A simple, economical, precise, and accurate new UV-visible spectrophotometric baseline manipulation method for simultaneous determination of tenofovir disoproxil fumarate (TE) and emtricitabine (EM) in combined tablet dosage form has been developed. The method is based on baseline manipulation (difference) spectroscopy where amplitudes at 261 and 289.9 nm were selected to determine TE and EM, respectively, in combined formulation, and distilled water was used as solvent. Both drugs obey Beer’s law in the concentration ranges of 4–20
Tenofovir (TE): 9[(
Pure drug sample of TE, purity 99.86% and EM, purity 99.92% was kindly supplied as a gift sample by Emcure Pharmaceutical Pvt., Ltd., Pune. These samples were used without further purification. Tablet used for analysis was TENVIR-EM (Batch no. X81241) manufactured by Cipla Ltd., Goa, India, containing TE 300 mg and EM 200 mg per tablet.
An UV-visible double beam spectrophotometer (Varian Cary 100) with 10 mm matched quartz cells was used. All weighing were done on electronic balance (Model Shimadzu AUW-220D).
Standard stock solution containing 200
Twenty tablets were weighed accurately and a quantity of tablet powder equivalent to 200 mg of TE (300 mg of EM) was weighed and dissolved in the 80 mL of distilled water with the aid of ultrasonicator for 15 min, and solution was filtered through Whatman paper no. 41 into a 100 mL volumetric flask. Filter paper was washed with the distilled water, adding washings to the volumetric flask and volume was made up to mark. The solution was suitably diluted with distilled water to get 200
The accuracy of the assay method was evaluated with the recovery of the standards from excipients. Recovery studies were carried out by applying the method to drug content present in tablet dosage form to which known amount of mix standard of TE and EM were added at 50%, 100%, and 150% levels. At each of the levels, three determinations were performed and results were obtained.
The precision of repeatability was studied by six replicate analyses of tablet solutions containing 12 and 18
The values of LOD and LOQ were calculated by using
To determine the robustness of the method, the final experimental conditions were purposely altered and the results were examined. The parameters considered (± values) for the study were, shaking time of solution (±2 min), wavelength of measurement (±1 nm), and concentration of TE in reference cell (±1
Specificity of the method was determined by comparing the absorbance values of standard mixture of drugs and formulation sample at specified wavelengths for both drugs. Mean of three absorbance values of standard mixture and formulation sample in the concentration range 4–20
The Beer Lambert law [
Overlain spectra of TE (4–20
Typical baseline manipulation spectrograph of TE and EM in combination when TE 20
In this method, composition of blank remains constant throughout the experiment. The method has certain advantages such as less time is required compared to other UV methods, less number of dilutions/solutions is required, and the method is suitable for binary mixtures of analytes.
Composition of the blank is changed to estimate different analytes form the mixture. The method has advantages same as SBM and it can be used for ternary mixtures of analytes. In this method, spectrum of sample containing mixture of three drugs shows two different peaks from which suitable analytical wavelength can be assigned to two analytes which is free from inferences by other analyte in mixture.
The newly developed method was validated according to the ICH guidelines with respect to specificity, linearity, accuracy, precision, and robustness.
Both drugs obey Beer’s law in the concentration ranges of 4–20
Optical characteristics of the method and result of formulation analysis, recovery study, and method sensitivity.
Parameter | Analyte | |
---|---|---|
TE | EM | |
|
261 | 289.9 |
Range ( |
4–20 | 6–30 |
Regression equation |
||
Slope (m) | 0.05974 | 0.022127 |
Intercept ( |
−0.23012 | −0.01772 |
Correlation coefficient | 0.999 | 0.999 |
Limit of quantitation (LOQ) | 1.58 | 4.5 |
Limit of detection (LOD) | 0.55 | 1.5 |
Formulation analysis (% assay, % RSD) | ||
F I | 99.75, 0.84 | 99.35, 1.01 |
F II | 101.2, 1.23 | 100.2, 0.97 |
Recovery study at the level (% R, % RSD) | ||
50% | 100.21, 0.54 | 99.5, 0.45 |
100% | 100.4, 0.94 | 101.4, 0.75 |
150% | 99.45, 0.54 | 99.78, 0.69 |
The assay for the marketed tablets was established with present spectrophotometric condition (band width 0.5 and scan speed 400 nm per minute) which was developed and it was found to be accurate and reliable. The average drug content was found in the range 99.35 to 101.2 for both drugs in two formulations, that is, formulation I and II. No interfering peaks were found in spectrograph, indicating the estimation of drug free from inference of excipients. Results of recovery study were in the range of 99.5–101.4% and percentage of RSD was always less than 0.69. The optical characteristic of the method and results for formulation analysis are presented in Table
The accuracy study was carried as per the procedure described in Section
Result of recovery studies for TE and EM.
Analyte name | Formulation study ( |
Recovery (accuracy) study | ||
---|---|---|---|---|
Base level amount | Amount spiked | Recovery level | % Recovery, | |
|
|
% RSD ( |
||
TE | 6 | 3 | 50% | 100.21, 0.54 |
6 | 6 | 100% | 100.4, 0.94 | |
6 | 9 | 150% | 99.45, 0.54 | |
| ||||
EM | 9 | 4.5 | 50% | 99.5, 0.45 |
9 | 9 | 100% | 101.4, 0.75 | |
9 | 13.5 | 150% | 99.78, 0.69 |
Precision data evaluated by using experimental design based
Intraday precision study for TE.
Analysts | Time | ||
---|---|---|---|
|
|
|
|
Analyst 1 | 99.5 | 100.3 | 99.34 |
Analyst 1 | 100.2 | 100.4 | 99.54 |
Analyst 1 | 101.1 | 101.1 | 100.4 |
Analyst 2 | 99.21 | 99.54 | 101.3 |
| |||
Source of variation | Sum of square | Degree of freedom | Mean of square |
| |||
Between time | 120086.5 | 2 | 60043.27 |
Between time and analyst | 120088.5 | 3 | 40029.5 |
Residual | −120083 | 6 | −20013.8 |
| |||
Total | 120092.3 | 11 | — |
Interday precision study for TE.
Source of variation | Sum of square | Degree of freedom | Mean of square |
---|---|---|---|
Between day | 119702.2 | 2 | 59851.08 |
Between day and analyst | 119701.7 | 3 | 39900.58 |
Residual | −119694 | 6 | −19949.1 |
| |||
Total | 119709.4 | 11 | — |
Effect of variation of experimental conditions was studied and results are presents as described previously. Results of the robustness study for TE & EM are presented in Table
Robustness study for TE and EM.
Factor | Level | Mean % assay, % RSD | |
---|---|---|---|
TE | EM | ||
Shaking time (±2 min) | 10 | 99.2, 0.97 | 99.34, 0.87 |
6 | 98.18, 0.25 | 98.42, 1.23 | |
Measurement wavelength (±1 nm) | 260 (EM), 288.9 (TE) | 99.45, 0.43 | 99.43, 0.54 |
262 (EM), 290.9 (TE) | 99.08, 0.48 | 99.65, 0.64 | |
Concentration of TE in blank (±1 |
21 | 99.09, 0.72 | 100.45, 1.34 |
19 | 100.3, 0.59 | 99.78, 0.45 |
Specificity was performed as described in the procedure section. The absorbances were measured of the standard mixture of drug and formulation sample at specified wavelength, that is, 261 for TE. Calculated
Results of specificity study for TE (
Concentration ( |
Abs. standard | Abs. sample | Difference ( |
|
---|---|---|---|---|
6 | 0.11504 | 0.1203 | 0.00526 |
|
12 | 0.2478 | 0.2234 | −0.0244 |
|
18 | 0.3681 | 0.3765 | 0.0084 |
|
24 | 0.4884 | 0.4787 | −0.0097 |
|
30 | 0.6087 | 0.5819 | −0.0268 |
|
Baseline manipulation method was compared with the laboratory developed first order derivative, ratio derivative method, and absorption corrected method. The results of ANOVA for TE are shown in Table
ANOVA table of comparison of results of reported spectrophotometric and proposed baseline manipulation methods for TE.
Source of variation | Sum of square | Degree of freedom | Mean square |
---|---|---|---|
Between samples | 3.8174 | 3 | 1.272 |
Within sample | 26.364 | 16 | 1.647 |
| |||
Total | 30.182 | — | — |
The developed method was found to be simple, sensitive, accurate, and precise and can be used for the routine quality control analysis of EM and TE. The concept of baseline manipulation method can be extended to other UV active drug molecules in formulation and in combination. As the method could effectively separate responses in spectra of the drugs from each other in a single spectrometric scan, it reduces human efforts and minimizes errors. The baseline manipulation method is equivalent to other methods such as area under curve or simultaneous estimation of drugs or other multicomponent method used for binary combination.
The authors would like to thank Emcure Pharmaceutical Ltd., Pune, for providing gift samples of drugs. Authors are also thankful to the Management and Principal of MAEER’s Maharashtra Institute of Pharmacy, Pune, for providing necessary facilities. The authors declare that they have no conflict of interests.