UV Spectrophotometric Estimation of Tenofovir Disoproxil Fumrate by area under Curve Methods in Bulk and Pharmaceutical Dosage Form
Rajan V. Rele*, Prathamesh P. Tiwatane
Central Research Laboratory, D.G. Ruparel College, Matunga, Mumbai 400016.
*Corresponding Author E-mail: jabourimess2@gmail.com
ABSTRACT:
Simple and precise UV spectrophotometric method by area under curve [AUC] - have been developed and validated for the estimation of tenofovir disoproxil fumrate in bulk and its tablet formulation. The standard and sample solutions of tenofovir disoproxil fumrate were prepared in distilled water. Tenofovir disoproxil fumrate was estimated area under curve (AUC) method, the zero-order spectrum of tenofovir disoproxil fumrate was measured in between 255 nm to 265 nm. Beer’s law was obeyed in the concentration range of 1 to 20 μg / ml with coefficient of correlation value 0.9999. These methods were tested and validated for various parameters according to ICH guidelines. The precision expressed as relative standard deviation were of 0.1560% respectively. The proposed method was successfully applied for the determination of tenofovir disoproxil fumrate in pharmaceutical formulation. Results of the analysis were validated statistically and were found to be satisfactory. The proposed method is simple, easy to apply, low-cost and require relatively inexpensive instruments.
This research article provides important insight into development, validation and application of reverse phase high pressure liquid chromatographic method for the assay of tenofovir disoproxil fumrate, bulk drug and pharmaceutical dosage form. It is a 1-(6-Aminopurin-9-yl)-prapan-2-yl-oxymethylphosphonic acid, drug used in the treatment of human immunodeficiency virus (HIV) Type-1 patients.
According to the literature review several methods has been developed for drug, like spectroscopy methods1-6. HPLC7-25, and HPTLC26. The newly developed method can be used for the routine analysis due to 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 and tenofovir disoproxil fumrate
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 tenofovir disoproxil fumrate was obtained from reputed firm with certificate of analysis.
Preparation of standard drug solutions:
10mg standard tenofovir disoproxil fumrate was weighed accurately and transferred to a 10ml volumetric flask and sonicated with 3ml of distilled water for 5 minutes. The volume was made up to the mark with distilled water to give a stock solution of tenofovir disoproxil fumrate of concentration 1000μg/ml. From this solution, 1ml of solution was pipetted out and transferred into 10ml 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 10mg of tenofovir disoproxil fumrate was weighed and transferred in 100ml 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 100 μg/ml. Such solution was used for analysis.
Experimental:
Method : Area under curve (AUC) method:
Area under curve method involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelengths such as λ1 and λ2. The area under curve between λ1 and λ2 were calculated by UV probe 2.42 software. In this method, 10 μg/ml solution of tenofovir disoproxil fumrate was scanned in the spectrum mode from 200 nm to 350 nm. From zero order spectrums the AUC calculation was done. The AUC spectrum was measured between 255 nm to 265 nm (Fig. 1).
Fig. 1. Area under curve spectrum of tenofovir disoproxil fumrate (10 μg/ml) showing area from 255 nm to 265 nm.
Into series of 10ml graduated flask, varying amount of standard solutions of tenofovir disoproxil fumrate was pipette out and volume was adjusted with distilled water. Solutions were scanned between 350 nm to 200nm in spectrum mode. The AUC calculations were done and the calibration curve for tenofovir disoproxil fumrate was plotted in the concentration range of 1 to 20μg/ml (Fig. 2).
Table 1: Values of results of optical and regression of drug
|
Parameter |
Area under curve (AUC) method |
|
Detection Wavelength (nm) |
250-265 |
|
Beer Law Limits (µg/ml) |
1-20 |
|
Correlation coefficient (r2) |
0.9999 |
|
Regression equation (y=b+ac) |
|
|
Slope (a) |
0.009 |
|
Intercept (b) |
0.004 |
Fig. 2. Calibration curve for tenofovir disoproxil fumrate by area under curve spectroscopy
Results of analysis are given in table 1.
Table 2: Results of recovery of tenofovir disoproxil fumrate for area under curve (AUC) method
|
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 |
2.005 |
100.27 |
0.0133 |
0.6635 |
|
2 |
2 |
4.014 |
100.37 |
0.0197 |
0.4910 |
|
2 |
6 |
8.013 |
100.17 |
0.0262 |
0.3280 |
|
2 |
8 |
9.972 |
99.72 |
0.1059 |
1.062 |
|
|
|
|
Mean =100.13 |
Mean =0.0413 |
Mean =0.6363 |
Validation:
Accuracy:
Accuracy of the proposed methods was carried as on the basis of recovery studies. It is performed by the standard addition method. Recovery studies were performed by adding standard drug at different levels to the pre-analyzed tablets powder solution and the proposed method was followed. From the amount of the drug estimated, the percentage recovery was calculated. The results of the analysis are shown in table (2).
Precision:
The method precision was established by carrying out the analysis of homogenous powder blend of tablets. The assay was carried out of drug by using proposed analytical method in six replicates. The values of relative standard deviation lie well within the limits indicated the sample repeatability of the method. The results obtained are tabulated in table 3.
Table 3: Precision- method precision
|
Experiment No. |
Weight of Tenofovir Disoproxil Fumrate taken in mg |
Weight of Tenofovir Disoproxil Fumrate found in mg |
|
1 |
10 |
10.009 |
|
2 |
10 |
10.019 |
|
3 |
10 |
10.038 |
|
4 |
10 |
10.019 |
|
5 |
10 |
10.047 |
|
6 |
10 |
10.009 |
|
|
Standard deviation |
0.0156 |
|
|
%RSD |
0.1560 |
Inter-day and intra-day precision:
An accurately weighed quantity of tablets powder equivalent to 10mg of tenofovir disoproxil fumrate was transferred to 10ml of volumetric flask. A 3 ml of distilled water was added and sonicated for 5 minutes and filtered. The filtrate and washing were diluted up to the mark with distilled water to give concentration as 1000μg/ml. A 1 ml of such solution further diluted to 10ml to give concentration as 100μg/ml Such solution was used for analysis.
Solution was scanned between 400nm to 200nm in spectrum mode. The area under curve of resulting solutions was measured at between 255nm to 265nm by using as blank as distilled water. The area under curve of final solutions was read after 0 hr., 3 hrs. and 6 hrs. in 10 mm cell at 255nm to 265nm. Similarly area under curve of the same solution was read on 1st, 2nd and 5th day. The amount of tenofovir disoproxil fumrate was estimated by comparison with standard at 250nm to 270nm, table 4.
Table 4: Summary of validation parameter for intra-day and inter-day
|
Sr. No. |
Parameters |
Area under curve (AUC) method |
|
(A) |
Intra-day precision (n=3) Amount found ± % RSD |
99.68% 0.1126 |
|
(B) |
Inter-day precision (n=3) Amount found ± % RSD |
99.86% 0.2138 |
|
(c) |
Ruggedness Analyst to analyst (n= 3) % RSD |
0.3865 |
Limit of Detection (LOD) and Limit of Quantification (LOQ)
The limit of detection (LOD) is defined as the lowest concentration of an analyte that an analytical process can reliably differentiate from back-ground levels. In this study, LOD and LOQ were based on the standard deviation of the response and the slope of the corresponding curve using the following equations-
LOD = 3.3 σ/S and LOQ = 10 σ/S
Where σ is the standard deviation of the signal to noise ratio of the sample and S is the slope of the related calibrations graphs.
The limit of quantification (LOQ) is defined as the lowest concentration of the standard curve that can be measured with an acceptable accuracy, precision and variability. The values of LOD and LOQ are given in table 5.
Table 5: Values of results of LOD and LOQ
|
parameters |
Values |
|
Limit of Detection (μg/ml) |
0.0512335 |
|
Limit of Quantification (μg/ml) |
0.1552529 |
Ruggedness:
The ruggedness of the method is defined as degree of reproducibility of results obtained by analysis of tenofovir disoproxil fumrate sample under variety of normal test conditions such as different laboratories, different analysts and different lots of reagents. Quantitative determination of tenofovir disoproxil fumrate was conducted spectrophotometrically on one laboratory. It was again tested in another laboratory using different instrument by different analyst. The assays obtained in two different laboratories were well in agreement. It proved ruggedness of the proposed methods.
RESULT AND DISCUSSION:
The area under curve UV-spectroscopic method is useful for routine analysis of tenofovir disoproxil fumrate in bulk drug and formulation. The method was validated according to International Conference on Harmonization guidelines for validation of analytical procedures. Tenofovir disoproxil fumrate has the absorbance maxima in the areas were measured between 255 nm to 265 nm. The polynomial regression data for the calibration plots showed good linear relationship in the concentration range of 1 to 20μg/ml and given in table1. Recovery studies were carried out by adding the pure drug to the previously analyzed tablet powder sample and shown in table 2. The percentage recovery value indicates non interference from excipients used in formulation. The reproducibility and accuracy of the method were found to be good, which was evidenced by low standard deviation. The most striking features of method is its simplicity and rapidity, not requiring tedious sample solutions preparations which are needed for other instrumental methods. From the results obtained it can be concluded that the proposed methods are fully validated and found to be simple, sensitive, accurate, precise, reproducible, rugged and robust and relatively inexpensive. So, the developed methods can be easily applied for the routine quality control analysis of tenofovir disoproxil fumrate in pharmaceutical formulation.
ACKNOWLEDGMENT:
Authors express sincere thanks to the Principal, Dr. Tushar M. Desai of D. G. Ruparel College, Mumbai.
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Received on 11.10.2019 Modified on 10.12.2019
Accepted on 16.01.2020 ©AJRC All right reserved
Asian J. Research Chem. 2020; 13(1): 07-11.
DOI: 10.5958/0974-4150.2020.00002.4