Simultaneous Spectrophotometric Estimation of Emtricitabine and Tenofovir disoproxil fumarate by Area Under Curve Method in Combined Dosage Form
Rajan V. Rele*
Central Research Laboratory, D.G. Ruparel College, Matunga, Mumbai 400016.
ABSTRACT:
The objective of the study was to develop a simple, accurate, precise and rapid a UV spectrophotometric method, area under curve method for the determination of emtricitabine and tenofovir disoproxil fumarate in combined dosage form. The method was further validated by ICH guidelines. The proposed area under curve method involves the measurement of area at selected analytical wavelength ranges and performing the analysis using “Cramer’s rule and Matrix method”. The two analytical wavelengths ranges were used i.e. 255-265nm and 272- 286nm for estimation of emtricitabine and tenofovir disoproxil fumarate respectively. The linearity of the proposed method was found in the concentration range of 2 – 20µg/ml (r2= 0.9999) for emtricitabine and tenofovir disoproxil fumarate respectively. The percentage mean recovery was found to be 100.129% emtricitabine and 100.30% for tenofovir disoproxil fumarate respectively. The method was statistically validated for its linearity, accuracy and precision as per ICH guidelines. Both intra and inter day variation showed less percentage (%) RSD values indicating high grade of precision of this method.
KEYWORDS: UV spectrophotometric estimation, emtricitabine, tenofovir disoproxil fumarate, validation.
INTRODUCTION:
Emtricitabine is 4-amino-5-fluoro-1-[(2R, 5S)-2 (hydroxyl -methyl)-1, 3-oxathiolan-5-yl]-1,2-dihydro- pyrimidin-2-one. It 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 methods1-9. HPLC10-30, and HPTLC31,. The newly developed method can be used for the routine analysis due simplicity 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 disoproxil fumarate
Structure of emtricitabine
MATERIALS AND METHOD:
Instrument and reagents:
Spectral scan was made on a Shimadzu UV-spectrophotometer, model 1800 (Shimadzu, Japan) with spectral band width of 0.5nm with automatic wavelength corrections by using a pair of 10mm quartz cells. All spectral measurements were done by using UV-Probe 2.42 software.
Reference standard of emtricitabine and tenofovir disoproxil fumarate were obtained from reputed firm with certificate of analysis.
Preparation of standard drug solutions:
100mg standard emtricitabine was weighed accurately and transferred to a 100ml volumetric flask and sonicated with 30ml of distilled water for 15 minutes. The volume was made up to the mark with distilled water to give a stock solution of emtricitabine of concentration 1000μg/ml. From this solution, 10ml of solution was pipetted out and transferred into 100ml volumetric flask. The volume was made up to mark with distilled water to give a working standard solution of concentration 100μg/ml.
Similarly 100mg standard tenofovir disoproxil fumarate was weighed accurately and transferred to a 100ml volumetric flask and sonicated with 30ml of distilled water for 15 minutes. The volume was made up to the mark with distilled water to give a stock solution of tenofovir disoproxil fumarate of concentration 1000 μg/ml. From this solution, 10ml of solution was pipetted out and transferred into 100ml volumetric flask. The volume was made up to mark with distilled water to give a working standard solution of concentration 100μg/ml.
Estimation from tablets:
Twenty tablets were weighed accurately and average weight of each tablet was determined. Powder equivalent to 20mg of emtricitabine and 30mg of tenofovir disoproxil fumarate was weighed and transferred in 100 ml of volumetric flask. A 30 ml of distilled water was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with distilled water to give concentration as 100μg/ml. Such solution was used for analysis.
Experimental:
Method: Area under curve method:
Area under curve method involves the calculation of integrated values of absorbance with respect to the wavelength between two selected wavelengths such as λ1 and λ2. The area under curve between λ1 and λ2 was calculated by UV probe 2.42 software.
(a) For emtricitabine:
For the selection of analytical wavelength range, 10 μg/ml solution of emtricitabine was scanned in the spectrum mode from 400 nm to 200 nm by using distilled water as blank. On examination of the spectra, 272-286 nm was selected as working wavelength range for emtricitabine.
(b) For tenofovir disoproxil fumarate:
For the selection of analytical wavelength range, 10 μg/ml solution of tenofovir disoproxil fumarate was scanned in the spectrum mode from 400 nm to 200 nm by using distilled water as blank. On examination of the spectra, 255-265 nm was selected as working wavelength range for emtricitabine.
Preparation of calibration curves:
Series of solutions containing 2 – 20 µg/ml of emtricitabine and tenofovir disoproxil fumarate were used to determine linearity of the proposed method respectively. The areas under curve of above solutions of emtricitabine and tenofovir disoproxil fumarate were measured at their respective selected analytical wavelength ranges. [Fig. 1 (a), 1(b)]. This area under curve (AUC) was then divide by concentration in mg/lit to get Xemt for emtricitabine and Xteno for tenofovir disoproxil fumarate.
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Fig. 1 (a): Spectrum showing area under curve of emtricitabine in the concentration of 10µg/ ml at 272-286nm.
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Fig. 1 (b): Spectrum showing area under curve of tenofovir disoproxil fumarate in the concentration of 10µg/ml at 255-265 nm. |
After measuring the area under curve of emtricitabine at 272-286 nm and 255-265 nm for tenofovir disoproxil fumarate by using UV-Probe software 2.42, the calibration curves were plotted of area under curve against concentrations [Fig. 2 (a), 2(b)].
Fig. 2 (a): Calibration curve of emtricitabine in the concentration range of 2-20 µg/ml.
Fig. 2 (b): Calibration curve of tenofovir disoproxil fumarate in the concentration range of 2-20 µg/ml.
Results of the analysis are given in table 1.
Table 1: Values of results of optical and regression of drugs
|
Parameter |
Emtricitabine |
Tenofovir disoproxil fumarate |
|
Detection Wavelength range (nm) |
272-286 |
255-265 |
|
Beer Law Limits (µg/ml) |
2-20 |
2 -20 |
|
Correlation coefficient(r2) |
0.9999 |
0.9999 |
|
Regression equation (y=b+ac) |
||
|
Slope (a) |
0.032 |
0.009 |
|
Intercept (b) |
0.002 |
0.003 |
Estimation from tablets:
Twenty tablets were weighed accurately and average weight of each tablet was determined. Powder equivalent to 20mg of emtricitabine and 30mg of tenofovir disoproxil fumarate was weighed and transferred in 100 ml of volumetric flask. A 30ml of distilled water was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with distilled water to give concentration as 20μg/ml of emtricitabine and 30μg/ml of tenofovir disoproxil fumarate respectively. Appropriate aliquot was pipetted out from the sample solution and was further diluted to obtain mixture containing 2µg/ml of emtricitabine and 3 µg/ml of tenofovir disoproxil fumarate respectively. The spectrum of sample solution containing Emtricitabine and Tenofovir disoproxil fumarate was recorded and areas under curves were recorded in wavelength ranges of 272-286nm and 255 -265nm. The areas under curves were analyzed by applying “Crammer’s rule and “Matrix method”. It is defines as “The total area under curve of mixture at particular wavelength range is equal to sum of area under curve of individual component at same wavelength range.” (Fig. 3).
Fig. 3: Spectrum showing area under curve of mixture at 255-265 nm and 272-286 nm.
X = AUC of component between selected wavelength ranges
Concentration of that component in mg/lit
C Emt= ( XT255-265 - AUCM272-286) - ( XT272-286 – AUCM255-265)
(XT255-265 * XT272-296) - (XT272-286 * XT233-265)
C Teno = ( XE255-265 - AUCM272-286) - ( XE272-296 – AUCM255-265)
(XT255-265 * XT272-296) - (XT272-286 * XT233-265)
Where,
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CEmt = |
Concentration of emtricitabine |
|
CTeno = |
Concentration of tenofovir disoproxil fumarate |
|
Xemt1 = |
Area under curve of emtricitabine at wavelength 255-265 nm |
|
Xemt2 = |
Area under curve of emtricitabine at wavelength 272-286 nm |
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Xteno1 = |
Area under curve of tenofovir disoproxil fumarate at wavelength 255-265 nm |
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XORN2 = |
Area under curve of tenofovir disoproxil fumarate at wavelength 272-286 nm |
|
AUCM = |
Area under curve of mixture |
Method Validation:
These methods were validated according to ICH guidelines.
Accuracy:
To ascertain the accuracy of proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% and 120%). Percent recovery for emtricitabine was found in the range of 100.30% and tenofovir disoproxil fumarate was found in the range of 100.13%. (Table 2).
Linearity:
The linearity of measurement was evaluated by analyzing different concentration of the standard solutions of Emtricitabine and Tenofovir disoproxil fumarate. For emtricitabine and tenofovir disoproxil fumarate drugs concentration range was found to be 2-20 µg/ml for linearity respectively.
Precision:
The method precision was established by carrying out the analysis of tablets powder blend containing 200mg of emtricitabine and 300mg of tenofovir disoproxil fumarate. The assay was carried out for the drugs by using proposed analytical method in six replicates. The values of relative standard deviation were well within limits 99.45% and 100.54% for emtricitabine and 99.65% and 100.34% for tenofovir disoproxil fumarate respectively indicating the sample repeatability of the method. The results obtained are tabulated in Table 3.
Intra-day precision was estimated by assaying tablets powder blend containing 200mg of emtricitabine and 300 mg of tenofovir disoproxil fumarate. The assay was carried out for the drugs by using proposed analytical method in six replicates. The results were average for statistical evaluation.
Inter-day precision was estimated by assaying tablets powder blend containing 200mg of emtricitabine and 300 mg of tenofovir disoproxil fumarate for three consecutive days (i.e. 1st, 3rd and 5th days). The statistical validation data for intra and inter day precision is summarized in Table 4.
Table 2 (a): Statistical evaluation of the data subjected to accuracy emtricitabine
|
Amount of Sample Added in (µg/ml) |
Amount of Standard Added in (µg/ml) |
Total amount recovered |
Percentage recovery (%) |
Standard deviation |
Percentage of relative standard deviation (C.O.V.) |
|
2 |
0 |
2.003077 |
100.1538 |
0.003972 |
0.198309 |
|
2 |
2 |
4.039121 |
100.978 |
0.034844 |
0.862657 |
|
2 |
6 |
5.962637 |
99.37729 |
0.028613 |
0.479873 |
|
2 |
8 |
8.057143 |
100.7143 |
0.027684 |
0.343598 |
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|
|
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Mean =100.3059 |
Mean =0.023778 |
Mean =0.471109 |
Table 2(b): Statistical evaluation of the data subjected to accuracy tenofovir disoproxil fumarate
|
Amount of Sample Added in (µg/ml) |
Amount of Standard Added in (µg/ml) |
Total amount recovered |
Percentage recovery (%) |
Standard deviation |
Percentage of relative standard deviation (C.O.V.) |
|
2 |
0 |
2.005442 |
100.27211 |
0.0133074 |
0.663564 |
|
2 |
2 |
4.014966 |
100.37415 |
0.0197162 |
0.491067 |
|
2 |
6 |
8.013605 |
100.17007 |
0.0262882 |
0.328045 |
|
2 |
8 |
9.972789 |
99.727891 |
0.1059712 |
1.062603 |
|
|
|
|
Mean =100.13605 |
Mean =0.0413207 |
Mean =0.63632 |
Table 3: Statistical evaluation of the data subjected to method of precision
|
Experiment no. |
Weight of emtricitabine taken in mg |
Weight of emtricitabine found in mg |
Weight of tenofovir disoproxil fumarate taken in mg |
Weight of tenofovir disoproxil fumarate found in mg |
|
1 |
10 |
9.969231 |
10 |
10.009 |
|
2 |
10 |
10.03077 |
10 |
10.019 |
|
3 |
10 |
10.000 |
10 |
10.038 |
|
4 |
10 |
10.0377 |
10 |
10.019 |
|
5 |
10 |
9.9692 |
10 |
10.047 |
|
6 |
10 |
9.9692 |
10 |
10.009 |
|
|
Standard deviation |
0.03199 |
|
0.015643 |
|
|
%RSD |
0.320031 |
|
0.156062 |
Table 4: Summary of validation parameter for intra-day and inter-day
|
Sr. No. |
Parameters |
Emtricitabine |
Tenofovir disoproxil fumarate |
|
1 |
Intra-day precision (N=3) amount found ± % R.S.D. |
100.25% 0.2541 |
100.14% 0.2615 |
|
2 |
Inter-day precision (N=3) amount found ± % R.S.D. |
99.27% 0.2417 |
99.38% 0.1795 |
Both intra- day and inter-day precision variation found to be less in % RSD values. It indicates high degree of precision of the method.
RESULT AND DISCUSSION:
The developed area under curve spectrophotometric method for simultaneous determination of Emtricitabine and Tenofovir disoproxil fumarate in tablet formulation was found to be simple and convenient for the routine analysis of two drugs. The proposed method is accurate, precise and reproducible. It is confirmed from validation data as given in tables 1 to 3. The % RSD was found to be less than 1, which indicates validity of method. Linearity was observed by linear regression equation method for emtricitabine and tenofovir disoproxil fumarate in different concentration range. The correlation coefficient of these drugs was found to be close to 1.00, indicating good linearity figure 2 (a) and 2 (b).
The assay results obtained by proposed method is shown in table 2 are in good agreement. Hence proposed method can be used for routine analysis of these two drugs in combined dosage form. Method is simple, accurate, precise, reliable, rapid, sensitive, reproducible and economical. It is validate as per ICH guidelines.
The proposed method is simple, precise, accurate and rapid for the determination of Emtricitabine and Tenofovir disoproxil fumarate in combined dosage form. This method can be adopted as an alternative to the existing methods. It can be easily and conveniently adopted for routine quality control analysis.
ACKNOWLEDGEMENT:
Authors express sincere thanks to the principal of D.G. Ruparel College, Dr. Tushar Desai, for encouragement and providing laboratory facilities.
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Received on 19.10.2020 Modified on 09.11.2020
Accepted on 23.11.2020 ©AJRC All right reserved
Asian J. Research Chem. 2021; 14(1):67-72.
DOI: 10.5958/0974-4150.2021.00011.0