 0.9992 respectively. The areas obtained were directly proportional to the concentration of analyte in the sample. The method can, therefor be termed as linear in the specified range.

Precision
The Intraday precision was assessed by analyzing samples of pharmaceutical formulation (n=3) The %RSD was found to be 0.80-0.95 % and 0.63-1.09 for EMT and TEN respectively. The Interday precision was determined using mean values and the % RSD for the analysis of

Fig. 2. Linearity overlay chromatogram of EMT and TEN
three samples of the pharmaceutical formulation on different days. The % RSD was found to be 1.11-1.46 % and 1.30-1.34 for EMT and TEN respectively. The precision evaluated as the repeatability in th % RSD was found to be 1.45% and 1.32% for EMT and TEN respectively. The results are shown in Table 2 and Table 3. The results obtained were well within the acceptance criteria. The method at selected chromatographic condition can therefore be termed as precise.

Accuracy
The accuracy of the method was determined by standard addition method at three different concentration level i.e 80%, 100% and 120%. The results of the accuracy were presented in Table 4 for EMT and TEN. It showed % recoveries for EMT and TEN to be in range of 100%-100.6% and 99.32%-100.83% respectively. The % recovery at each level, mean% recovery, % RSD met the established acceptance criteria.

Robustness
The robustness data for the method was given tin Table 5. The %RSD is below 2% which is within the given limit of acceptance criteria. The method at selected chromatographic condition is therefore robust.

LOD and LOQ
The LOD and LOQ were obtained by using the mean of the slope and the standard deviation of the intercept of the independent curves. The limit of detection and the limit of quantification were found to be 4.80 μg/ ml and 14.7 μg/ ml respectively for EMT and 0.11 μg/ ml and 0.33μg/ ml respectively for TEN.

System suitability and specificity
The % RSD values calculated in the system suitability test for the different parameters tested were within the acceptable range (RSD < 2.0%) as presented in Table 6. This result showed that the system is suitable for the analysis of the EMT and TEN. These results indicate that the analytical method was found to be specific as there was no interference of any excipients or impurities.

Forced Degradation Studies
Stress degradation studies were performed as per the ICH guideline. Table 7 shows results of degradation studies performed on EMT and TEN. It was observed that both the drugs EMT and TEN have significant degradation in acidic, basic, oxidation, photo degradation and thermal degradation. Results indicated, in acid and base EMT found more degradation and TEN showed more degradation with oxidative stress. As per ICH guidelines peak, purity angle should be less than peak purity threshold. Hence, degradation products of EMT and TEN was not interfere in the analysis of EMT and TEN using proposed method. So proposed method was also used for determination of stability of EMT and TEN in pharmaceutical dosage form. Chromatogram resulted in the various stress study was presented in Fig. 3.

CONCLUSION
In the present research, EMT and TEN was simultaneously estimated by RP-HPLC. The results of the validation studies in the present research work indicated that the proposed method was specific, robust, selective, linear and high precision characteristics without any interference from the excipients and degradation products. RP-HPLC method for selected combination of drug was not till reported. Therefore, the developed method was successfully used for quantitative analysis of EMT and TEN in synthetic mixture of EMT and TEN.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
As per international standard or university standard written ethical approval has been collected and preserved by the author(s).