A Stability Indicating Method for estimating Emtricitabine and Tenofovir disoproxil fumarate simultaneously in Bulk and Combined dosage form by RP-HPLC

 

Rubi Shaheen¹*, S. H. Rizwan²

¹PG Student, Department of Pharmaceutical Analysis, Deccan School of Pharmacy, Hyderabad, Telangana, India

²Professor, Department of Pharmaceutical Analysis, Deccan School of Pharmacy, Hyderabad, Telangana, India.

 

ABSTRACT:

Emtricitabine and Tenofovir disoproxil fumarate simultaneously estimated in bulk and combined dosage form by high performance liquid chromatography (HPLC) which is simple, specific, accurate and economical method. Separation using this method was achieved on a phenomenex C18 (50mm x 2.1mm ID) 1.8µm, using mobile phase sodium phosphate: acetonitrile: methanol at a ratio of 50:30:20 eluted at a flow rate of 1 ml/min. The retention time for Emtricitabine and Tenofovir disoproxil fumarate is found to be 2.3 min and 3.5 min. Quantitation was achieve with photo diode array detector at wavelength of 270 nm based on peak area with the linear calibration curve at concentration of 50-150 µg/ml for emtricitabine and 25-75 µg/ml for Tenofovir disoproxil fumarate. % Recovery was found to be 101.9% and 100.5%. The LOD were found to be 0.795 µg/ml and 2.4 µg/ml and LOQ was found to be 2.4 µg/ml for Emtricitabine and 7.0 µg/ml for Tenofovir disoproxil fumarate. The above drug combination is subjected to hydrolytic, photolytic and thermal stress environment. It was observed that Emtrıcıtabıne mostly degrade under dry heat (thermal degradatıon) where as Tenofovir disoproxil fumarate degrades mostly when subjected to peroxıde degradatıon. The method was found to be specific and can be employed for the routine quality control analysis of both the drugs individually and in combined dosage form.

 

 

 

INTRODUCTION:

Emtricitabine (EMT) chemically is 2',3'-dideoxy-5-fluoro-3'-thiacytidine 4-amino-5-fluoro-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one¹. It has molecular formula of C₈H₁₀FN₃O and a molecular weight of 247.28 g/mol. Soluble in methanol, water and acetonitrile. EMT is an analogue of cytidine. The drug works by inhibiting reverse transcriptase, the enzyme that copies HIV RNA into new viral DNA.

 

Tenofovir disoproxil fumarate (TDF) chemically is Bis{[(isopropoxycarbonyl) oxy] methyl} ({[(2R)-1-(6-amino-9H-purin-9-yl)-2-propanyl] oxy} methyl) phosphonate. It has molecular Formula of C₁₉H₃₀N₅O₁₀P and Molecular Weight of 519.443 g/mol. Soluble in methanol, water and slightly soluble in acetonitrile². It selectively inhibits viral reverse transcriptase, a crucial enzyme in retroviruses such as human immunodeficiency virus (HIV), while showing limited inhibition of human enzymes, such as DNA polymerases α, β, and mitochondrial DNA polymerase γ³.

 

The literature survey reveals that there are few HPLC method available for the determination of individual EMT and TDF in bulk and dosage forms^4-7. The present work is to develop precise, accurate and economic stability indicating method for estimating EMT and TDF simultaneously in bulk and combined dosage form.

 

MATERIAL AND METHOD:

Chemicals and reagents:

TDF and EMT was a gift samples from Hetero Healthcare Ltd., India. The commercial fixed dose combination product (Tenof-EM containing 300 mg Tenofovir sisoproxil fumarate and 200 mg Emtricitabine) was procured from the local Pharmacy. HPLC grade acetonitrile, water and methanol was procured from Fisher Scientific and other chemicals like sodium phosphate, sodium hydroxide (NaOH), hydrochloric acid (HCl) were procured from Merck.

 

Instrumentation and Chromatographic Condition:

HPLC experiment was carryed out on shimadzu LC 2010 with photodiode array detector using auto sampler. Data gathering and processing was prepared using spin chrome (LC Solution) software. The analytical column used for separation was phenomenex C18 (50mm x2.1 mm ID) 1.8 µm, other equipment used were ultra-sonicator (PCI digital), analytical balance (Wensar weighing scales), pH meter (Systronics digital). Mobile phase composed of sodium phosphate buffer (PH 2.8), acetonitrile and methanol at a ratio of 50:30:20, peak symmetry and reproducibility were obtained on phenomenex C18 (50mm×2.1mmID) 1.8µm column. The optimized wavelength for detecting the analyte was found to be 270 nm; flow rate of 1 ml/min gave optimized separation and peak symmetry. The retention time was found to be 2.4 and 3.7 min.

 

Preparation of sodium phosphate buffer:

Take accurately weighed 7.8 g of sodium dihydrogen phosphate in 900 ml of HPLC water; adjust to pH 2.8 with phosphoric acid dilute to 1000 ml with HPLC water.

 

Preparation of mobile phase:

Take a combination of sodium phosphate buffer (pH2.8), acetonitrile, methanol in proportion of 50:30:20 which are filtered through 0.45 μm membrane filters. This prepared solution was used as mobile phase. This solution was also used for specificity blank solution.

 

Standard solution of EMT:

Weigh exactly about 20 mg of EMT in a 100 ml volumetric flask then diluted with 70 ml of mobile phase and sonicate and volume was made up with the mobile phase. Now the stock solution is ready. Further, 0.1 ml of the stock solution is taken into 10 ml volumetric flask and volume was made up with mobile phase to get final concentration (20 µg/ml of EMT).

 

Standard solution of TDF:

Weigh exactly about 30 mg of TDF in a 100 ml volumetric flask, then diluted with 70 ml of mobile phase and make up the volume with the mobile phase after sonication. Now the stock solution is ready. Further, take 0.1 ml of the stock solution to 10 ml volumetric flask and make up the volume with mobile phase to get final concentration (30 µg/ml of TDF).

 

Stock solution:

Crush about 20 tablets and weigh the quantity of powder equivalent to 200 mg of EMT and 300 mg of TDF, add it to 100 ml volumetric flask and to this add 70 ml of mobile phase then make up the volume with mobile phase after sonicating for about 30 min. Further, take      0.1 ml of this stock solution into 10 ml volumetric flask and make up the volume using mobile phase to get final concentration of 20 µg/ml of EMT and 30 µg/ml of TDF.

 

Injected the two replicate injections of standard and sample solution and calculated the assay by using formula:

 

                   AT     WS    DT      P        AV

% Assay = ––– × ––– × ––– × ––– × –––

                    AS     DS    WT    100     LC

 

Where, AS:  Average area counts of standard preparation          

AT:  Average area counts of sample preparation

WS: Weight of working standard taken in mg                   

WT: Weight of sample in assay preparation

DT: Dilution of sample preparation                                   

 DS: Dilution of standard preparation

P: potency of standard                                                        

AV: Average weight of tablets in mg

LC: Label claim

 

Preparation of 0.1N Hydrochloric Acid (HCl):

Take 1 L volumetric flack to this add 20 ml of distilled water then add 8.3 ml of analytical grade hydrochloric acid and make up to the mark of flask with distilled water.

 

Preparation of 0.1N Sodium Hydroxide (NaOH):

Take 4 g of sodium hydroxide and transfer it in 100 ml volumetric flask and dissolved it in small quantity of water and finally make up to the mark of the flask with distilled water.

 

Stability studies:

Hydrolytic Studies:

Hydrolytic decomposition was carried out in HCl and NaOH by refluxing at 80⁰C till sufficient degradation of drug was attained. The concentration of the drug was     1 ml. 0.1N HCl and 0.1N NaOH were used for acidic and alkaline degradation respectively.

 

Acidic degradation:

To 1 ml of stock solution of EMT and TDF, 1 ml of HCl was added and solution was kept for 30 minute at 80⁰C. The resultant solution was neutralized by NaOH before diluting, then diluted to obtain 10 µg/ml of EMT and           10 µg/ml of TDF solution and 10 µl solutions of both the drugs were injected into the system and the chromatograms were recorded to assess the stability of sample.

 

Alkaline degradation:

To 1 ml of stock solution of EMT and TDF, 1 ml of NaOH was added and solution was kept for 30 min at 80⁰C. The resultant solution was neutralized by HCl before diluting, then diluted to obtain 10 µg/ml of EMT and 10 µg/ml of TDF and 10 µl solutions of both the drugs were injected into the system and the chromatograms were recorded to assess the stability of sample.

 

Oxidative degradation:

To 1 ml of stock solution of EMT and TDF, 1 ml of 20% hydrogen peroxide was added separately. The solutions were kept for 30 min at 80⁰C. The resultant solution was diluted to obtain 10 µg/ml of EMT and 10 µg/ml of TDF and 10 µl solutions of both the drugs were injected into the system and the chromatograms were recorded to assess the stability of sample.

 

Photolytic Degradation:

To study the effect of photolysis, 1 ml of stock solution was exposed to UV light for 4 hrs. The resultant solution was diluted to obtain 10 µg/ml of EMT and 10 µg/ml of TDF and 10 µl solutions of both the drugs were injected into the system and the chromatograms were recorded to assess the stability of sample.

 

Thermal degradation:

The standard drug solution was placed in oven at 105⁰C for 6 hours to dry and to check degradation caused by heat. The resultant solution was diluted to obtain 10 µg/ml of EMT and 10 µg/ml of TDF and 10 µl solutions of both the drugs were injected into the system and the chromatograms were recorded to assess the stability of sample.

 

RESULT AND DISCUSSION:

Optimization of mobile phase:

Optimum analytical conditions were set after the optimization procedure was performed. The best peak shape and maximum separation was achieved with mobile phase composition sodium phosphate buffer   (PH 2.8), acetonitrile and methanol at a ratio of 50:30:20, peak symmetry and reproducibility were obtained on phenomenex C18 (50mm × 2.1mmID)      1.8 µm column. The optimized wavelength for detecting the analyte was found to be 270 nm; flow rate of              1 ml/min gave optimized separation and peak symmetry. The retention time was found to be 2.4 and 3.7 min. Chromatogram of EMT and TDF was shown in fig.1 and optimized chromatographic conditions as shown in the table 1

 

Figure 1.A typical chromatogram for EMT and TDF

 

Table 1.Optimized Chromatographic Conditions

 

Estimation of EMT and TDF in tablet dosage forms:

The drug was estimated using above calculated regression equation. The assay limit of the content was found to be in the range of 100 % and 102 %. The parameters calculated for system suitability are:

A number of theoretical plates, tailing factor, resolution, retention time and peak area. The results of the tablet dosage form are shown in table 2 below.

 

Table 2.Result of Marketed formulation analysis

% RSD- Percentage relative standard deviation (mean n=6)

 

Method validation:

Method was validated to specificity, linearity, repeatability and recovery. The validation protocol was performed according to the ICH guideline (8).

System Precision:

System suitability was performed to measure that this specific method could generate results of acceptable accuracy and precision. Injections of standard solutions were given for five times and chromatograms were observed for: The reproducibility of retention time, adequate sensitivity to calculate LLOQ, chromatographic separation.

 

The system suitability was carried out after method development. For parameters like plate number (N), resolution (R), tailing factor (TF), relative standard deviation (RSD) for peak area using repetitive injections was measured. The system suitability parameters were found to be within limits. The results were shown in table 3-4.

 

Table 3. Results for system suitability of EMT

Retention time – Rt, Theoretical plates-TP, Tailing factor -TF, SD- Standard deviation, %RSD- Percentage relative standard deviation

 

Table 4. Results for system suitability of TDF

Retention time – Rt, Theoretical plates-TP, Tailing factor -TF, SD- Standard deviation, %RSD- Percentage relative standard deviation

 

Linearity

The linearity of the method was determined by preparing five different concentrations of both EMT and TDF in the range of 50-150 µg/ml and 25-75 µg/ml respectively. The calibration curve was obtained by plotting peak area versus concentration. Linearity was check for three       

 

different days and the results were obtained as shown in tables 5 and fig. 2-3. The calibration curve was linear and correlation coefficient is 0.999 for both the drugs.

 

Accuracy:

For accuracy determination, three different concentrations were prepared separately i.e., 80%, 100% and 120% by spike using known amount of the drug analyte and % recovery was calculated. Recovery was found to be 101.9% for EMT and 100.5% for TDF with the value of RSD less than 2 indicating that the proposed method is accurate for simultaneous estimation of both the drugs. The results were shown in tables 6.

 

Table 5. Linearity of EMT and TDF

Slope-m, correlation coefficient- R², Y-intercept-C

 

Figure 2. Calibration curve for Emtricitabine

 

Figure 3. Calibration Curve of Tenofovir Disoproxil Fumarate

 

Table 6. Recovery results for EMT and TDF

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

LOD is the lowest concentration of the substance that can be detected but the amount cannot be quantified by this method.

The minimum concentration at which the analyte can be detected is determined from the linearity curve by applying the following formula

 

LOD = 3.3 × σ / s

 

LOQ is the lowest concentration of the substance that can be estimated quantitatively it can be determined from linearity curve by applying the following formula

 

LOQ =10 × σ / s

 

Where, 

σ = the standard deviation of the response

S = the slope of the calibration curve

 

LOD for EMT and TDF were found to be 0.795 µg/ml and 2.4 µg/ml and LOQ were found to be 2.4 µg/ml for EMT and 7.0 µg/ml for TDF.

 

Robustness:

The analysis was performed in different condition (Effect of variation of flow and wavelength) to find the variability of test results. The following conditions are checked for variation of results. The sample was analysed at flow rate 0.8 ml/min and 1.2 ml/min instead of 1 ml/min, remaining condition are same and wavelength 268 nm and 272 nm instead of 270 nm, remaining condition are same. 10 µl of the above sample was injected twice and chromatograms were recorded. Results of the robustness % RSD of peak area was found to be less than 2.

 

Table 7. Results for Method precision of EMT and TDF

Rt – Retention time, Avg- average, Std.Dev- Standard deviation, %RSD- Percentage relative standard deviation

Method Precision and Intermediate Precision/Ruggedness:

The method precision is determined by injecting six standard solutions. The areas of all the injections were taken to calculate standard deviation and % RSD. The intermediate precision was determined by injecting six working standard solution on different days by different operator.

 

The area of all the injections were taken and standard deviation, % RSD, % assay was calculated. Method precision and intermediate precision results show that the method was precise and % RSD was less than 2. The results were shown in table 7-8.

 

Table 8. Results for Ruggedness/Intermediate precision for EMT and TDF

% RSD- Percentage relative standard deviation

 

Stability studies:

10 µl of each solution was injected into the system and the chromatograms were recorded to assess the stability of sample. The drug was subjected to various force degradation condition. It was observed that EMT mostly degrade under dry heat (thermal degradatıon) where as TDF degrade mostly when ıt subjected to peroxıde degradatıon. The results are shown in table 9 and degradation pattern shows in figure 4-5.

 

 

Figure 4. Degradation pattern for TDF

 

Table 9. Degradation pattern in term of percentage for EMT and TDF

  std-standard

 

Figure 5. Degradation pattern for EMT

 

CONCLUSION:

The combination of EMT and TDF is used in the treatment of HIV infection. The present work focuses on developing of stability indicating method for EMT and TDF as per ICH guidelines. The proposed method gives reliable assay results with short analysis time, using sodium phosphate buffer, acetonitrile, methanol as a mobile phase. The optimum wavelength for detection was found to be 270 nm. EMT was eluted at 2.4 min and TDF was eluted at 3.7 min. Calibration curve is linear in the range of 50-150 µg/ml and 25-75 µg/ml respectively. Accuracy studies showed that the percentage mean recovery is 101% and 100.5% respectively. The amount of EMT and TDF present in the taken formulation (Tenof-EM) was found to be 100.3% and 102.5% respectively. Forced degradation studies were carried under various stress conditions. It was observed that EMT mostly degrades under dry heat (thermal degradatıon) where as TDF degrade mostly when ıt subjected to peroxıde degradatıon. From the above experimental results and parameters it was concluded that, this newly developed methods for estimation of Emtricitabine and Tenofovir Disoproxil Fumarate which is simple, rapid, precise, accurate and reproducible. Forced degradation studies carried out are helpful to determine stability of this drug in combined dosage form.

 

LIST OF SYMBOLS AND ABBREVIATIONS:

% - Percentage

v/v – volume/volume

µg/ml- microgram/microlitre

EMT- Emtricitabine

TDF- Tenofovir disoproxil fumarate

Rt - Retention time

m- Slope

R²- correlation coefficient

C- Y-intercept

std-  standard

SD- Standard deviation

%RSD- Percentage relative standard deviation

ACKNOWLEDGEMENTS:

The authors are thankful to Deccan School of Pharmacy for providing chemicals and instruments and Hetero Healthcare Ldt., Hyderabad, India for providing the samples for research.

 

CONFLICT OF INTEREST STATEMENT:

The authors declared no conflict of interest in the manuscript.

 

REFERENCE:

1.      Drug profile for Emtricitabine. Available from http://www.drugbank.ca/drugs/DB00879

2.      Drug profile of Tenofovir disoproxil fumarate. Available from http://www.drugbank.ca/drugs/DB09299

3.      E.Littler, X.Zhou. Deoxyribonucleic Acid Viruses: Antivirals for Herpes viruses and Hepatitis B Virus. Comprehensive Medicinal Chemistry II. 2007; 7:295-327.

4.       MD Abdul Sattar and Suneetha Achanta. Analytical Method Development and Validation for Determination of Emtricitabine and Tenofovir Disoproxil Fumarate Using Reverse Phase HPLC Method in Bulk and Tablet Dosage Form. J. Pharm. Sci.& Res. 2018; 10(5):1207-1212.

5.      Vishnu P. Choudhari, Sanket R. Parekar, Subhash G. Development and Validation of UV- Visible Spectrophotometric Baseline Manipulation Method for Simultaneous Quantitation of Tenofovir Disoproxil Fumarate and Emtricitabine in Pharmaceutical Dosage Form. Journal of Spectroscopy. 2013; 146580: 6.

6.      Anna Pratima G. Nikalje, Zibran Syed, DileepBhosale. A Simple, Reliable, Rapid and Stability Indicating Ultra Performance Liquid Chromatographic Method for the Quantitation of Emtricitabine. Am. J. PharmTech Res. 2013; 3(1): 621-632.

7.      Budagam lavanya, Perumalla Hariprasad, Allumellu Venkatapraveen, Dudipala Prasannalakshmi, Maram Ramireddy. Simultaneous estimation of emtricitabine and tenofovir disproxil fumerate by HPLC method. Der Pharmacia Lettre. 2012; 4(6): 1855-1862.

8.      International Conference on Harmonisation (ICH). In: Q2 (R1) Validation of Analytical Procedures: Text and Methodology.  Geneva, 2005.

 

Received on 10.02.2020                    Modified on 02.03.2020

Accepted on 12.04.2020                   ©AJRC All right reserved