VALIDATED METHOD FOR THE SIMULTANEOUS DETERMINATION OF EMTRICITABINE, BICTEGRAVIR AND TENOFOVIR ALAFENAMIDE IN PHARMACEUTICAL DOSAGE FORM USING UPLC

VALIDATED METHOD FOR THE SIMULTANEOUS DETERMINATION OF EMTRICITABINE, BICTEGRAVIR AND TENOFOVIR ALAFENAMIDE IN PHARMACEUTICAL DOSAGE FORM USING UPLC

Vamsi Dadi * and G. Sowjanya

Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy, GITAM (Deemed to be University), Rushikonda, Visakhapatnam, Andhra Pradesh, India.

ABSTRACT: The proposed study aimed to develop and validate a method for the simultaneous determination of emtricitabine, bictegravir, and tenofovir alafenamide in pharmaceutical dosage form using ultra-ultra dosage performance liquid chromatography (UPLC). The drugs were separated using HSS C18 (100 x 1.8mm, 1.7m) column with a mobile phase composition consisting of 0.1% orthophosphoric acid (pH 2.2) and acetonitrile in the ratio 70:30%v/v at a flow rate of 0.3ml/min. The column temperature was maintained at 30°C, and a detection wavelength of 260nm was used. The retention times were found to be 0.62mins for emtricitabine, 0.89 min for bictegravir and 1.75 min for tenofovir alafenamide. The developed method was validated in accordance with the international conference on harmonization (ICH) guidelines and found to be accurate, precise, specific, and robust. The method obeyed Beer’s law in the 50 – 300µg/ml concentration range for emtricitabine, 12.5 – 75µg/ml for bictegravir and 6.25 – 37.5µg/ml for tenofovir alafenamide, with a correlation coefficient of 0.999. The developed method can be used for the routine quantitative analysis in quality control for the determination of emtricitabine, bictegravir, and tenofovir alafenamide in the pharmaceutical dosage form.

Keywords: Emtricitabine, Bictegravir, Tenofovir alafenamide, UPLC, Method validation

INTRODUCTION: Emtricitabine ^1-3Fig. 1A, chemically designated as 4-amino-5-fluoro-1-[(2R, 5S) – 2 - (hydroxymethyl) - 1, 3 – oxathiolan – 5 -yl] pyrimidin-2-one is an antiviral drug. It has a molecular formula of C₈H₁₀FN₃O₃S with a molecular weight of 247.25 g/mol and has a pKa value of 2.65.

It is a white to off-white powder and soluble in water. It is used to treat HIV infection, which inhibits the activity of the human immunodeficiency virus (HIV) reverse transcriptase enzyme resulting in deoxyribonucleic acid (DNA) chain termination.

FIG. 1A: CHEMICAL STRUCTURE OF EMTRICITABINE

Bictegravir ^4-6 Fig. 1B, chemical name  2,5-Methanopyrido [1’,2’:4,5] pyrazino [2,1-b] [1,3] oxazepine-10-carboxamide, 2,3,4,5,7,9,13,13a-octahydro – 8 – hydroxyl - 7, 9 – dioxo – N -[(2,4,6-trifluorophenyl) methyl]-, sodium salt (1:1), (2R,5S,13aR)- is an antiviral drug. It has a molecular formula of C₂₁H₁₇F₃N₃NaO₅ with molecular weight of 471.4g/mol has a pKa value of 9.81. It is an off-white to yellow solid having solubility in water.

FIG. 1B: CHEMICAL STRUCTURE OF BICTEGRAVIR

It is used to treat HIV infection by acting as an HIV – I integrase strand transfer inhibitor, which results in the inhibition of HIV replication into the human genome. Tenofovir alafenamide ^7-9 Fig. 1C with chemical nameL-alanine, N-[(S)-[[(1R)-2-(6-amino-9H-purin-9-yl)-1­methylethoxy] methyl] phenoxyphosphinyl]-, 1-methylethyl ester, (2E)-2-butenedioate (2:1) is an antiviral drug. It has an empirical formula of C₂₁H₂₉O₅N₆P•½(C₄H₄O₄) and a formula weight of 534.50g/mol. Tenofovir alafenamide is a white to off-white or tan powder and has solubility in water. It is used to treat chronic hepatitis B infection by stopping or slowing the growth of the virus.

FIG. 1C: CHEMICAL STRUCTURE OF TENOFOVIR ALAFENAMIDE

The literature review reveals that there are very few methods developed for the simultaneous estimation of emtricitabine, bictegravir, and tenofovir alafenamide in their bulk and pharmaceutical dosage forms, such as reversed-phase high-performance liquid chromatography (RP-HPLC) ^10-15, UPLC ^{16, 17} and liquid chromatography-tandem mass spectrometry (LC-MS/MS) ¹⁸. The objective of the present study was to develop and validate a UPLC method for the simultaneous determination of emtricitabine, bictegravir, and tenofovir alafenamide in the pharmaceutical dosage form.

MATERIAL AND METHODS:

Reagents and Chemicals: The emtricitabine, bictegravir, and tenofovir alafenamide working standards were received as gift samples from Hetero Drugs Pvt. Ltd., Hyderabad, India. The tablets (Biktarvy) were purchased from a local pharmacy. All the solvents used were of HPLC grade and purchased from Merck, Mumbai, India. All the chemicals used for developing the method were of analytical reagent (AR) grade and purchased from Sigma Aldrich, India.

Instrumentation and Chromatographic Conditions: Water ACQUITY UPLC^19-21system equipped with Binary solvent manager, a sample manager with HSS C18 (100 × 1.8mm, 1.7µ) column maintained at 30°C, a solvent tray, and ultra violet (UV) detector with detection wavelength set at 260nm was used for the simultaneous determination of emtricitabine, bictegravir and tenofovir alafenamide in pharmaceutical dosage form. All the parameters of UPLC were controlled by empowering software. Electronic balance, digital pH meter, and ultrasonic bath sonicator were other instruments used. The mobile phase used in this method was 0.1% orthophosphoric acid (pH 2.2) and acetonitrile in the ratio 70:30%v/v on isocratic mode at a flow rate of 0.3ml/min.

Preparation of Standard and Sample Solutions: Accurately weighed and transferred 100mg of Emtricitabine, 25mg of Bictegravir, and 12.5mg of Tenofovir working Standards into a 50 ml clean dry volumetric flasks, added 10ml of diluent, sonicated for 10 minutes and made up to final volume with diluents (2000µg/ml Emtricitabine, 500µg/ml Bictegravir and 250µg/ml of Tenofovir). 1ml from the above stock solutions was taken into a 10ml volumetric flask and made up to 10ml (200µg/ml Emtricitabine, 50µg/ml Bictegravir and 25µg/ml of Tenofovir) Accurately weighed equivalent weight of the combination powder (Biktarvy tablets) sample transfer into a 100 ml volumetric flask, 50ml of diluents was added and sonicated for 25 min, further, the volume was made up with diluent and filtered by milli-Q filters (2000µg/ml Emtricitabine, 500µg/ml Bictegravir and 250 µg/ml of Tenofovir). 1ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (200µg/ml Emtricitabine, 50µg/ml Bictegravir and 25µg/ml of Tenofovir).

Method Validation ²²:

System Suitability: For determining the system suitability, a standard solution was injected five times into the UPLC and calculated the system suitability parameters such as % relative standard deviation, plate count, tailing factor and resolution.

Linearity: Serial dilutions of standard drugs in the concentration range of 50 – 300µg/ml for emtricitabine, 12.5 – 75µg/ml for bictegravir, and 6.25 – 37.5µg/ml for tenofovir alafenamidewere prepared and injected into the UPLC. Linearity graphs were plotted between concentration and peak areas.

Accuracy: The solutions were prepared in three different concentration levels of 50%, 100%, and 150%, injected into UPLC, and % recoveries were calculated.

Precision: Intra and Inter-day precision studies determined the method's precision. The standard solution was injected six times on the same day (intra-day) and on different days (inter-day), and the % RSD was calculated.

Specificity: The specificity of the method was determined by injecting the placebo solution and comparing it with the standard solution for the interference with drug peaks.

Limit of Detection (LOD) and Limit of Quantitation (LOQ): LOD and LOQ are determined by using standard deviation (SD) and slope of the calibration curve. The limiting values are calculated as per the following equations:

LOD = (3.3 × SD)/ Slope and

LOQ = (10 × SD)/ Slope.

Robustness: The robustness of the method was determined by varying the optimum chromatographic conditions such as mobile phase ratio (±10%), flow rate (±0.1mL/min), and column oven temperature (±5ºC). The system suitability parameters were calculated and recorded.

Solution Stability: To prove the stability of the drugs, the standard solution, and the spiked solution were kept at room temperature for 24 h.

Forced Degradation Studies ²³: To test the stability of drugs under stress conditions, the standard solution and the spiked solution were subjected to the forced degradation conditions such as acidic condition (2N Hydrochloric acid, 30 min at 60°C), alkaline condition (2N sodium hydroxide, 30mins at 60°C), oxidative condition (20% hydrogen peroxide, 30 min at 60°C), thermal condition (105°C for6h) and photolytic condition (placing the beaker in UV Chamber for 7days or 200 Watt-hours/min photostability chamber).

RESULTS AND DISCUSSION: To develop the UPLC method for the simultaneous estimation of emtricitabine, bictegravir, and tenofovir alafenamide, initially many mobile phase compositions were tried to elute the drugs. Finally, a mobile phase consisting of 0.1% orthophosphoric acid (pH 2.2) and acetonitrile in the 70:30%v/v on isocratic mode at a flow rate of 0.3ml/min was selected as optimum conditions based on the peak parameters. A standard solution containing a concentration 200µg/ml of emtricitabine, 50µg/ml of bictegravir and 25µg/ml of tenofovir alafenamide were prepared and scanned in the range of 200-400nm for detecting the maximum absorption wavelength and was found to be 260nm based on the overlain UV spectrum as shown in Fig. 2.

FIG. 2: OVERLAY UV SPECTRUM OF EMTRICITABINE, TENOFOVIR AND BICTEGRAVIR

Based on the system suitability parameters, HSS C18 (100 × 1.8mm, 1.7µ) column was selected. The retention time for the drugs were found to be 0.62 min for emtricitabine, 0.89 min for bictegravir and 1.75 min for tenofovir alafenamide.

The system suitability parameter results were summarized in Table 1, and the standard and sample chromatograms for the drugs were shown in Fig. 3 and 4.

TABLE 1: SYSTEM SUITABILITY AND VALIDATION PARAMETER RESULTS

FIG. 3: STANDARD CHROMATOGRAM

FIG. 4: SAMPLE CHROMATOGRAM

The drugs obeyed beer’s law in the 50–300µg/ml concentration range for emtricitabine, 12.5–75µg/ml for bictegravir and 6.25–37.5µg/ml for tenofovir alafenamide, the correlation coefficient was found to be within limits. The linearity plots were shown in Fig. 5A, 5B, and 5C.

FIG. 5A: LINEARITY PLOT OF EMTRICITABINE        

FIG. 5B: LINEARITY PLOT OF BICTEGRAVIR

FIG. 5C: LINEARITY PLOT OF TENOFOVIR ALAFENAMIDE

The % recovery for emtricitabine was 99.98% - 101.01%, bictegravir was found to be 99.35% - 100.22%, and tenofovir alafenamide was found to be 99.99% - 101.06%indicating that the method was accurate. The % relative standard deviation (%RSD) for intra-day precision and intermediate precision was found to be 0.6 and 0.4 respectively for emtricitabine, 0.5 and 0.5 respectively for bictegravir and 1.1 and 1.5 respectively for tenofovir alafenamide, indicating that the method was precise. To determine the specificity of the method, the placebo solution was prepared and observed for the interfering peaks with the drug peaks. As there are no peaks interfering with drug peaks, the method was found to be specific. The placebo chromatogram was shown in Fig. 6.

FIG. 6: PLACEBO CHROMATOGRAM

The LOD and LOQ was found to be 3.66 µg/ml and 11.11 µg/ml respectively for emtricitabine, 0.54 µg/ml and 1.63 µg/ml respectively for bictegravir and 0.16µg/ml and 0.49µg/ml respectively for tenofovir alafenamide. The method was found to be robust and stable when stored for 24 h as the results fall within the acceptance criteria. It was found that the amount recovered from both fresh and stored solutions was highly similar. From the forced degradation studies, it was known that the drugs were found to be stable when exposed to different stress conditions, as the net degradation was found to be within limits. The results were summarized and shown in Table 2 and the chromatograms were presented in FIG. 7A, 7B, 7C, 7D, and 7E.

TABLE 2: FORCED DEGRADATION STUDIES RESULTS FOR EMTRICITABINE, BICTEGRAVIR AND TENOFOVIR

FIG. 7A: ACID DEGRADATION CHROMATOGRAM

FIG. 7B: BASE DEGRADATION CHROMATOGRAM

FIG. 7C: OXIDATIVE DEGRADATION CHROMATOGRAM

FIG. 7D: PHOTOLYTIC DEGRADATION CHROMATOGRAM

FIG. 7E: THERMAL DEGRADATION CHROMATOGRAM

CONCLUSION: A specific, accurate method was developed to simultaneously estimate emtricitabine, bictegravir and tenofovir alafenamide in pharmaceutical dosage form using UPLC. The method was validated by using various validation parameters, and the method was found to be linear, precise, accurate, specific and robust. The run time was 2 min, enabling rapid quantitation of many samples in routine and quality control analysis of capsule formulations.

ACKNOWLEDGMENT: The authors are thankful to the Hetero drugs Pvt. Ltd., Hyderabad, for providing the standard drugs as the gift samples. They are also grateful to the Spectrum labs, Hyderabad, for providing the required facilities to carry out this work.

CONFLICTS OF INTERESTS: The authors declare that they have no conflict of interest.

REFERENCES:

1. Saag MS: Emtricitabine, A new antiretroviral agent with activity against HIV and hepatitis B virus.Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 2006; 42: 126-131.
2. Frampton JE and Perry CM: Emtricitabine: A review of its use in the management of HIV infection. Drugs 2005; 65: 1427-1448.
3. Molina JM and Cox SL: Emtricitabine: a novel nucleoside reverse transcriptase inhibitor. Drugs of today (Barcelona, Spain: 1998) 2005; 41: 241-252.
4. Flexner C: Antiretroviral agents and treatment of HIV infection. In, Brunton LL, Hillal-Dandan R, Knollman BC, eds. Goodman & Gilman’s the pharmacological basis of therapeutics. 13th ed. New York: McGraw-Hill 2018; 1137-57.
5. Markham A: Bictegravir: first global approval. Drugs 2018; 78: 601-6.
6. Surgers L and Lacombe K: Hepatoxicity of new antiretrovirals: a systematic review. Clinics and Researchin Hepatology and Gastroenterology 2013; 37: 126-33.
7. Ray AS, Fordyce MW and Hitchcock MJ: Tenofovir alafenamide: A novel prodrug of tenofovir for the treatment of Human Immunodeficiency Virus. Antiviral research 2016; 125: 63-70.
8. Maggi P, Montinaro V, Mussini C, Di Biagio A, Bellagamba R and Bonfanti P: Novel antiretroviral drugs and renal function monitoring of HIV patients.
9. AIDS reviews 2014; 16: 144-151.
10. Fernández-Montero JV, Vispo E and Soriano V: Emerging antiretroviral drugs. Expert Opinion on Pharmacotherapy 2014; 15: 211-219.
11. Meenakshi R and Shyam Sunder R: Method development and validation by RP-HPLC for simultaneous estimation of emtricitabine, bictegravir, tenofovir alafenamide in fixed dosage form. Indo American Journal of Pharmaceutical Sciences 2018; 05(07): 7123-7131.
12. Tej Kumar Kokkirala and DuvvuriSuryakala: RP-HPLC method development and validation for the estimation of Emtricitabine, Bictegravir and Tenofovir alafenamide in bulk and pharmaceutical dosage form. Journal of Taibah University for Science 2019; 13(1): 1137-1146.
13. Vijay Swaroop Singh G and Divakar TE: A Novel Stability Indicating RP-HPLC Method Development and Validation for Simultaneous Estimation of Bictegravir, Emtricitabine and Tenofovir in Pure and Fixed Dose Combination.Asian Journal of Organic and Medicinal Chemistry 2019; 4(1): 7-13.
14. Vendra Sri Surya Deepthi and Devanaboyina Narendra: Method Development and Validation for Simultaneous Estimation of Emtricitabine, Bictegravir and Tenofovir Alafenamide by RP-HPLC. International Journal of Pharmaceutical Sciences Review and Research 2019; 58(2): 54-59.
15. Tanuja A, Ganapaty S and Varanasi SN Murthy: Development and Validation of Stability Indicating RP-HPLC Method for the Simultaneous Estimation of Bictegravir, Emtricitabine and Tenofovir Alafenamide Fumarate. Turkish J of Pharmaceutical Sciences 2020.
16. Anil Vikas Yamana and Chandra Sekhar KB: Simultaneous HPLC method development and validation for quantification emtricitabine, bictegravir and tenofovir alafenamide in combined dosage form with stability studies. Journal of Global Trends in Pharmaceutical Sciences 2020; 11(3): 8226 – 8234.
17. Pushpa Kumari K and Gowri Sankar D: Stability indicating UPLC method for simultaneous estimation of emtricitabine, bictegravir and tenofovir alafenamide. Indian Drugs 2020; 57(1): 44-50.
18. Kishore Konam, KanalaSomasekhar Reddy: Stability Indicating RP-UPLC Method for Simultaneous Estimation of Emtricitabine, Tenofovir and Bictegravir in Bulk and Pharmaceutical Dosage Form. International Journal of Life science and Pharma Research 2020; 10(5):90-101.
19. Appala Raju N: Bioanalytical method development and validation for simultaneous determination of bictegravir, tenofovir and emtricitabine in human plasma by LC-MS/MS. World Journal of Pharmacy and Pharmaceutical Sciences 2018; 7(12): 441-463.
20. Ashok Chakravarthy V, Sailaja BBV and Praveen Kumar A: Development and validation of a dissolution method for Frovatriptan tablets by reversed phase UPLC. International Journal of Pharmacy and Pharmaceutical Sciences 2015; 7(4): 125–130.
21. Kishor Kumar L and Mule: Rapid analytical method for assay determination for prochlorperazineedisylate drug substances by Ultra Performance Liquid Chromatography. International Journal of Current Pharmaceutical Research 2017; 9(4): 118-122.
22. Krishnaphanisri P and Raja S: Development and validation of new RP-UPLC method for the determination of Cefdinir in bulk and dosage form. International Journal of Pharmacy and Pharmaceutical Sciences 2018; 10(1): 178-184.
23. ICH: Q2 (R1), Validation of analytical procedures: text and methodology 2005.
24. Jahnavi Bandla and Ganapaty S: Stability Indicating UPLC Method Development and Validation for the Determination of Crizotinib in Pharmaceutical Dosage Forms. International Journal of Pharmaceutical Sciences and Research 2018; 9(4): 1000-06.
    

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    How to cite this article:

    Dadi V and Sowjanya G: Validated method for the simultaneous determination of emtricitabine, bictegravir and tenofovir alafenamide in pharmaceutical dosage form using UPLC. Int J Pharm Sci & Res 2022; 13(11): 4536-43. doi: 10.13040/IJPSR.0975-8232.13(11).4536-43.

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