Simultaneous Determination of Emtricitabine and Tenofovir disoproxil fumarate in Pharmaceutical Dosage by Reverse Phase High Performance Liquid Chromatography
Rajan V. Rele*, Sandip P. Patil
Central Research Laboratory, D.G. Ruparel College, Matunga, Mumbai 400016.
*Corresponding Author E-mail: drvinraj@gmail.com
ABSTRACT:
A simple, rapid and accurate high performance liquid chromatography method is described for simultaneous determination of emtricitabine and tenofovir disoproxil fumarate from active pharmaceutical ingredients. The separation of drug was achieved on Hypersil BDS C18 (150 x 4.6 mm i.d.) with 5 µ particle size column showed most favorable chromatographic pattern over the other columns. The mobile phase consisted of a mixture of buffer and methanol (85:15 % v/v). The buffer was mixtures of 0.1 % (v/v) ortho-phosphoric acid solution adjusted the pH 3.5 with tri-ethyl amine. The detection was carried out at wavelength 260 nm. The mixture of buffer of pH 3.5 and methanol (85:15% v/v) was used as a diluent. The method was validated for system suitability, linearity, accuracy, precision, robustness, stability of sample solution. The method has been successfully used to analyze emtricitabine and tenofovir disoproxil fumarate from active pharmaceutical ingredients.
KEYWORDS: Emtricitabine, Tenofovir disoproxil fumarate, methanol, tri-ethyl amine, ortho phosphoric acid.
INTRODUCTION:
Emtricitabine is 4-amino-5-fluoro-1-[(2R,5S)-2 (hydroxyl-methyl)-1,3-oxathiolan-5-yl]-1,2-dihydro- pyrimidin-2-one. EMT is a nucleoside reverse transcriptase inhibitor (NRTI) for the treatment of HIV infection in adults. 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 is, 1-(6-Aminopurin-9-yl)-prapan-2-yl-oxymethylphosphonic acid, drug used in the treatment of human immunodeficiency virus (HIV) Type-1 patients.
Such Combination of Emtricitabine and Tenofovir disoproxil fumarate is used for the treatment of HIV infection.
According to the literature review several methods has been developed for drug, like spectroscopy methods1-10. HPLC11 -31, and HPTLC32, The newly developed HPLC method can be used for the routine analysis due to retention time as 1.73 and 3.83 minutes hence large no. of sample can be analyzed in short period. In the proposed methods optimization and validation of this method are reported.
Structure of tenofovir and tenofovir disoproxil fumarate
Structure of emtricitabine
MATERIALS AND METHOD:
Chemical and reagents:
Reference standard of emtricitabine and tenofovir disoproxil fumarate were obtained from reputed firm with certificate of analysis. Tri-ethylamine, methanol and ortho phosphoric acid were used of analytical grade and the HPLC grade water was used from Millipore. Standard and sample solutions were prepared in diluent [mixture of buffer of pH 3.5 and methanol (85:15% v/v)].
Instrumentation:
The HPLC system used was MERCK Hitachi HPLC system equipped with auto sampler (D 7200 separation module) and UV detector (D- 7400). The chromatogram was recorded and peaks quantified by means of PC based EZ Chrom Elite software.
A SHIMADZU analytical balance (0.01mg) was used.
Preparation of Standard preparation:
Standard solution:
A 10mg of standard emtricitabine and 15mg of tenofovir disoproxil fumarate were weighted accurately and transferred in 10ml volumetric flask. About 5ml of diluent [mixture of buffer of pH 3.5 and methanol (85:15 % v/v)] was added and sonicated for 10 minutes. The volume was adjusted up to the mark with diluent to give concentration as 1000μg/ml of emtricitabine and 1500 μg/ml of tenofovir disoproxil fumarate respectively. The working standard solution was prepared by diluting 1ml of 1000μg/ml of emtricitabine and 1500μg/ml. of tenofovir disoproxil fumarate solution to 10ml with diluent to get concentration 100μg/ml of Emtricitabine and 150μg/ml of Tenofovir disoproxil fumarate respectively.
Sample preparation:
Twenty tablets were weighed accurately and average weight of each tablet was determined. A powder equivalent to 1000mg of standard emtricitabine and 1500 mg of tenofovir disoproxil fumarate were weighted accurately and transferred in 10ml volumetric flask to give concentration as 1000μg/ml of emtricitabine and 1500μg/ml. of tenofovir disoproxil fumarate respectively. The working standard solution was prepared by diluting 1ml of 1000μg/ml of emtricitabine and 1500μg/ml of tenofovir disoproxil fumarate solution to 10ml with diluent to get concentration 100μg/ml of emtricitabine and 150μg/ml. of tenofovir disoproxil fumarate respectively.
Chromatographic condition:
Chromatographic separation was performed at ambient temperature on a reverse phase Hypersil BDS C18 (150 x 4.6mm i.d.) with 5µ particle size column. The mobile phase was a mixture of buffer of pH 3.5 and methanol (85:15% v/v). The buffer was mixtures of 0.1% (v/v) ortho-phosphoric acid adjusted the pH 3.5 with tri-ethyl amine. The flow rate of the mobile phase was adjusted to 1ml /min. The detection was carried out at wavelength 260nm. (Fig.1) The injection volume of the standard and sample solution was set at 1.0µl.
Figure 1: Overlay UV spectra of emtricitabine and tenofovir disoproxil fumarate
Method validation:
System suitability:
System performances of developed HPLC method were determined by injecting standard solutions. Parameter such as theoretical plates (N), symmetry, and area were determined. The results are shown in table 1 which indicates good performance of the system.
Table 1: System suitability parameters evaluated on standard solution of emtricitabine and tenofovir disoproxil fumarate
|
Name |
Retention Time |
Area |
Area % |
USP Plate Count |
Symmetry |
Resolution |
|
Tenofovir disoproxil fumarate |
1.737 |
449892 |
7.46 |
2260 |
1.84 |
0.00000 |
|
Emtricitabine |
3.853 |
5580487 |
92.54 |
3456 |
1.42 |
8.17277 |
Specificity:
Specificity is the ability of the method to resolve the active ingredients. Hence blank, standard Emtricitabine was injected to prove specificity. The typical chromatogram of the standard and sample assayed are given in figure 2 and 3 respectively.
Figure 2: Typical chromatogram of emtricitabine and tenofovir disoproxil fumarate (standard)
Figure 3: Typical chromatogram of emtricitabine and tenofovir disoproxil fumarate (sample)
Linearity
Under the experimental conditions described above, linear calibration curve were obtained throughout the concentration range studied. Regression analysis was done on the peak area (y) v/s concentration (x). The regression analysis data obtained is tabulated in table no. 2.
Table 2: Statistical evaluation of the data subjected to regression analysis
|
Parameters |
Emtricitabine |
Tenofovir disoproxil fumarate |
|
Correlation Coefficient (r) |
0.9999 |
0.9998 |
|
% Intercept (y) |
12205 |
6307 |
|
Slope (m) |
52692x |
2997 |
Accuracy:
The accuracy method was determined by applying proposed method to synthetic mixture containing known amount of drug corresponding to 80 %, 100 % and 120 %. The accuracy was then calculated as the percentage of analyte recovered by the assay. The results of the recovery analysis are enclosed under table no.3, 4.
Precision:
The method precision was established by carrying out the analysis of emtricitabine and tenofovir disoproxil fumarate. The assay was carried out of the drug using analytical method in five replicates. The value of relative standard deviation lies well with the limits. The results of the same are tabulated in the table no. 4.
Robustness
The robustness of the method was determined to check the reliability of an analysis with respect to deliberate variations in method parameters.
The typical variations are given below:
Variation in the flow rate by + 0.2 ml /min
Variation in wavelength ± 5 nm
The results of the analysis of the samples under the conditions of the above variation indicated the nature of robustness of the method.
Table 3: Statistical evaluation of the data subjected to accuracy of emtricitabine
|
Level |
Test |
Wt in mg |
Area |
Quantity added in μg /ml |
Quantity recovered in μg /ml |
% Recovery |
Mean recovery |
|
|
80% |
1 |
10.41 |
4426364 |
79.6 |
78.69 |
98.86 |
99.21 |
|
|
2 |
10.52 |
4445611 |
79.6 |
79.03 |
99.29 |
|||
|
3 |
10.12 |
4454547 |
79.6 |
79.19 |
99.49 |
|||
|
100% |
1 |
10.11 |
5456686 |
99.5 |
97.01 |
97.49 |
97.59 |
|
|
2 |
10.08 |
5463947 |
99.5 |
97.13 |
97.62 |
|||
|
3 |
10.04 |
5465888 |
99.5 |
97.17 |
97.66 |
|||
|
120% |
1 |
10.21 |
6573385 |
119.4 |
116.86 |
97.87 |
97.57 |
|
|
2 |
10.15 |
6559049 |
119.4 |
116.60 |
97.66 |
|||
|
3 |
10.17 |
6526856 |
119.4 |
116.03 |
97.18 |
|||
|
Mean recover of all level |
98.12 |
|||||||
Table 4: Statistical evaluation of the data subjected to accuracy of tenofovir disoproxil fumarate.
|
Level |
Test |
Wt in mg |
Area |
Quantity added in μg /ml |
Quantity recovered in μg /ml |
% Recovery |
Mean recovery |
|
|
80% |
1 |
15.12 |
363213 |
120.88 |
122.38 |
101.24 |
101.56 |
|
|
2 |
15.21 |
364670 |
120.88 |
122.87 |
101.65 |
|||
|
3 |
15.17 |
365204 |
120.88 |
123.05 |
101.80 |
|||
|
100% |
1 |
15.19 |
452640 |
151.1 |
152.51 |
100.94 |
101.38 |
|
|
2 |
15.23 |
455063 |
151.1 |
153.33 |
101.48 |
|||
|
3 |
15.09 |
456144 |
151.1 |
153.69 |
101.72 |
|||
|
120% |
1 |
15.05 |
545063 |
181.32 |
183.65 |
101.29 |
101.46 |
|
|
2 |
15.18 |
536992 |
181.32 |
180.94 |
99.79 |
|||
|
3 |
15.29 |
555957 |
181.32 |
187.33 |
103.31 |
|||
|
Mean recovery of all level |
101.47 |
|||||||
Table 4: Statistical evaluation of the data subjected to method precision of emtricitabine and tenofovir disoproxil fumarate
|
Test |
Amount of test sample in mg. |
Area |
% Assay |
Amount of test sample in mg. |
Area |
% Assay |
|
|
Emtricitabine |
Tenofovir disoproxil fumarate |
||||
|
Test -1 |
10.05 |
5465888 |
98.73 |
15.35 |
458986 |
100.75 |
|
Test -2 |
10.01 |
5427638 |
98.43 |
15.29 |
451167 |
99.42 |
|
Test -3 |
9.94 |
5432774 |
99.21 |
15.21 |
452640 |
100.27 |
|
Test -4 |
9.97 |
5441443 |
99.07 |
15.32 |
455063 |
100.08 |
|
Test -5 |
9.95 |
5456686 |
99.55 |
15.31 |
456144 |
100.39 |
|
Test -6 |
9.89 |
5463947 |
100.29 |
15.28 |
457987 |
100.99 |
|
|
Mean Assay |
99.21 |
Mean Assay |
100.32 |
||
|
|
SD |
0.655 |
SD |
0.548 |
||
|
|
RSD |
0.660 |
RSD |
0.547 |
||
Method application:
Twenty tablets were weighed accurately and average weight of each tablet was determined. A powder equivalent to 1000mg of standard emtricitabine and 1500 mg of tenofovir disoproxil fumarate were weighted accurately and transferred in 10ml volumetric flask to give concentration as 1000μg/ml of Emtricitabine and 1500μg/ml. of Tenofovir disoproxil fumarate respectively. The working standard solution was prepared by diluting 1ml of 1000μg/ml of emtricitabine and 1500μg/ml. of tenofovir disoproxil fumarate solution to 10ml with diluent to get concentration 100μg/ml of emtricitabine and 1500μg/ml. of tenofovir disoproxil fumarate respectively. From this solution 1.0µl was injected specific conditions. The analyte peak was identified by comparison with that of respective standard. The (%) assay results were expressed in table no. 4. It indicates the amount of emtricitabine and tenofovir disoproxil fumarate in the product meets the requirement.
RESULT AND CONCLUSION:
The reproducibility, repeatability and accuracy of the proposed method were found to be satisfactory which is evidenced by low values of standard deviation and percent relative standard deviation. The accuracy and reproducibility of the proposed method was confirmed by recovery experiments, performed by adding known amount of the drug to the pre-analyzed active pharmaceutical ingredient and reanalyzing the mixture by proposed method. Thus the proposed RP-HPLC method is used for estimation of emtricitabine and tenofovir disoproxil fumarate from active pharmaceutical ingredient. It is more precise, accurate, linear, robust, simple and rapid method. Hence the proposed RP-HPLC method is strongly recommended for the quality control of the raw material, active pharmaceutical ingredient and pharmaceutical formulation.
ACKNOWLEDGMENT:
Authors express sincere thanks to the principal, of D.G. Ruparel College, Mumbai for encouragement and providing laboratory facilities.
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Received on 29.07.2021 Modified on 30.08.2021
Accepted on 21.09.2021 ©AJRC All right reserved
Asian J. Research Chem. 2021; 14(6):412-416.
DOI: 10.52711/0974-4150.2021.00070