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New simple spectrophotometric method for determination of the antiviral mixture of emtricitabine and tenofovir disoproxil fumarate
⁎Corresponding author. Tel.: +20 3 4871351; fax: +20 3 4873273. tbelaleg@yahoo.com (Tarek S. Belal)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
A new simple spectrophotometric method was recently developed for the determination of binary mixtures without prior separation. The method is based on generation of ratio spectra of compound X by using standard spectrum of compound Y as a divisor, then the peak to peak (or peak to trough) amplitudes between two selected wavelengths in the ratio spectra are found proportional to concentration of X without interference from compound Y. The method was applied for the determination of the antiviral mixture of emtricitabine (EMT) and tenofovir disoproxil fumarate (TEN). For the determination of EMT, a solution of 15 μg/mL TEN was used as divisor to generate the ratio spectra, and the peak to trough amplitudes between 228.5 and 260.5 nm were plotted against EMT concentration. Similarly by using 10 μg/mL EMT as divisor, the peak to trough amplitudes between 229 and 260 nm were found proportional to TEN concentration. The reliability and analytical performance of the proposed spectrophotometric procedure were statistically validated with respect to linearity, range, precision, accuracy, detection and quantification limits. Calibration curves were linear in the range of 2–40 μg/mL for both drugs with correlation coefficients >0.9997. The proposed method was successfully applied for the simultaneous determination of the two drugs in several laboratory prepared mixtures and in their combined tablet dosage form.
Keywords
Emtricitabine
Tenofovir
Spectrophotometric analysis
Binary mixture
Ratio spectra
Peak to peak measurement
1 Introduction
One of the main challenges facing analytical chemists is the spectrophotometric determination of two or more compounds in the same sample without preliminary separation. Several spectrophotometric methods have been used for resolving mixtures of compounds with overlapping spectra such as derivative spectrophotometry (Wahbi and Ebel, 1974; Morelli, 1994) Vierordt’s method (Görög, 1995), dual wavelength spectrophotometry (Shibata et al., 1971), pH-induced difference spectrophotometry (Davidson and Stenlake, 1974), H-point standard addition method (HPSAM) (Sabry and Khamis, 2000) and several more sophisticated computerized and chemometric spectrophotometric methods (Korany et al.,1986, 1990; Wahbi et al., 1989; El-Gindy et al., 2010).
Generation of absorbance ratio spectra has been the basis of several analytical procedures for the simultaneous spectrophotometric determination of compounds in binary and ternary mixtures. First, binary mixtures were resolved using the ratio-spectra derivative method (Salinas et al., 1990). Then, this method was modified and extended for the determination of ternary mixtures. Examples of these modified versions are the derivative ratio spectra zero-crossing method (Berzas Nevado et al., 1992; Abdel-Hay et al., 2008), the double divisor ratio spectra derivative method (Dinç and Onur, 1998) and the successive derivative ratio spectra method (Afkhami and Bahram, 2005). Furthermore, the ratio spectra were exploited for the development of other mathematical methods for resolution of binary and ternary mixtures such as the mean centering of ratio spectra method (Abdelwahab et al., 2012), the ratio subtraction method (El-Bardicy et al., 2008) and the convoluted double divisor ratio spectra using combined trigonometric Fourier functions method (Youssef and Maher, 2008). In most of these methods, at least two mathematical processes (e.g. division followed by derivative curve generation) are needed in order to get measurable amplitude that is correlated to the concentration of only one compound without interference from the others in the mixture. Obviously, some methods need more sophisticated mathematical treatment to unambiguously determine the target compound in the presence of interferences (El-Bardicy et al., 2008; Youssef and Maher, 2008).
Emtricitabine (EMT) and tenofovir disoproxil fumarate (TEN) are reverse transcriptase inhibitors with antiviral activity against HIV-1 and hepatitis B virus. Fixed dose combination products of the two antiviral drugs for the treatment of HIV infection have been developed in order to improve patient adherence and avoid monotherapy, thereby decreasing the risk of acquired drug resistance (Sweetman, 2009; Truvada webpage). Several HPLC methods were found in the literature for the assay of this mixture in human plasma or in tablet dosage forms. Detection of both drugs in these methods was achieved either depending on UV-detection (Karunakaran et al., 2010; Pendela et al., 2011) or tandem mass spectrometry (Gomes et al., 2008; Delahunty et al., 2009; Yadav et al., 2010). HPTLC was also applied for the determination of this binary mixture (Joshi et al., 2009; Bhirud and Hiremath, 2013). Finally, few spectrophotometric methods were published for the simultaneous estimation of EMT and TEN in their combined tablets. These reports recommended the use of area under the curve and dual wavelength methods (Ghorpade et al., 2010), simultaneous equations and absorbance ratio methods (Ingale et al., 2010) and the absorbance correction method and first order derivative spectrophotometry (Karunakaran et al., 2011).
Recently, a new ratio spectra peak to peak measurement method was developed for the determination of binary mixtures without prior separation (Belal et al., 2013). The method employs only one step (dividing the mixture spectra by a standard divisor spectrum), followed by peak to peak measurement in the produced ratio spectra. The method eliminates the derivative step, and does not require searching for zero-crossing points or any complicated mathematical or chemometric treatment of data. This work describes the application of this new method for the quantification of the binary mixture of EMT and TEN where the two compounds were simultaneously determined in several laboratory prepared mixtures and in tablets without prior separation.
2 Theoretical background
Consider a mixture of two compounds X and Y. The absorption spectrum of the mixture-measured in 1 cm pathlength- is defined by the equation:
3 Experimental
3.1 Apparatus
Spectrophotometric measurements were performed using a Specord S600 UV/VIS diode array spectrophotometer (scan speed 6000 nm/min and wavelength interval 0.5 nm), associated with WinAspect software version 2.3 (Analytik Jena AG, Germany).
3.2 Materials
Authentic samples of emtricitabine (EMT) and tenofovir disoproxil fumarate (TEN) were kindly provided by Gilead Pharmaceuticals, USA. High purity distilled water was used for the preparation of all stock and working solutions. The pharmaceutical preparation assayed in the study is Truvada® tablets (Gilead Sciences Inc., Canada) labeled to contain 200 mg EMT and 300 mg TEN per tablet.
3.3 General procedure
Stock solutions of 200 μg/mL EMT and 200 μg/mL TEN were prepared in distilled water and stored refrigerated at 4 °C. The working solutions were prepared by dilution of EMT and TEN stock solutions with distilled water to reach the concentration range of 2–40 μg/mL for both drugs. The absorbance spectra were recorded against distilled water in the range of 200–300 nm and stored. The EMT stored spectra were divided (amplitude by amplitude at each wavelength) by the spectrum of the divisor (15 μg/mL TEN). The peak to trough amplitudes in the EMT ratio spectra between 228.5 and 260.5 nm were measured and plotted against the corresponding concentrations to construct the calibration graph. Similarly, the stored absorbance spectra of TEN were divided by the spectrum of 10 μg/mL EMT. The peak to trough amplitudes in the TEN ratio spectra between 229 and 260 nm were measured and plotted against the corresponding concentrations.
3.4 Applications
3.4.1 Analysis of laboratory prepared mixtures
Aliquots from the previously mentioned stock solutions of EMT and TEN were used to prepare a series of standard laboratory-prepared mixtures of the two drugs. The mixtures were diluted to volume with distilled water. Their absorbance spectra were recorded against distilled water, and the stored spectra were divided by the spectrum of 15 μg/mL TEN (for determination of EMT content), and the spectrum of 10 μg/mL EMT (for TEN determination). The peak to trough amplitudes were measured (Table 1), and concentrations were determined using the corresponding calibration graphs.
| Parameter | EMT | TEN |
|---|---|---|
| Wavelengths (nm) | 228.5 and 260.5 | 229.0 and 260.0 |
| Concentration range (μg/mL) | 2–40 | 2–40 |
| Intercept (a) | 0.1414 | 0.0959 |
| Saa | 0.0292 | 0.0297 |
| Slope (b) | 0.2204 | 0.1631 |
| Sbb | 0.0019 | 0.0015 |
| RSD% of the slope (Sb%) | 0.8621 | 0.9197 |
| Correlation coefficient (r) | 0.99977 | 0.99976 |
| Sy/xc | 0.0454 | 0.0507 |
| Fd | 13244 | 12475 |
| Significance F | 2.90 × 10−11 | 3.47 × 10−11 |
| LODe(μg/mL) | 0.4372 | 0.6009 |
| LOQf(μg/mL) | 1.3249 | 1.8210 |
3.4.2 Analysis of commercial tablets
A total of 5 Truvada® tablets were weighed and finely powdered. To an accurately weighed quantity of the powder equivalent to 40 mg EMT and 60 mg TEN, 50 mL of distilled water was added, sonicated for 15 min, and then filtered into a 100-mL volumetric flask. The residue was washed with two 10-mL portions of distilled water, and the washing solutions were added to the filtrate and diluted to volume with distilled water. Aliquots of the tablets’ aqueous extract were diluted with distilled water to obtain final concentrations within the specified range (2–40 μg/mL for both drugs) then treated as under General Procedure. Recovery values were calculated from similarly treated standard solutions. For standard addition assay, sample solutions were spiked with aliquots of EMT and TEN stock solutions to obtain total concentrations within the previously specified range then treated as under General procedure. Recovered concentrations were calculated by comparing the analyte response with the increment response attained after addition of the standard.
4 Results and discussion
4.1 Development of the spectrophotometric method
The absorbance (zero-order) spectra of EMT and TEN over the range of 200–300 nm in distilled water are shown in Fig. 1. Obviously, the UV bands of both drugs are considerably overlapped that conventional spectrophotometric measurement cannot be used for their simultaneous determination. EMT shows an absorption maximum at about 283 nm which corresponds to minimum interference from TEN (Fig. 1), however, TEN still contributes to the absorption of the mixture at this wavelength, therefore the EMT peak at 283 nm cannot be used for its selective determination in the presence of TEN. On the other hand, the slope region in the absorption spectrum of EMT above 290 nm shows zero interference from TEN; therefore it was exploited through application of the first derivative spectrophotometry for the determination of EMT at 298 nm (Karunakaran et al., 2011). Alternatively, to overcome the mutual interference of each compound in the determination of the other, a simple one-step correction procedure based on the absorbance ratio spectra was developed. A study was carried out to examine the effect of divisor concentration on the ratio spectra of EMT and TEN. When the concentration of divisor is increased or decreased, the resulting absorbance ratio values are proportionally decreased or increased, respectively, although the positions of the peaks and troughs remain unaffected by changing the divisor concentration. The best results in terms of signal to noise ratio, sensitivity, accuracy and precision were obtained by using 15 μg/mL TEN (for EMT) and 10 μg/mL EMT (for TEN) as divisors.
The ratio spectra of different EMT standards at increasing concentrations in distilled water, obtained by dividing each by the spectrum of 15 μg/mL TEN in the same solvent are illustrated in Fig. 2. The peak to trough amplitudes between 228.5 and 260.5 nm on the generated ratio spectra are proportional to EMT concentration. Fig. 3 shows the ratio spectra of a standard solution of EMT and a mixture solution containing the same concentration of EMT. The difference between the 2 spectra is the constant interference value (the term CX/
in Eq. (2)). This interference was eliminated in the Salinas method (Salinas et al., 1990) by recording the first derivative of the mixture ratio spectrum. Alternatively, the constant interference can be eliminated by measurement of the absorbance ratio difference between two selected wavelengths λ1 and λ2. Ideally, these selected wavelengths should correspond to the peak and the trough in the ratio spectrum in order to achieve the highest sensitivity. Fig. 3 shows that the peak to trough amplitude in the mixture spectrum is equal to that in standard EMT spectrum, therefore EMT can be quantified in the mixture without interference from TEN.
For the determination of TEN, an analogous procedure was followed. Fig. 4 shows the ratio spectra of different standards of TEN using 10 μg/mL EMT as divisor. The peak to trough amplitudes between 229 and 260 nm were measured and found proportional to TEN concentration. Fig. 5 presents the ratio spectra of a standard solution of TEN and a mixture solution containing the same concentration of the drug. It is evident that the measured peak to trough amplitude in the mixture spectrum is equal to that in standard TEN spectrum.
4.2 Validation of the proposed spectrophotometric method
4.2.1 Linearity and concentration ranges
The linearity of the proposed spectrophotometric procedure was evaluated by analyzing a series of different concentrations of each drug (n = 7). The assays were applied following the established experimental conditions. The measured peak to peak amplitudes at the selected wavelengths were found to be proportional to concentrations of the analyte. The linear regression equations were generated by least squares treatment of the calibration data. Table 1 presents the performance data and statistical parameters including linear regression equations, concentration ranges, correlation coefficients, standard deviations of the intercept (Sa), slope (Sb) and standard deviations of residuals (Sy/x). Regression analysis shows good linearity as indicated from the correlation coefficient values (>0.9997). In addition, deviation around the slope can be further evaluated by calculation of the RSD% of the slope (Sb%) which were found to be less than 1.0%. The analysis of variance test for the regression lines reveals that, for equal degrees of freedom, an increase in the variance ratio (F values) means an increase in the mean of squares due to regression and a decrease in the mean of squares due to residuals. The greater the mean of squares due to regression, the steeper is the regression line. The smaller the mean of squares due to residuals, the less is the scatter of experimental points around the regression line. Consequently, regression lines with high F values (low significance F) are much better than those with lower ones. Good regression lines show high values for both r and F statistical parameters (Armitage and Berry, 1994).
4.2.2 Limits of detection and quantification
According to ICH guidelines (ICH, 2005), the approach based on the standard deviation of intercept value and the slope of the calibration graph was used for determining the limits of detection and quantification. The LOD and LOQ values for the two drugs are presented in Table 1.
4.2.3 Precision and accuracy
In order to assess the within-day (intra-day) precision, as percentage relative standard deviation (RSD%) and the within-day accuracy, calculated as percentage relative error (Er%) for the proposed method, three replicate determinations at three different concentration levels were carried out on the same day. The between-day (inter-day) precision and accuracy were assessed similarly by calculating the RSD% and Er% values for three replicate determinations of the same concentration levels on three consecutive days. Found concentrations were calculated using the corresponding regression equations and they were satisfactory. The RSD% and Er% values are listed in Table 2. These values did not exceed 2% thus indicating high precision and accuracy for the proposed spectrophotometric method.
| Analyte | Nominal value (μg/mL) | Within-day | Between-day | ||||
|---|---|---|---|---|---|---|---|
| Found ± SDa(μg/mL) | RSD (%)b | Er (%)c | Found ± SDa (μg/mL) | RSD (%)b | Er (%)c | ||
| EMT | 5 | 4.93 ± 0.03 | 0.61 | −1.40 | 4.91 ± 0.09 | 1.83 | -1.80 |
| 10 | 10.05 ± 0.18 | 1.79 | 0.50 | 9.98 ± 0.19 | 1.90 | -0.20 | |
| 20 | 19.83 ± 0.30 | 1.51 | −0.85 | 19.89 ± 0.35 | 1.76 | -0.55 | |
| TEN | 5 | 4.91 ± 0.02 | 0.41 | −1.80 | 4.92 ± 0.06 | 1.22 | -1.60 |
| 10 | 9.97 ± 0.13 | 1.30 | −0.30 | 10.03 ± 0.17 | 1.70 | 0.30 | |
| 20 | 19.97 ± 0.19 | 0.95 | −0.15 | 19.86 ± 0.31 | 1.56 | -0.70 | |
4.2.4 Stability of solutions
The stability of TEN and EMT standard and sample working solutions in water during handling was verified by keeping them at room temperature for 6 h. No significant spectrophotometric changes were noticed. The stock solutions were also stable when kept refrigerated at 4 °C for at least one week.
4.3 Applications of the proposed method
4.3.1 Analysis of laboratory-prepared synthetic mixtures
The validity of the proposed method was examined by preparing several laboratory-prepared mixtures of the two compounds at various concentrations within the specified linearity range. The mixtures were prepared in different ratios both above and below the normal ratio expected in commercial tablets. The laboratory-prepared mixtures were analyzed according to the previously described procedure. The analysis results including recovered concentrations, RSD% and Er% values shown in Table 3 were satisfactory thus validating the precision and accuracy of the developed method and demonstrating its capability to resolve and quantify the two drugs in different ratios.
| Nominal value (μg/mL) | Found ± SDa (μg/mL) | RSD (%)b | Er (%)c | ||||
|---|---|---|---|---|---|---|---|
| EMT | TEN | EMT | TEN | EMT | TEN | EMT | TEN |
| 15 | 5 | 14.69 ± 0.14 | 5.06 ± 0.07 | 0.95 | 1.38 | −2.07 | 1.20 |
| 20 | 10 | 19.63 ± 0.25 | 9.81 ± 0.07 | 1.27 | 0.71 | −1.85 | −1.90 |
| 15 | 10 | 14.72 ± 0.14 | 9.94 ± 0.16 | 0.95 | 1.61 | −1.87 | −0.60 |
| 20 | 20 | 19.67 ± 0.20 | 19.79 ± 0.37 | 1.02 | 1.87 | −1.65 | −1.05 |
| 10 | 20 | 9.78 ± 0.16 | 19.79 ± 0.15 | 1.64 | 0.76 | −2.20 | −1.05 |
4.3.2 Analysis of commercial tablets
The developed spectrophotometric procedure was applied for the assay of the antiviral combination in its commercial product (Truvada® tablets). The active ingredients were extracted with distilled water then dilution was made with the same solvent to reach concentration levels within the specified range. The studied drugs were directly quantified without any interference from the inactive ingredients (croscarmellose sodium, lactose monohydrate, magnesium stearate, microcrystalline cellulose, and pregelatinized starch) [22], which indicates the selectivity of the method. Recoveries were calculated using both external standard and standard addition methods. The assay results revealed satisfactory accuracy and precision as indicated from% recovery, SD and RSD% values (Table 4). Furthermore, a reference dual wavelength spectrophotometric method was applied for the estimation of EMT and TEN in their combined formulation (Ghorpade et al., 2010). Recovery data obtained from the developed spectrophotometric method were statistically compared with those of the reference method using Student’s t- and the variance ratio F-tests. In both tests, the calculated values did not exceed the theoretical ones at the 95% confidence level which indicated that there were no significant differences between the recoveries obtained from the developed method and those of the reference method (Table 4). It is evident from these results that the proposed method is applicable to the assay of this drug combination with minimum sample preparation, cost and time-effectiveness and satisfactory level of accuracy and precision.
5 Conclusion
This work presents a novel simple spectrophotometric procedure for the analysis of binary drug mixtures without prior separation. The method eliminates the derivative step in the conventional ratio-spectra derivative method (Salinas method), and does not require searching for zero-crossing points as in traditional derivative spectrophotometry. It is also much simpler than the ratio subtraction method as it does not involve the sequential order of manipulations of division, constant subtraction and multiplication. The proposed method does not include the tedious search for two wavelengths where the interfering compound shows the same absorptivity as in the dual wavelength method. Besides, it does not require any sophisticated mathematical or chemometric treatment for the absorption data. The method involves the generation of absorbance ratio spectra followed by measurement of the peak to peak (peak to trough) amplitudes. The proposed method was applied for the simultaneous determination of the two HIV-1 reverse transcriptase inhibitors Emtricitabine and Tenofovir in their drug combination. The proposed method is time saving, economic and environmentally-friendly (green method) since no chemical reagents or organic solvents were used, and only water was used as solvent for preparation of all solutions in this study. Compared to the previously reported spectrophotometric methods for this mixture, the proposed method is advantageous regarding simplicity of operation and sensitivity of measurement. The developed method does not require elaborate treatment, sophisticated experimental setup or consumption of organic solvents usually associated with HPLC and HPTLC methods of analysis, however the developed method lacks the specificity advantage of separation methods, and obviously cannot be applied in complex multi-component mixtures. The applicability of the developed method was evaluated through the determination of this antiviral drug combination in several laboratory-prepared mixtures and in pharmaceutical tablets with good accuracy and precision, therefore it can be considered as an alternative tool for the routine analysis of this fixed dose combination with minimum sample preparation.
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