STABILITY INDICATING RP-HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF EMITRICITABINE, TENOFOVIR, COBICISTAT AND ELVITEGRAVIR

STABILITY INDICATING RP-HPLC METHOD FOR THE SIMULTANEOUS DETER-MINATION OF EMITRICITABINE, TENOFOVIR, COBICISTAT AND ELVITEGRAVIR

P. D. Chaithanya Sudha ^*, P. Sohail and Ushashree Avulapati

Department of Pharmaceutical Analysis, Krupanidhi College of Pharmacy Bengaluru - 560035, Karnataka, India.

ABSTRACT: A stability-indicating RP-HPLC method for the simultaneous determination of emtricitabine (EMT), tenofovir (TNF), cobicistat (COB) and elvitegravir (ELV) in solid dosage forms. The waters 2695, High-Performance Liquid Chromatographic system with column kromasil C₁₈, 250 × 4.6 mm, 5 µ. The detector used is PDA detector at 288 nm. The mobile phase used in this method is pH-3.5 phosphate buffer and acetonitrile in the ratio of 60:40% V/V. Flow rate used for this proposed method is 1.0 ml/min. The retention times observed are 2.304 min, 2.691 min, 3.185 min and 4.537 min for emtricitabine, tenofovir, cobicistat, and elvitegravir respectively. The linearity calculated was found to be within the range. The % recoveries for EMT, TNF, COB and ELV were within the acceptance criteria. These drugs were found to be stable at forced degradation studies and results are within the limits. The proposed method can be used for the quality control of the combination in the pharmaceutical dosage forms.

Emtricitabine (EMT), Tenofovir (TNF), Cobicistat (COB), Elvitegravir (ELV), RP-HPLC, Degradation studies, and validation

INTRODUCTION: Emtricitabine is NRTI and is an analog of cytidine which inhibits the reverse transcriptase enzyme that copies the HIV RNA into a new viral DNA. By interrupting this process the drug lowers the amount of the HIV or viral load in the patient’s body and increases the number of immune system cells. Emtricitabine is used in prevention and treatment of HIV infection. It is a nucleoside reverse transcriptase inhibitor. It is used in a combination with tenofovir and efavirenz and is approved by USFDA.

Tenofovir alafenamide is NRTI used to treat chronic hepatitis B and to prevent HIV. It causes premature termination of DNA transcription and prevents viral replication. This drug shows high anti-HIV activity in the male genital tract. Tenofovir inhibits the activity of the HIV-1 reverse transcriptase by competing with the natural substrate, deoxyadenosine 5’ triphosphate and by DNA chain termination.

FIG. 1: CHEMICAL STRUCTURE OF EMITRICITABINE

IUPAC Name: 4-amino-5-flouro-1-(2R, 5S)-2- (hydroxymeythyl)-1, 3-oxathiolan-5-yl-1,2-dihydro - pyrimidine-2-one.

Molecular Formula: C₈H₁₀N₃O₃SF.

Molecular Weight: 247.248 g/mol.

Category: Anti-viral agent.

Tenofovir disproxal is used in the prevention of HIV infection and to treat chronic hepatitis-B. It is used alone and in combination with tenofovir and/or efavirenz and/or emtricitabine. It is a nucleoside reverse transcriptase inhibitor.

FIG. 2: CHEMICAL STRUCTURE OF TENOFOVIR ALAFENAMIDE

IUPAC Name: Bis-(isopropoxycarbonyl) oxy-methyl (2R)-1-(6-amino-9H-purine-9-yl)-2- pro-panyl oxy-methyl) phosphonate.

Molecular Formula: C₉H₁₄N₅O₄P.

Molecular Weight: 287.213 g/mol.

Category: Anti-viral agent.

Cobicistati is used for the treatment of HIV. It is a pharmacokinetic enhancer that inhibits the cytochrome P450 forms. In combination with the other retroviral agents increases systemic exposure.

FIG. 3: CHEMICAL STRUCTURE OF COBICISTAT

IUPAC Name: (1, 3-thiazol-5-yl) methyl, N-(2R, 5R)-5-(2S)-2-methyl (2-propan-2-yl)-1, 3-thiazol-4 -yl methyl carbamoyl amino-4-(morpholin-4-yl) butanamido-1, 6-diphenylhexan-2-yl carbamate.

Molecular Formula: C₄₀H₅₃N₇O₅S₂.

Molecular Weight: 776.03 g/mol.

Elvitegravir is an integrase strand inhibitor used to treat HIV infection. This is mainly used with other antiretroviral agents.

FIG. 4: CHEMICAL STRUCTURE OF ELVITEGRAVIR

IUPAC Name: 6-(3-chloro-2-fluorophenyl) methyl -1-(2S)-1-hydroxy-3-methyl butan-2-yl-7- methoxy -4-oxo-1, 4-dihydroquinoline-3-carboxylic acid.

Molecular Formula: C₂₃H₂₃C_lFNO_5.

Molecular Weight: 447.883 g/mol.

Genovya is the combination of the above four drugs which indicates a complete regimen for the treatment of HIV-1 infection in adults and pediatric patients.

MATERIALS AND METHODS:

Standard Preparation: Accurately weighed and transferred 50 mg of emtricitabine, 2.5 mg of tenofovir alafenamide, 37.5 mg of cobicistat and 37.5 mg of elvitegravir working standards into a 25 ml clean dry volumetric flasks, add 10 ml of diluent, sonicated for 10 min and makeup to the final volume with diluents. 1000 µg/ml of emtricitabine, 50 µg/ml of tenofovir alafenamide 750 µg/ml of cobicistat and 750 µg/ml of elvitegravir). 1 ml of the above stock solution was transferred to a 10ml volumetric flask and made up with diluent. 100 µg/ml of emtricitabine, 5 µg/ml of tenofovir alafenamide 75 µg/ml of cobicistat and 75 µg/ml of elvitegravir).

Sample Preparation: 5 tablets were weighed and calculate the average weight of each tablet then the weight equivalent to 1 tablet was transferred into a 100 mL volumetric flask, 60 mL of diluent added and sonicated for 25 min, further the volume made up with diluents and filtered. 2000 µg/ml of emtricitabine, 100 µg/ml of tenofovir alafenamide, 1500 µg/ml of cobicistat and 1500 µg/ml of elvitegravir). 0.5 ml of filtered sample stock solution was transferred to a 10 ml volumetric flask and made up with diluent. 100 µg/ml of emtricitabine, 5 µg/ml of tenofovir alafenamide, 75 µg/ml of cobicistat and 75 µg/ml of elvitegravir).

0.01N KH₂PO₄ Buffer Preparation: Accurately weighed 1.36 gm of potassium dihydrogen orthophosphate in a 1000 ml of volumetric flask add about 900 ml of milli-Q water added and degas to sonicate and finally make up the volume with water then pH adjusted to 3.5 with dil. Ortho-phosphoric acid solution.

Mobile Phase: The mobile phase is prepared by mixing Buffer and ACN in the ratio 60:40% V/V.

Diluent Preparation: 0.01N KH₂PO₄: Acetonitrile (50:50).

Instrumentation and Chromatographic Con-ditions: The waters 2695, High-Performance Liquid Chromatographic system is used for the method development. The following chromate-graphic conditions are used.

Chromatographic Conditions:       

Flow rate: 1.0 ml/min

Column:  Kromasil C₁₈, 250 × 4.6 mm, 5m.

Detector wavelength: 288.0 nm

Column temperature: 30 °C

Injection volume: 10.00 mL

Run time: 8.0 min

Diluent: 0.01N KH₂PO₄: Acetonitrile (50:50)

Method Development: To optimize chromate-graphic conditions, the effect of chromatographic variables such as different columns, mobile phase composition, and flow rates were studied at constant conditions such as appropriate wavelength 288 nm, injection volume of 10 μl and run time of about 8 min throughout the trials to achieve the best possible separation and resolution. The conditions which produce the best resolution tailing factor, USP plate count were selected for the estimation. The resulting chromatograms were recorded and chromatographic parameters such as tailing factor, USP plate count and resolution were calculated.

Forced Degradation Studies: A stress study was conducted to demonstrate the effective separation of degradations from the main analyte peaks of the sample when exposed to the following stress conditions. All the stressed samples were suitable diluted to required concentration with diluent and injected twice into the HPLC system by using optimized chromatographic conditions and the chromatograms were recorded and evaluated for peak purity. The % of degradation of emtricitabine, tenofovir, cobicistat, and elvitegravir were calculated.

TABLE 1: STRESS CONDITIONS FOR THE PROPOSED RP-HPLC METHOD

Method Validation:

System Suitability Studies: The system stability test was carried out by injecting five replicate injections of 10 μL of the standard solutions of emtricitabine, tenofovir, cobicistat, and elvitegravir into the chromatographic system by using optimized chromatographic conditions. The system suitability parameters were evaluated for tailing factor, % relative standard deviation for retention time and peak areas, resolutions and theoretical plates were determined.

Linearity of Detector Response: The linearity of an analytical method was established by preparing a series of linearity solutions (25-150% level) by diluting aliquots of 0.25, 0.5, 0.75, 1.0 and 1.25 ml were taken from stock solution of concentration 100 µg/ml of emtricitabine, 5 µg/ml of tenofovir, 75 µg/ml of cobicistat and 75 µg/ml of elvitegravir and then diluted up to mark with diluent. Such that the final concentrations were in the range 25-150 µg/ml emtricitabine, 1.25-7.5 µg/ml tenofovir, 18.75-112.5 µg/ml cobicistat and  18.75-112.5 µg/ml elvitegravir. The volume of 10 μl of each sample was injected five times for each concentration level in triplicate into the chromatographic system and the chromatographs were recorded. The calibration curve was constructed by plotting the peak area versus drug concentration. A linear relationship between peak area vs. concentration was observed in the range of study (concentration in µg/ml on X-axis and peak area response on Y-axis) from this calculate the correlation coefficient, slope and intercept.

Precision:

System Precision repeatability / Intra-Day Precision: The system precision study was demonstrated by injecting 10 µl solution of standard preparations six times into the chromate-graphic system and chromatograms were recorded. Calculated peak areas for emtricitabine, tenofovir, cobicistat, and elvitegravir results were expressed as % RSD.

Method Precision: The method precision of the test method was conducted by 10 µl solution of sample preparations six times into the chromatographic system and chromatograms were recorded. Calculated peak areas for emtricitabine, tenofovir, cobicistat, and elvitegravir results were expressed as % RSD.

Intermediate/Inter-Day Precision Ruggedness: The intermediate precision of the method was carried out by injecting 10 µl solution of standard preparations six times into the chromatographic system on different days and chromatograms were recorded. Calculated peak areas for emtricitabine, tenofovir, cobicistat, and elvitegravir and results were expressed as % RSD.

Accuracy Recovery: The accuracy of the method was studied by % recovery across its range by making 3 different concentrations at 50%, 100% and 150% Levels using standard addition method where sample preparations were spiked with known amount of standard and then each concentration was injected triplicate into the chromatographic system and chromatograms were recorded. The % recoveries obtained from each Level for emtricitabine, tenofovir, cobicistat, and elvitegravir calculated.

Robustness: The robustness of the proposed method was determined by deliberately varying the chromatographic conditions such as mobile phase compositions, flow rate, wavelength, and column temperature.

The standard solutions prepared as per the test method were injected triplicate into the chromatograph at variable conditions such as flow rate at ± 0.1 ml/min, mobile organic phase composition by  ± 10%, wavelength by ± 5 nm and column temperature by ± 5 °C. System suitability parameters were evaluated from the obtained chromatograms.

Specificity Interference Studies: Specificity of the method for the interference of the blank and the placebo was conducted by injecting blank, placebo, standard and sample solutions in triplicate as per test method. The specificity of the proposed RP-HPLC method also assessed by comparing the chromatograms obtained from blank, placebo, standard and sample solutions.

RESULTS:

System Suitability Studies: System Suitability chromatography obtained is shown in Fig. 5 and the results of the proposed method are presented in Table 2.

FIG. 5: CHROMATOGRAM OF SYSTEM SUITABILITY SOLUTION

TABLE 2: SYSTEM SUITABILITY TEST PARAMETERS

Linearity of Detector Response: The detector response was found to be linear in the concentration range of 25-150 µg/ml of emtricitabine, 1.25-7.5 µg/ml of tenofovir, 18.75-112.5 µg/ml of cobicistat and 18.75-112.5 µg/ml of elvitegravir respectively as shown in the Fig. 6, 7 and peak areas are measured. The calibration curves of emtricitabine, tenofovir, cobicistat, and elvitegravir are shown in Fig. 12-15 respectively. The linearity studies and regression characteristics of the proposed method are presented in Tables 3 and 4.

TABLE 3: LINEARITY STUDIES OF THE PROPOSED METHOD

TABLE 4: REGRESSION CHARACTERISTICS OF THE PROPOSED METHOD

Precision:

System Precision Repeatability / Intra-Day precision: The % RSD of the peak areas for emtricitabine, tenofovir, cobicistat, and elvitegravir shown in Table 5. Standard chromatograms of repeatability studies are shown in Fig. 16-21.

TABLE 5: RESULTS OF REPEATABILITY STUDIES (INTRA-DAY PRECISION)

Method Precision: The % RSD of the peak areas for emtricitabine, tenofovir, cobicistat, and elvitegravir are shown in Table 6.

Intermediate/Intra-Day Precision: The % RSD was determined for retention time, and peak areas of emtricitabine, tenofovir, cobicistat, and elvitegravir are shown in Tables 7-11.

Accuracy Recovery: The % recoveries obtained from each concentration level for emtricitabine, tenofovir, cobicistat, and elvitegravir.

TABLE 6: RESULTS OF METHOD PRECISION STUDIES

TABLE 7: RESULTS OF INTERMEDIATE (INTER-DAY) PRECISION STUDIES

TABLE 8: DATA OF ACCURACY STUDIES OF EMITRICITABINE

^*mean of three determinations

TABLE 9: DATA OF ACCURACY STUDIES OF TENOFOVIR

^*mean of three determinations

TABLE 10: DATA OF ACCURACY STUDIES OF COBICISTAT

^*mean of three determinations

TABLE 11: DATA OF ACCURACY STUDIES OF ELVITEGRAVIR

*mean of three determinations

Robustness: System suitability parameters from the obtained chromatograms were evaluated and are reported in Tables 12-15.

Specificity Interference Studies: The chromate-grams obtained from the blank, placebo, standard and sample solutions were recorded and results of specificity studies are reported in Table 16.

Chromatograms of blank and placebo solutions showed no peaks at the retention times of emtricitabine, tenofovir, cobicistat and elvitegravir and the retention times of the analyses in standard and sample solutions were found to be same. So, the proposed RP-HPLC method was said to be specific and free from interference due to the recipient’s presence in the tablets.

Forced Degradation Studies: The chromatograms of the stressed samples were evaluated for peak purity as shown in Fig. 22-27. These stress degradation results of Emtricitabine, Tenofovir, Cobicistat, and Elvitegravir reported in Tables 16-20.

TABLE 12: DATA OF ROBUSTNESS STUDIES OF EMITRICITABINE

TABLE 13: DATA OF ROBUSTNESS STUDIES OF TENOFOVIR

TABLE 14: DATA OF ROBUSTNESS STUDIES OF COBICISTAT

TABLE 15: DATA OF ROBUSTNESS STUDIES OF ELVITEGRAVIR

TABLE 16: RESULTS OF SPECIFICITY STUDIES

TABLE 17: FORCED DEGRADATION STUDIES OF EMITRICITABINE

TABLE 18: FORCED DEGRADATION STUDIES OF TENOFOVIR

TABLE 19: FORCED DEGRADATION STUDIES OF COBICISTAT

TABLE 20: FORCED DEGRADATION STUDIES OF ELVITEGRAVIR

DISCUSSION:

System Suitability Studies: The % RSD for the peak areas of responses and retention times of five replicates injections of standard solutions of emtricitabine, tenofovir, cobicistat, and elvitegravir were found to be less than 2%.

The theoretical plates were more than 2000 for all the combinations. The tailing factor was found to be less than 2.0 and resolution between adjacent peaks was found to be more than 5.0.

Linearity of Detector Response: The detector response was found to be linear in the concentration range of 25-150 µg/ml of emtricitabine, 1.25-7.5 µg/ml of tenofovir, 18.75-112.5 µg/ml of cobicistat and 18.75-112.5 µg/ml of elvitegravir. The correlation coefficient values were found to be within the limits.

Precision:

System Precision Repeatability / Intra-Day Precision and Method Precision: The % RSD was determined for retention time and peak areas of emtricitabine, tenofovir, cobicistat and elvitegravir obtained from 6 replicates injections in the precision studies are consistent as evidenced by the values of % RSD below 2%. Hence, it can be calculated that the proposed RP-HPLC method was précised.

Accuracy Recovery: The mean % recovery from spiked samples was found to be in the range of 99.8-100.3% emtricitabine, 98.78-100.96% tenofovir, 99.4-100.38% cobicistat and 99.27-100.39% elvitegravir respectively, which were within the acceptance limit. The excellent mean recoveries and standard deviation suggested that the good accuracy of the proposed RP-HPLC method.

System Suitability: It was found that the system suitability parameters were within the limits at all variable conditions. From the results obtained, it can be concluded that the proposed RP-HPLC method is robust towards small variations.

Forced Degradation Studies: In all forced degradation samples, it was found that the peak purity of all 4 drugs passed peak purity and also found that the degradant peaks were well separated from the main analyte peaks. No significant degradant was observed under Thermal, Photolytic and neutral degradation studies emtricitabine, tenofovir, cobicistat, and elvitegravir were degraded below 5% without any major degradants.

Limit of Detection (LOD) and Quantification (LOQ): The LOD values were found to be 1.03 µg/ml, 0.03 µg/ml, 1.07 µg/ml and 0.44 µg/ml for emtricitabine, tenofovir, cobicistat, and elvitegravir respectively and LOQ values to found to be 3.13 µg/ml, 0.09 µg/ml, 3.23 µg/ml and 1.34 µg/ml for emtricitabine, tenofovir, cobicistat and elvitegravir respectively. These low LOD and LOQ values indicate that the proposed RP-HPLC method is sensitive.

CONCLUSION: The results of this investigation reveal that by applying the proposed RP-HPLC method, the retention times of emtricitabine, tenofovir, cobicistat and elvitegravir were found to be 2.304, 2.691, 3.185 and 4.137 min respectively. Quantitative linearity was obeyed in the concentration       range of 25-150 µg/ml, 1.25-7.5 µg/ml, 18.75-112.5 µg/ml and 18.75-112.5 µg/ml with correlation coefficient value of 0.999. The % RSD values obtained from the precision studies were also found to be less than 2, which indicates the precise method. The high % recoveries indicate that the proposed method was highly accurate. The low values of LOD and LOQ indicate the high sensitivity of the proposed method. The absence of interfering peaks observed in the chromatogram of blank and placebo interference studies indicates specific methods and degradants formed during stress degradation studies were also well separated and not interfere with estimation of the drugs by the proposed stability-indicating RP-HPLC method. From this study, it is concluded that the proposed stability indicating RP-HPLC method was found to be simple, accurate, precise, rapid and useful for the routine analysis of emtricitabine, tenofovir, cobicistat, and elvitegravir in bulk and pharmaceutical dosage forms. The obtained results were satisfactory as per ICH guidelines.

ACKNOWLEDGEMENT: Nil

CONFLICTS OF INTEREST: Nil

REFERENCES:

1. Pranitha D, Vanitha C, Francis P, Alagar Raja M, Vishnu Vardan P, Surendar M and David B: Simultaneous estimation of emtricitabine, tenofovir disoproxil fumarate and rilpivirine in bulk form by RP-HPLC method. J Pharm Res 2012; 5(8): 4600-02.
2. Anandakumar K, Kannan K and Vetrichelvan T: Development and validation of emtricitabine and tenofovir disoproxil fumarate in pure and fixed dose combination by UV spectroscopy. Digest Journal of Nanomaterial and Biostructure 2011; 6: 1085-90.
3. Narendra D, Satyanarayana T and Ganga RB: HPLC method development and validation for simultaneous estimation of tenofovir and emtricitabine in combined pharmaceutical dosage form. International J of Research in Pharmaand Biomedical Sciences 2012; 3: 361-67.
4. Palavan C, Ramprasad LA, Srinivasu P and Sheshagirirao JVLN: A new RP-HPLC method development for the simultaneous estimation of emtricitabine, efavirenz and tenofovir in tablet dosage form. American Journal of Pharma Tech Research 2013; 3: 547-54.
5. Ravendrababu VV, Pankaj KS and Singvi I: A new gradient liquid chromatographic method for simultaneous estimation of tenofovir disoproxil fumarate, cobicistat, emtricitabine and elvitegravir in bulk and tablet dosage form. Asian Journal of Chemistry 2014; 26: 6233-37.
6. Joshi M, Nikalje AP, Shahed M and Dehghan M: HPTLC method for the simultaneous estimation of emtricitabine and tenofovir in tablet dosage form. Indian Journal of Pharmaceutical Sciences 2009; 71: 95-97.
7. Manish G, Avinash K, Ravi Y and Tiwari P: Development and validation of UPLC method for emtricitabine, tenofovir and efavirenz in pharmaceutical preparation; Analytical Chemistry. An Indian Journal 2010; 9: 247-51.
8. Matt MK, Pilli NR and Rao JVNL: A validated liquid chromatography and tandem mass spectrometric method for simultaneous quantification of tenofovir, emtricitabine and efavirenz in human plasma and its pharmacokinetic application. Acta Chromatographica 2015; 1: 27-39.
9. Raju N and Begum S: Simultaneous RP-HPLC method for the estimation of the emtricitabine, tenofovir disoproxil fumerate and efavirenz in tablet dosage forms. Res J Pharm Tech 2008; 1: 522-5.
10. Delahunty T, Bushman L, Robbins B and Fletcher CV: The simultaneous assay of tenofovir and emtricitabine in plasma using LC/MS/MS and isotopically labeled internal standards. J Chromatogr B Analyt Technol Biomed Life Sci 2009; 877: 1907-14.
11. Mangoankar K and Desai A: Simultaneous estimation of emtricitabine, tenofovir disoproxil fumarate and efavirenz from tablets by reverse phase high-performance liquid chromatography method. Indian Drug 2008; 45(3): 188-92.
12. Joshi M, Nikalje AP, Shahed M and Dehghan M: HPTLC method for the simultaneous estimation of emtricitabine and tenofovir in tablet dosage form. Indian Journal of Pharmaceutical Sciences 2009; 71(1): 95-97.
13. Kaul N, Agrawal H, Paradkar AR and Mahadik KR: International conference on harmonization guidance in practice: stress degradation studies on lamivudine and development of a validated specific stability-indicating HPTLC assay method. Chromatographia 2004; 60(3-4): 213-21.
14. Jullien V, Tréluyer JM, Pons G and Rey E: Determination of tenofovir in human plasma by high-performance liquid chromatography with spectrofluorimetric detection. Journal of Chromatography B 2003; 785(2): 377-81.
15. Rezk NL, Crutchley RD and Kashuba ADM: Simultaneous quantization of emtricitabine and tenofovir in human plasma using high-performance liquid chromatography after solid-phase extraction. Journal of Chromatography B 2005; 822(1-2): 201-08.
16. Delahunty, L. Bushman, and C. V. Fletcher, Sensitive assay for determining plasma tenofovir concentrations by LC/MS/MS, Journal of Chromatography B, vol. 830, no. 1, pp.6–12, 2006.
17. Barkil ME, Gagnieu MC and Guitton J: Relevance of a combined UV and single mass spectrometry detection for the determination of tenofovir in human plasma by HPLC intherapeutic drug monitoring. Journal of Chromatography B 2007; 854(1-2): 192-97.
    

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    How to cite this article:

    Sudha PDC, Sohail P and Avulapati U: Stability indicating RP-HPLC method for the simultaneous determination of emitricitabine, tenofovir, cobicistat and elvitegravir. Int J Pharm Sci & Res 2020; 11(3): 1452-66. doi: 10.13040/IJPSR.0975-8232.11(3).1452-66.

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