Vol. 4, No. 6 (2014): 812-822 Research Article
Open Access
I
ISSSSNN::22332200--6688110 0
A validated stability indicating RP-HPLC method for
estimation of Emtricitabine, Tenofovir and
Rilpivirine in bulk and pharmaceutical dosage form
Sreedhar Lade
1* and Y. Rajendra Prasad
21 University College of Pharmaceutical Sciences, Acharya Nagrjuna University, Nagarjuna Nagar, Guntur- 522 510 (A.P.) India. 2 University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam-530 003, (A.P.) India.
* Corresponding author: Sreedhar Lade; e-mail: sreedharlade7@gmail.com
ABSTRACT
A new simple, precise, selective, accurate and rapid reverse phase high performance liquid chromatographic stability indicating method had been developed and validated for simultaneous quantitative determination of Emtricitabine, Tenofovir and Rilpivirine in bulk and pharmaceutical dosage form. The chromatographic separation was achieved with Hypersil BDS C-18, (250×4.6 mm) and 5µm particle size column. The optimized mobile phase consisting of phosphate buffer: Acetonitrile (45:55%v/v), to this added 1 ml of triethyl amine and adjusted the mobile phase to pH 4.0 with o-phosphoric acid. The flow rate was 1.0 mL/min and eluents were detected at 280 nm using PDA detector. The retention time of emtricitabine, tenofovir and rilpivirine were found to be 2.692, 4.402 and 5.725 respectively. The percentage recoveries for three molecules were found to be in the range of 99 -101%. The calibration curve was constructed between peak area vs concentration and demonstrated good linear in the range of 50 -300 μg/ml for Emtricitabine, 75-450 μg/ml for Tenofovir and 6.25-37.5 μg/ml for Rilpivirine. Degradation
studies were studied for Emtricitabine, Tenofovir and Rilpivirine under various stress conditions such as acid hydrolysis, base hydrolysis, oxidation, thermal, photochemical and UV. All the degradation peaks were resolved effectively using developed method with different retention times. The developed method was validated according to ICH guidelines. As the method could effectively separates the degradation products from active ingredient, it can be used for routine analysis of drug both in bulk and pharmaceutical dosage form.
Keywords:
Emtricitabine, Tenofovir and Rilpivirine, Acetonitrile, Buffer, RP-HPLC.INTRODUCTION
Emtricitabine (FTC), with trade name Emtriva (formerly Coviracil), is a nucleoside reverse transcriptase inhibitor (NRTI) for the treatment of HIV infection in adults and children. Emtricitabine is an analogue of cytidine. Emtricitabine is commercially available and is approved by the FDA for treatment of HIV infection. Emtricitabine exhibits clinical activity against the hepatitis B virus (HBV). The chemical name of Emtricitabine is 4-amino-5-fluoro-1-[(2R,5S )-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydro pyrimidin-2-one. Its molecular formula is C22H18N6 HCl and its molecular weight is 402.88.
Tenofovir disoproxil fumarate (TDF or PMPA1), belongs to a class of antiretroviral drugs known as nucleotide analogue reverse transcriptase inhibitors
(NRTIs), which block reverse transcriptase, an enzyme crucial to viral production. The chemical name of Tenofovir disoproxil fumarate is 9-[(R)-2[[bis[[(isopropoxycarbonyl)oxy]methoxy]phosphinyl] methoxy]propyl] adenine fumarate (1:1). C19H30N5O10P
C4H4O4 and a molecular weight of 635.52.
Rilpivirine is a non-nucleoside reverse transcriptase inhibitor (NNRTI) of human immunodeficiency virus type 1 (HIV-1). It is a diarylpyrimidine, a class of molecules that resemble pyrimidine nucleotides found in DNA. Because of its flexible chemical structure, resistance of rilpivirine is less likely to develop than other NNRTI’s. The chemical name for rilpivirine hydrochloride is 4-[[4-[[4-[(E)-2-cyanoethenyl]-2,6-dimethylphenyl]amino]2-pyrimidinyl]amino]benzo
nitrile monohydrochloride. Its molecular formula is C22H18N6 HCl and its molecular weight is 402.88.
Figure 1. Chemical structure of Emtricitabine.
Figure 2. Chemical structure of Tenofovir.
Figure 3. Chemical structure of Rilpivirine.
A stability indicating Reversed phase high performance liquid chromatographic method for simultaneous determination of Emtricitabine, Tenofovir disoproxil fumarate and Rilpivirine Hcl in bulk drugs and Pharmaceutical Dosage form.
MATERIALS AND METHODS
Chemicals and solvents
Emtricitabine, Tenofovir disoproxil fumarate and Rilpivirine Hcl were obtained as gift samples from spectrum research labs, Hyderabad, India. The commercial Pharmaceutical Preparation Complera containing 200 mg, 25 mg and 300 mg of Emtricitabine, Rilpivirine Hcl and Tenofovir disoproxil fumarate respectively (Marketed by Gilead Sciences) were procured from local pharmacy. Acetonitrile, and water used are of HPLC grade.
Instrumentation
The chromatographic separations were performed using HPLC-Waters alliance (Model-2695) consisting of an in-built auto sampler, a column oven and 2996 PDA
detector. The data was acquired through Empower-2-software. The column used was Hypersil BDS, C18 (250×4.6mm i.d, 5µm particle size). Meltronics sonicator was used for enhancing dissolution of the compounds. Elico pH meter was used for adjusting the pH of buffer solution. All weighing was done on Sarotorious balance (model AE-160).
Chromatographic conditions
The mobile phase consists of buffer:Acetonitrile in the ratio of 45:55 v/v%. The mobile phase was pumped from solvent reservoir in the ratio of 45:55 %v/v to the column in the flow rate of 1.0 ml/min whereas run time set was 10 min. The separation was performed on Hypersil BDS C18 column and the column was maintained the temperature of 30°C and the volume of each injection was 10 µL. Prior to injection, the column
was equilibrated for at least 30 min with mobile phase flowing through the system. The eluents were monitored at 280 nm.
Preparation of buffer solution: (PH:4.0)
Accurately weighed 1.36gm of Potassium dihyrogen Ortho phosphate in a 1000ml of Volumetric flask add about 900ml of milli-Q water added and degas to sonicate and finally make up the volume with water then added 1ml of Triethylamine then pH adjusted to 4.0 with dil. Orthophosphoric acid solution.
Preparation of mobile phase:
Buffer and Acetonitrile are in the ratio 45:55, filtered through 0.45 µ filter under vacuum.
Preparation of standard solution: (200, 300 and 25 PPM)
Accurately weighed and transferred 20 mg , 30 and 2.5 mg of Emitricitabine, Tenofovir and Rilpivirine working standards each into a 10 ml clean dry volumetric flask, added 7ml of diluent, sonicated for 30 minutes and made up to the final volume with diluent. From the above stock solution, 1 ml was pipetted out into a 10 ml Volumetric flask and then made up to the final volume with diluent to obtained desired concentrations (200µg/ml Emitricitabine, 300µg/ml Tenofovir and 25µg/ml Rilpivirine).
Preparation of sample solution:
5 tablets were weighed and calculated the average weight of each tablet and transferred to a 500 mL volumetric flask, 260 mL of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered. From the filtered solution 1ml was pipetted out into a 10 ml volumetric flask and made up to 10ml with diluent.
Label Claim: 200mg Emitricitabine + 300mg of Tenofovir + 25mg of Rilpivirine
Validation of Proposed method:
Specificity, Forced degradation studies, Linearity, Accuracy, Limit of detection and Limit of quantification.
Linearity:
Aliquots of 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 ml were taken from stock solution of concentration 2 mg/ml Emtricitabine, 3 mg/ml Tenofovir, and 0.25 mg/ml Rilpivirine and then diluted up to mark with diluent. Such that the final concentrations were in the range
50ppm-300ppm for Emtricitabine, 75ppm-450ppm for Tenofovir and 6.25ppm-37.5ppm for Rilpivirine. Volume of 10µl of each sample was injected in five times for each concentration level and 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. The observations and calibration curve were shown in Table 2 and Fig. 4, 5.
Table 1. Optimized Chromatographic conditions and system suitability parameters for proposed HPLC method for Emtricitabine, Tenofovir and Rilpivirine
Parameter Chromatographic conditions Instrument : Waters 2695, High performance Liquid Chromatography
Flow rate : 1ml/min
Column : Hypersil BDS C18, 250 x 4.6 mm, 5µ. Detector wave length : 280nm
Column temperature : 30°C Injection volume : 10µL Run time : 10 min
Diluent : Water:Acetonitrile (50:50) Mode of separation : Isocratic mode
Table 2. Accuracy
S.NO
Conc. of Emtricitabine
in ppm
Emtricitabine Area
Conc. of Tenofovir in
ppm
Tenofovir area
Conc. of Rilpivirine in
ppm
Rilpivirine area
1 50 1053532 75 168954 6.25 515704
2 100 2006616 150 334451 12.5 1073271
3 150 3192503 225 497556 18.75 1528496
4 200 4272452 300 653256 25 2049140
5 250 5191551 375 846333 31.25 2611210
6 300 6231250 450 992012 37.5 3086424
Figure 5. HPLC Chromatogram of samples.
Table 3. System Precision
INJECTIONS Emtricitabine Tenofovir Areas Rilpivirine
1 4936742 648585 2051395
2 4917903 652983 2064263
3 4971900 645401 2025834
4 4956069 647814 2033924
5 4952029 651941 2015130
6 4910208 647973 2013173
AVG 4940809 649116 2033953
S.D 23677.22 2829.1 20371.3
%RSD 0.48 0.44 1.00
System precision:
Precision is the measure of closeness of the data values to each other for a number of measurements under the same analytical conditions. Standard solution of
Emtricitabine (200μg /ml), Tenofovir (300μg/ml) and
Rilpivirine (25μg/ml) were prepared as per test
method and injected for 6 times. Results are shown in Table-3.
Precision
Intraday and interday precision study of Emtricitabine, Tenofovir and Rilpivirne were carried out by estimating corresponding responses for 6 times on the same day and on consecutive days for the
concentration of 200μg/ml for Emtricitabine, 300μg/ml for Tenofovir and 25μg/ml for Rilpivirine. The percent
relative standard deviation (%RSD) was calculated which was within the acceptable criteria of not more than 2. The results were shown in tables 3 and 4.
Table 4a. Intra-day precision
Sample
Preparations Emtricitabine Tenofovir %Assay Rilpivirine
Sample-1 99.66 98.93 100.32
Sample-2 99.47 99.67 99.68
Sample-3 99.01 99.17 100.47
Sample-4 100.66 100.62 100.67
Sample-5 100.00 98.88 99.03
Sample-6 100.71 99.66 99.23
AVG 99.92 99.49 99.90
S.D 0.6756 0.6515 0.6875
%RSD 0.68 0.65 0.69
Table 4b. Inter-day precision
Sample
Preparations Emtricitabine Tenofovir %Assay Rilpivirine
Sample-1 101.27 98.92 100.14
Sample-2 100.75 101.03 101.92
Sample-3 100.22 101.04 101.43
Sample-4 100.44 100.27 101.63
Sample-5 99.03 100.22 101.38
Sample-6 99.52 100.22 100.54
AVG 100.20 100.28 101.17
S.D 0.8182 0.7744 0.6862
Accuracy (Recovery studies)
To determine the accuracy in sample preparation, method of standard additions was made for measuring the recovery of the drugs. A fixed amount of sample was taken and standard drug was added at 50, 100 and 150% levels. The results were analyzed and the results were found to be within the limits. The accuracy was expressed as the percentage of the analytes recovery. The results were shown in table 5.
Specificity
The specificity of the method was performed by injecting blank solution (without any sample) and then a drug solution of 10µl injected into the column, under Optimized chromatographic conditions, to demonstrate the separation of three molecules Emtricitabine, Tenofovir and Rilpivirine from any of the impurities, if present. As there was no interference of impurities and also no change in the retention time, the method was found to be specific.
Table 5a.Robustness_Emtricitabine
Sr.No Parameter Optimized Used Peak Area Retention
Time (min) Plate count Tailing Factor
1 Flow Rate (±0.1 ml/min) 1.0 ml/min
0.9 ml/min 3699782 2.985 6698 1.24
1.0 ml/min 4936742 2.689 6032 1.27
1.1 ml/min 4315222 2.447 6671 1.24
2 Column Temperature
(±5ºC) 30ºC
25ºC 4994809 2.691 6978 1.25
30ºC 4936742 2.689 6032 1.27
35 ºC 4994030 2.686 7043 1.24
3 Mobile phase composition Buffer:ACN::45:55v/v 40:60 45:55 2266221 4936742 2.691 2.689 6940 6032 1.26 1.27
50:50 3350456 2.700 6756 1.25
Table 5b. Robustness_Tenofovir
Sr.No Parameter Optimized Used Peak Area Retention
Time (min) Plate count Tailing Factor
1 Flow Rate (±0.1 ml/min) 1.0 ml/min
0.9 ml/min 735412 4.878 7818 1.32
1.0 ml/min 648585 4.507 7280 1.29
1.1 ml/min 577164 3.980 7504 1.30
2 Column Temperature
(±5ºC) 30ºC
25ºC 659027 4.351 8014 1.28
30ºC 648585 4.507 7280 1.29
35 ºC 659340 4.305 8134 1.27
3 Mobile phase composition Buffer:ACN::45:55v/v 40:60 45:55 370312 648585 4.307 4.507 8148 7280 1.25 1.29
50:50 665550 4.483 7567 1.33
Table 5c. Robustness_Rilpivirine
Sr.No Parameter Optimized Used Peak Area Retention
Time (min) Plate count Tailing Factor
1 Flow Rate (±0.1 ml/min) 1.0 ml/min
0.9 ml/min 849093 6.344 11443 1.03
1.0 ml/min 2051395 5.918 6569 1.54
1.1 ml/min 1821382 5.158 6752 1.56
2 Column Temperature
(±5ºC) 30ºC
25ºC 2070379 5.504 7230 1.52
30ºC 2051395 5.918 6569 1.54
35 ºC 2070379 5.504 7230 1.52
3 Mobile phase composition Buffer:ACN::45:55v/v 40:60 45:55 939732 2051395 5.555 5.918 7157 6569 1.50 1.54
50:50 1362362 5.851 7037 1.52
Limit of Detection and Limit of Quantification
LOD and LOQ were calculated using the following formula LOD = 3.3(SD)/S and LOQ = 10 (SD)/S, where SD = standard deviation of response (peak area) and S= slope of the calibration curve. Limit of Detection and
Limit of Quantification were found to be 0.0128μg/ml and 0.0390μg/ml respectively for Emtricitabine, 0.380μg/ml and 1.154μg/ml respectively for Tenofovir and 0.0016μg/ml and 0.0049μg/ml respectively as per
ICH guidelines. The results were shown in table 6.
Robustness
Robustness was carried by varying three parameters from the optimized chromatographic conditions such as making small changes in flow rate (±0.1ml/min), mobile phase composition (±5%) and column
temperature (±5ºC). It was observed that the small changes in these operational parameters did not lead to changes of retention time of the peak of interest and the %RSD was not more than 2. The degree of reproducibility of the results proven that the method is robust. The results were shown in table 7.
System suitability
The system suitability was determined by making six replicate injections from freshly prepared standard solutions. The observed RSD values were well within
Forced degradation studies
Forced degradation studies were performed to demonstrate the optimized method is stability indicating. To prove the method which can be able to measure accurately active pharmaceutical ingredient in presence of degradants which are expected to be formed during different types of degradations applied to the drug sample.
For forced degradation analysis, aliquots of stock (2mg/ml, 3mg/ml and 2.5mg/ml) were separately treated with 1ml of 2N HCl (Acid stability), 1ml of 2N NaOH (Alkaline stability), 1ml of 20% H2O2 (Oxidative degradation), exposure of standard drug solution at 105ºC for 6 hrs (dry heat degradation), photo stability degradation (exposure of drug at 200 watt/m2) and
neutral degradation (refluxing with water at 60ºC for 6 hours. Stability of these samples was compared with fresh sample on the day of analysis. The HPLC chromatograms of degraded products show no interference at the analyte peaks, hence the method was specific and stability indicating. The chromatograms were shown in figures 5 to 8 and the results were shown in table8. The detailed degradation for each condition is as follows:
Oxidation:
To 1 ml of stock solution of Emitricitabine and Tenofovir and Rilpivirine, 1 ml of 20% hydrogen peroxide (H2O2) was added separately. The solutions were kept for 30 min at 600c. For HPLC study, the
resultant solution was diluted to obtain 200µg/ml, 300µg/ml and 25µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.
Acid Degradation Studies:
To 1 ml of stock solution Emitricitabine and Tenofovir and Rilpivirine, 1ml of 2N Hydrochloric acid was added and refluxed for 30mins at 600c .The resultant solution
was diluted to obtain 200µg/ml, 300µg/ml and 25µg/ml solution and 10 µl solutions were injected into the system and the chromatograms were recorded to assess the stability of sample.
Alkali Degradation Studies:
To 1 ml of stock solution Emitricitabine and Tenofovir and Rilpivirine, 1 ml of 2 N sodium hydroxide was added and refluxed for 30mins at 600c. The resultant
solution was diluted to obtain 200µg/ml, 300µg/ml and 25µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.
Dry Heat Degradation Studies:
The standard drug solution was placed in oven at 1050c
for 6 h to study dry heat degradation. For HPLC study, the resultant solution was diluted to 200µg/ml, 300µg/ml and 25µg/ml solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.
Photo Stability studies:
The photochemical stability of the drug was also studied by exposing the 2mg/ml, 3mg/ml and 0.25mg/ml solution to UV Light by keeping the beaker in UV Chamber for 7days or 200 Watt hours/m2 in
photo stability chamber. For HPLC study, the resultant
solution was diluted to obtain 200µg/ml, 300µg/ml and 25µg/ml solutions and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.
Neutral Degradation Studies:
Stress testing under neutral conditions was studied by refluxing the drug in water for 6hrs at a temperature of 60º. For HPLC study, the resultant solution was diluted to 200µg/ml, 300µg/ml and 25µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.
Table 6a. Forced Degradation studies_Emtricitabine
Sr.No Injection %Assay %Degradation Purity Angle Purity
Threshold Purity Flag
1 Controlled sample 99.92 -- 0.763 1.342 No
2 Acid Degradation 91.78 8.15 0.800 1.294 No
3 Base Degradation 89.01 10.91 1.532 2.457 No
4 Peroxide Degradation 93.63 6.29 1.261 2.322 No
5 Thermal Degradation 92.95 6.97 0.369 1.303 No
6 UV Degradation 95.61 4.31 1.05 1.688 No
7 Water Degradation 96.60 3.32 1.018 1.207 No
Table 6b. Forced Degradation studies_Tenofovir
Sr.No Injection %Assay %Degradation Purity Angle Purity
Threshold Purity Flag
1 Controlled sample 99.49 --- 0.561 1.447 No
2 Acid Degradation 90.46 9.03 1.05 1.671 No
3 Base Degradation 94.87 4.62 0.365 0.472 No
4 Peroxide Degradation 93.33 6.16 0.376 0.530 No
5 Thermal Degradation 94.69 4.80 0.450 0.810 No
6 UV Degradation 95.71 3.78 0.450 0.638 No
Table 6c. Forced Degradation studies_Rilpivirine
Sr.No Injection %Assay %Degradation Purity Angle Purity
Threshold Purity Flag
1 Controlled Sample 99.90 --- 0.322 1.847 No
2 Acid Degradation 91.59 8.31 0.289 0.822 No
3 Base Degradation 93.20 6.7 0.317 0.439 No
4 Peroxide Degradation 95.19 4.71 0.295 0.483 No
5 Thermal Degradation 95.27 4.63 0.910 0.985 No
6 UV Degradation 96.26 3.64 1.03 1.454 No
7 Water Degradation 97.44 2.46 0.171 0.377 No
RESULTS AND DISCUSSION
The main aim for development of chromatographic method was to get reliable method for quantification of Emtricitabine, Tenofovir and Rilpivirine from bulk and pharmaceutical dosage form and should be applicable for the degradation products also. Different chromatographic conditions were employed for the analysis. Finally the analysis was performed by using Phosphate Buffer: Acetonitrile in the ratio of 45:55 %v/v at a flow rate 1.0 ml/min. Samples were analysed at 280 nm at an injection volume of 10 μL and
separation was carried by using Hypersil BDS, C18, (150 x 4.6 mm, 5µ)., column. The retention time and tailing factor were calculated. The retention time of Emtricitabine, Tenofovir and Rilpivirine was found to be 2.740, 5.002 and 5.895 min respectively. The proposed column was selected which resulted in a sharp and symmetrical peak with 1.14 tailing factor and theoretical plates of 5539 for Emtricitabine, 0.94 tailing factor and theoretical plates of 6990 for Tenofovir and 1.30 tailing factor and theoretical plates of 6479 for Rilpivirine. The calibration curve was linear over the concentration range of 50-300 (ppm) (Emtricitabine), 75-400(ppm) (Tenofovir) and 6.25-37.5(ppm)
(Rilpivirine). Six different concentrations of Emtricitabine, Tenofovir and Rilpivirine in the given range were prepared and injected into HPLC. The linearity of the method was statistically confirmed. RSD values for accuracy and precision studies obtained were less than 2% which revealed that developed method was accurate and precise. The system suitability parameters were given in table-5.
Forced degradation studies concluded that the all the degradant peaks obtained during degradation were well resolved from the main drugs i.e. Emtricitabine, Tenofovir and Rilpivirine. And the peak purity was above threshold i.e. purity angle was less than purity threshold as per Empower-2 software. Hence the method is found to be stability indicating. The slope (m) and intercept(c) obtained were shown in the table-5.
Therefore proposed validated stability indicating method was successfully applied to determine Emtricitabine, Tenofovir and Rilpivirine in Bulk and Pharmaceutical dosage form.
Figure 6b. Base Degradation
Figure 6c. Peroxide Degradation
Figure 6e. UV Degradation
Figure 6f. Water degradation
HPLC Chromatogram of LOQ
Table 7. Characteristics of HPLC method
Drug Parameters defined Obtained value
Emtricitabine
Linearity range (ppm) Regression coefficient(r2)
Intercept Slope LOD (ppm) LOQ (ppm) Tailing factor Plate count
50-300 ppm 0.999 3026 20883 0.0128 0.0390 1.27 6032
Tenofovir
Linearity range (ppm) Regression coefficient(r2)
Intercept Slope LOD (ppm) LOQ (ppm) Tailing factor Plate count
75-400 ppm 0.999 766 2214 0.380 1.154 1.29 7280
Rilpivirine Linearity range (ppm)
Regression coefficient(r2)
Intercept Slope LOD (ppm) LOQ (ppm) Tailing factor Plate count
6.25-37.5 ppm 0.999 12365 82430 0.0016 0.0049 1.54 6569
Figure 7. Linearity of Emtrictabine
Figure 9. Linearity of Rilpivirine
CONCLUSION
The developed method is accurate, simple, rapid and selective and proved to be stability indicating for the simultaneous estimation of Emtricitabine, Tenofovir and Rilpivirine in Bulk and pharmaceutical dosage form. The sample preparation is simple, the analysis time is short and the elution is by isocratic method. The retention time of Emtricitabine, Tenofovir and Rilpivirine were found to be 2.740, 5.002 and 5.895 min respectively. The excipients of the commercial sample analyzed did not interfere in the analysis, which proved the specificity of the method for these drugs. Forced degradation studies of different conditions shows that all the degradants were well resolved from these main drug peaks and able to quantify the Emtricitabine, Tenofovir and Rilpivirine in presence of degradants and excepients proved that the method is found to be stability indicating. Hence the proposed method can be conveniently adopted for the routine quality control analysis in the bulk and combined formulations.
Acknowledgements
The authors gratefully acknowledge Spectrum Pharma Research Solutions, Hyderabad, India for providing necessary facilities to carry out this research work.
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