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  • 1 King Saud University, Riyadh 11495, Saudi Arabia
  • 2 Alexandria University, El-Messalah, Alexandria 21521, Egypt
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A stability-indicating capillary electrophoresis method coupled to a diode array detector (DAD) was developed and validated for the simultaneous determination of emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) in combined tablets. This proposed method utilized a fused silica capillary (effective length, 62 cm; internal diameter [ID], 75 μm) and a background electrolyte (BGE) consisting of phosphate solution (pH 9.5, 50 mM). The separation was achieved at a voltage of 25 kV and a temperature of 21 °C using paracetamol as an internal standard. The described method was linear over the range of 5–200 μg/mL for both drugs (r = 0.9992). Intra- and inter-day relative standard deviation (RSD) (n = 9) was 0.41%. The limits of detection for FTC and TDF were 1.25 and 1.00 μg/mL, respectively. The average percentage recoveries of FTC and TDF from their tablet formulations were 99.66 ± 0.73 and 99.48 ± 0.33, respectively. The two drugs were subjected to thermal, photolytic, hydrolytic, and oxidative stress conditions, and then the stressed samples were analyzed by the proposed method. Degradation products produced as a result of stress studies did not interfere with the detection of FTC and TDF. The assay can thus be considered stability indicating.

Abstract

A stability-indicating capillary electrophoresis method coupled to a diode array detector (DAD) was developed and validated for the simultaneous determination of emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) in combined tablets. This proposed method utilized a fused silica capillary (effective length, 62 cm; internal diameter [ID], 75 μm) and a background electrolyte (BGE) consisting of phosphate solution (pH 9.5, 50 mM). The separation was achieved at a voltage of 25 kV and a temperature of 21 °C using paracetamol as an internal standard. The described method was linear over the range of 5–200 μg/mL for both drugs (r = 0.9992). Intra- and inter-day relative standard deviation (RSD) (n = 9) was 0.41%. The limits of detection for FTC and TDF were 1.25 and 1.00 μg/mL, respectively. The average percentage recoveries of FTC and TDF from their tablet formulations were 99.66 ± 0.73 and 99.48 ± 0.33, respectively. The two drugs were subjected to thermal, photolytic, hydrolytic, and oxidative stress conditions, and then the stressed samples were analyzed by the proposed method. Degradation products produced as a result of stress studies did not interfere with the detection of FTC and TDF. The assay can thus be considered stability indicating.

Introduction

Human immunodeficiency virus (HIV) infection ranks the fourth among the world's fatal diseases and is the leading cause of death in Africa. There were 35.0 million people living with HIV in 2013, up from 29.8 million in 2001 [1]. Recently, HIV and acquired immunodeficiency syndrome (AIDS) perception has changed greatly from a fatal disease to a manageable chronic illness. This is due to the dramatic improvement in morbidity and mortality rates, as well as the quality of life. The development of antiretroviral (ARV) drugs has played an important role in the control of HIV virus. Among which are nucleoside reverse transcriptase inhibitors (NRTIs) and nucleotide reverse transcriptase inhibitors (NtRTIs) [2, 3].

NRTIs work through inhibition of reverse transcriptase, the enzyme responsible for copying HIV RNA into viral DNA, by blocking a critical step in the HIV reproduction. Emtricitabine (FTC), 4-amino-5-fluoro-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl] pyrimidin-2-one (Figure 1a), is an example of NRTIs, which is an analogue of cytidine [3].

Figure 1.
Figure 1.

Chemical structure of the studied compounds: (a) emtricitabine and (b) tenofovir disoproxil fumarate

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2017.00057

Tenofovir disoproxil fumarate (TDF) [[(2R)-1-(6-aminopurin-9-yl)propan-2-yl]oxymethyl-(propan-2-yloxy-carbonyl-oxymethoxy)phosphoryl]oxymethyl propan-2-yl carbonate, (E)-but-2-enedioic acid (Figure 1b), is an example of the NtRTIs. TDF is an acyclic nucleoside phosphonate diester analogue of adenosine monophosphate (pro-drug of tenofovir). It is the first NtRTI approved for use in combination with other ARV agents in the treatment of HIV-1 infections which block reverse transcriptase, an enzyme crucial to viral production [4, 5]. It inhibits the activity of HIV reverse transcriptase by competing with the natural substrate deoxyadenosine 5′-triphosphate, after incorporation into DNA, by DNA chain termination [4, 6].

TDF–FTC combination (Truvada®) was recommended in WHO (2013) guidelines as first-line treatment for adolescents, adults, pregnant and breast-feeding women, and people co-infected with tuberculosis (TB) or hepatitis B (HBV). This combination results in a better patient compliance and a better HIV RNA suppression than using each drug separately, and it also improves medication adherence. FTC and TDF make ideal companions as part of a combination drug regimen [7–10]. Moreover, FTC and TDF are indicated in conjunction with other ARV drugs such as non-nucleoside reverse transcriptase inhibitors (NNRTIs) or protease inhibitors (PIs) for treating HIV in 12 years old and adults, also combined with safer sex practices for pre-exposure prophylaxis (PrEP) in high-risk adults [7].

Different methods have been reported in the literature for the determination of FTC and/or TDF in pharmaceutical preparations. These methods include high-performance liquid chromatography–ultraviolet (HPLC–UV) [11–17], liquid chromatography–mass spectrometry (LC–MS) [18–20], ultra-performance liquid chromatography (UPLC) [21], high-performance thin-layer chromatography (HPTLC) [22–24], and spectrophotometry [25–30].

To our knowledge, the use of capillary electrophoresis (CE) to determine FTC and TDF in combined tablets has not been reported in the literature so far. However, separation of nucleoside reverse transcriptase inhibitor triphosphates (NRTI-TPs), by CE, including FTC and TDF, has been studied previously. This study was concerned with the separation of NRTI-TPs in peripheral blood mononuclear cells (PBMCs) from human whole blood [31]. Since the method was not sensitive enough for actual analysis, only spiked samples were assayed. The separation was performed using a background electrolyte (BGE) consisting of carbonate and borate buffers (33 mM carbonate, 27 mM borate, pH 9.44), at a positive voltage with the cathode being at the detector end. However, nucleoside reverse transcriptase inhibitor di-phosphates (TFV-DP) degraded, producing several peaks in the electropherogram. In addition, the same authors investigated the separation of the same NRTIs mixtures under a negative applied voltage and suppressed EOF conditions (BGE consisting of phosphate buffer, pH 2.70). These conditions provided better separation with TFV-DP, being eluted as single peaks. While qualitative data were published, the validation study was not included in this work. Moreover, it did not focus on the degradation of the investigated drugs, and hence, it was not concerned as stability indicating.

CE has emerged in recent years as a powerful analytical technique for the separation and quantitation of a large variety of pharmaceutical compounds. CE possesses many unique advantages over HPLC, the universal method of analysis, such as simplicity, small sample volumes, high separation efficiency and resolution, low consumption of reagents, short analysis time, easy conditioning of column, cost savings, minimal band broadening, and greater peak capacity with millions of theoretical plates [32, 33]. Although miniaturization has now been introduced in HPLC, it does not reach the level of the miniaturization and simplicity in CE.

Stability testing forms an integral part of drug product development. This is extremely important to provide evidence on how the quality of a drug substance varies with time under the influence of a variety of environmental factors (temperature, humidity, and light). It is also essential to establish the storage conditions, retest periods, and shelf lives. The main aspect of a drug product that plays an important role in shelf-life determination is the assay of the active drug in the presence of degradants. The assay of a drug product in stability test samples needs to be carried out using a stability-indicating method, as recommended by the International Conference on Harmonization (ICH) guidelines [34].

The aim of this study was the development and validation of a CE technique coupled with diode array detector (DAD) for the analysis of FTC and TDF combination in their tablets. The method also enabled the determination of the studied drugs in presence of their degradation products under different stress conditions. Thus, it was considered as the first stability-indicating CE method for the determination of this combination.

Experimental

Materials and Reagents

Reference standard samples of FTC and TDF were purchased from Atlantic Research Chemicals Ltd. (Bude, Cornwall, United Kingdom). Paracetamol (PAR) internal standard (IS) was obtained from BDH Chemicals Ltd. (Poole, England). Truvada® tablets (Batch number L175994D) were obtained from Gilead Sciences, Inc. (Foster City, CA, USA). The tablets are labeled to contain 200 mg FTC and 300 mg TDF per tablet. Analytical grade sodium dihydrogen orthophosphate anhydrous, phosphoric acid, sodium hydroxide, hydrochloric acid, hydrogen peroxide, borax (disodium tetraborate decahydrate), and methanol were purchased from Merck (Darmstadt, Germany). Deionized water and a Millipore membrane filter (0.2 μm) from Nihon, Millipore (Yonnezawa, Japan) were used throughout the experiments.

Instrumentation and Electrophoretic Conditions

The employed CE system consisted of an Agilent CE instrument (Agilent Technologies Deutschland, Waldbronn, Germany) equipped with a diode array detector (DAD) and a data handling system comprised of an IBM personal computer and Agilent ChemStation software.

Detection was performed at 254 nm. A deactivated fused silica capillary was obtained from Agilent Technologies (Fullerton, CA) and had the following dimensions: total length, 70 cm; effective length, 62 cm; and internal diameter [ID], 75 μm. The temperature of the capillary and the sample was maintained at 21 °C. The background electrolyte solution (BGE) consisted of phosphate solution (50 mM, pH 9.5). Samples were injected into the capillary by injection pressure at the anodic side at 30 millibar (mbar) for 40 s. The electrophoresis was conducted by applying high voltage (25 kV) to the capillary, with the cathode at the detector end.

The capillary was washed between runs with deionized water (2 min) and then equilibrated with the running solution (5 min) to ensure reproducibility of run to run injections. Positive pressure up to 1400 mbar was used for rinsing. Before sample injection, the capillary was conditioned with 0.1 M sodium hydroxide (5 min), deionized water (5 min), and running electrolyte (10 min).

For pH measurements, a pH meter (Mettler Toledo AG 8603, Switzerland) was used. An ultrasonic water bath sonicator, model 3510E-DTH (Danbury, USA), was used for sonication. A water bath (GFL, D-309938 Burgwedel, Germany), a UV irradiator model (UVP, UVGL-55 Handheld UV Lamp, Cambridge, UK), and an electric oven (Memmert, D-91126 Schwabach, Germany) were used in the stress degradation studies.

Standard Solutions and Calibration Graphs

Stock solutions containing 1000 μg/mL of each of FTC, TDF, and PAR (IS) were prepared in methanol. These solutions were stable for at least 3 weeks if kept in the refrigerator. An intermediate stock solution of 100 μg/mL PAR was prepared by dilution in BGE. Suitable aliquots of the standard stock solutions of FTC and TDF (25–1000 μL) were transferred into a series of 5 mL volumetric flasks. Volumes of 1.25 mL PAR intermediate stock solution (100 μg/mL) were added to each flask, and volumes were completed to the mark with the BGE solution to yield final concentrations of 5, 10, 30, 60, 100, and 200 μg/mL of each of FTC and TDF and so that each solution contains a final concentration of 25 μg/mL PAR. All solutions were filtered before being injected. Triplicate injections of each concentration were performed. The peak area ratio of each concentration to the IS was plotted against the corresponding standard concentration to obtain the calibration graphs of each compound, and the corresponding regression equations were derived.

Analysis of Laboratory-made Mixtures

Separate volumes of 100, 400, and 750 μL of the stock solutions (1000 μg/mL) of both FTC and TDF were transferred into a series of 5 mL volumetric flasks to get three concentration levels (low 20, medium 80, and 150 μg/mL), respectively. Volumes of 1.25 mL IS intermediate stock solution (100 μg/mL) were added to each flask. The final volume was then completed to the mark with the BGE solution to yield laboratory-made mixtures at three different concentration levels within the linearity range of each compound and so that each mixture contains 25 μg/mL PAR. Triplicate injections of each mixture solution were made. The peak area ratio of each concentration to the IS was calculated. The concentration of each drug was obtained using the calibration graph or the corresponding regression equation obtained as described in the previous section.

Tablets Preparation

Twenty of Truvada® tablets were weighed and finely powdered. An accurately weighed portion equivalent to one tablet content (200 mg FTC and 300 mg TDF) was transferred into a 100 mL volumetric flask. A 10 mL volume of methanol was added, and the solution was stirred and sonicated for 20 min. It was then made up to volume with BGE, mixed well, and centrifuged at 3000 rpm for 10 min. Suitable dilutions were made to prepare tablet solutions corresponding to 100 μg/mL FTC and 150 μg/mL TDF, along with 25 μg/mL PAR, IS. The solutions were filtered through a Millipore membrane filter (0.2 μm) before injection. The solutions were injected five times, and the peak area ratio of each concentration to the IS was calculated. The concentration of each drug was obtained using the calibration graph or the corresponding regression equation.

Accelerated Degradation Studies

Forced degradation studies were carried out on FTC, TDF standards, and their mixtures. All degradation experiments were performed using 0.5 mL of stock FTC and TDF (1000 μg/mL). After exposing the solutions to the particular degradation conditions, 1.25 mL PAR (100 μg/mL) was added, and final dilution with BGE was made to get final concentrations corresponding to 100 μg/mL of each drug.

Acid and Base Hydrolysis

FTC and TDF mixture solution was mixed with 1 mL 0.1 N HCl or 0.1 N NaOH for acid and base hydrolysis, respectively. These mixtures were heated in a boiling water bath at (100 °C) for 1 h. Prior to CE–DAD analysis, samples were withdrawn at an appropriate time and neutralized.

Oxidative Degradation

FTC and TDF mixture solution was treated with 0.5 mL 3% (v/v) H2O2 and then heated in a water bath at 80 °C for 3 h.

Dry Heat Testing

Bulk drug powders 0.01 g of FTC and TDF (1-mm-thick layer in a Petri dish) were heated to a temperature of 100 °C for 2 h. The heated powder of each drug was separately transferred into 10 mL volumetric flasks and diluted to the mark with methanol to prepare drug solutions equivalent to 1000 μg/mL of each drug. Volumes of 0.5 mL of these stock solutions were transferred into a series of 5 mL volumetric flasks, along with 1.25 mL of PAR (100 μg/mL) to get final solutions with concentrations corresponding to 100 μg/mL of each drug.

Neutral Hydrolysis

This testing was conducted to study the degradation behavior of the drugs in neutral conditions. FTC and TDF mixture solutions were mixed with 1 mL of water and then heated in a boiling water bath at 100 °C for 1 h.

Photochemical Degradation

Each drug (FTC and TDF) in methanolic solution was exposed to direct daylight for 3 days at room temperature. Moreover, the stability of both drugs was also investigated by UV irradiation of FTC and TDF mixture solution at short wavelength at 254 nm and long wavelength at 366 nm (for 2 h). As a control, a parallel set of samples was kept in the dark at refrigerator temperature.

The stressed samples were detected at different wavelengths using the DAD to ensure that no additional degradation products were formed with different extinction values than the parent drugs. Peak purity was assessed using the DAD to ensure that the analyte peak did not contain more than one substance.

Results and discussion

Optimization of the CE Conditions

Buffer Selection and Effect of Buffer pH

Buffer pH determines the magnitude of the electroosmatic flow (EOF) due to its effect on the percent of analytes ionization [36, 37]. Increase buffer pH leads to an increase in the dissociation of silanol groups at the internal surface of the capillary. This effect could be explained in terms of the increase in EOF [36]. The pH value of BGE is a critical factor in the separation of acidic and basic drugs. FTC and TDF are acidic drugs with pKa values of 2.65 and 3.75, respectively. In order to investigate the effect of pH on the separation of these drugs, two buffer systems were used. Initially, BGE consisting of phosphate buffer solutions in the pH range (2.5–9.5) was tested at the interval of one pH unit. BGE with pH values of less than 4.0 resulted in an increase in the migration time of both FTC and TDF, neither of them was eluted till 40 min. While at pH values 5.0–7.0, FTC appeared as a broad peak. Increasing the pH to 9.5 resulted in a significant improvement of FTC peaks in terms of peak sharpness and symmetry. Then, borate buffer solutions in the pH range (7.0–9.2) were also tested at the interval of one pH unit. BGE at this range resulted in an increase in the migration time for both drugs and FTC appeared as a broad peak. The best results regarding peak shape and response were obtained using phosphate solution (pH 9.5). Phosphate solutions of pH values of 9.0, 9.2, and 9.5 were further tested to be sure of the obtained results.

Therefore, phosphate solution of pH 9.5 was found to be optimum for the analysis since it produced the most symmetric and best-resolved peaks, as well as the highest sensitivity, within a reasonable run time (<15 min). The obtained electropherogram is shown in Figure 2.

Figure 2.
Figure 2.

A typical electropherogram of a standard mixture of 100 μg/mL FTC (1), 25 μg/mL PAR, IS (2), and 100 μg/mL TDF (3), a); and the absorption spectra of FTC, b), and of TDF, c)

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2017.00057

Effect of Buffer Strength

Buffer concentration also has a significant effect on the separation performance through its influence on the EOF and the current produced in the capillary where small changes of ionic strength may influence the EOF, effective mobilities, and resolution. An increase in BGE concentration (ionic strength) may have a positive effect (suppression of electromigration dispersion of analytes and reduction of EOF) but too high concentration of BGE causes an increase in BGE conductivity resulting in peak broadening due to increased joule heating and temperature gradient inside the capillary [32, 33, 38].

The effect of BGE strength was studied using varying concentrations of phosphate solutions (25, 50, and 75 mM) at a constant pH of 9.5. Experimental trials showed that BGE consisting of phosphate solution (50 mM) resulted in an increase in the symmetry and sharpness of both FTC and TDF peaks where lower or higher strength values resulted in less peak sharpness with increased tailing. Thus, a concentration of 50 mM phosphate solution at pH 9.5 was selected as optimum with a good peak shape and good response.

Effect of Organic Modifiers

Organic modifiers affect buffer viscosity, zeta potential, dielectric constant, and migration time. Methanol is usually added to the BGE solution to increase the solubility of the solute or other additives and also to affect the ionization degree of the sample components. However, its contribution to the separation process is complex [32, 33].

To improve the peak shape of the analytes, the effect of methanol as an organic modifier was examined. Methanol was added at various concentrations (5, 10, 15%, v/v) to BGE, 50 mM phosphate solution (pH 9.5). It was noticed that increasing the amount of organic modifier (methanol) resulted in an increase in the migration time of both drugs, together with significant broadening of FTC peak. Best results regarding peak shape and response were obtained without the use of methanol. Hence, final separation was carried out without adding any organic modifier to the BGE.

Effect of Voltage

Both the electroosmotic and electrophoretic velocities are directly proportional to the field strength, so the use of high voltages will result in the shortest times for the separation. The theory predicts that short separation times will give the highest efficiencies since diffusion is the most important feature contributing to band broadening. The limiting factor here is joule heating. Experimentally, the optimal voltage is determined by performing runs at increasing voltages until deterioration in resolution is noted [32, 33, 38–43]. In order to determine the optimum voltage required for the best resolution of the analytes, applied voltage was studied in the range 20–30 kV (normal mode with EOF and detection near the cathodic end of capillary) under the separation conditions selected previously. The increase of applied voltage led to shorter analysis times, sharper peaks, and higher efficiencies. However, higher voltages (30 kV) also exhibited higher currents and increased joule heating. On the other hand, lower voltages at (20 kV) resulted in long migration time, along with broad and less intense peaks for both compounds. As a representative example of different factors affecting the CE separation, the effect of voltage on the elution characteristics of the studied drugs is shown in Figure 3.

Figure 3.
Figure 3.

Effect of the applied voltage on the electrophoretic separation of a standard mixture of 100 μg/mL FTC (1), 25 μg/mL PAR, IS (2), and 100 μg/mL TDF (3)

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2017.00057

The voltage of 25 kV (normal mode) was found to be optimal for the analysis. Both compounds were well-resolved as sharp peaks within a migration time of less than 15 min.

Effect of Applied Injection Pressure

The applied injection pressure was gradually increased from 10 to 40 mbr. Increasing the applied injection pressure to more than 20 mbr resulted in a significant peak broadening for FTC. Twenty millibar was found to be optimal for the analysis since both analytes were well-resolved as sharp peaks. Also, injection pressures of less than 20 mbar resulted in peak distortion, particularly for FTC.

Effect of Injection Time

Sample injection time was varied from 10 to 100 s. Increasing the injection time from 10 to 40 s resulted in an increase in the response of all the peaks until 40 s injection time, above which broadening of FTC peak was recorded. Also, lower injection times resulted in less intense peaks. An injection time of 40 s offered the best results in terms of response and peak shape. Thus, it was selected as optimum for sample injection.

Effect of Cartridge Temperature

Precise temperature control is important in CE [32, 33, 41–44]. As the temperature increases, the viscosity decreases; thus, the electrophoretic mobility also increases [32, 41–44]. The effect of temperature on CE migration of FTC and TDF was investigated at 15, 21, and 25 °C. An increase of the capillary temperature resulted in a decrease of migration times due to lower electrolyte viscosity. A temperature of 21 °C was selected as optimum for the analysis in terms of offering best compromise between peak shape, resolution, and analysis time.

Selection of Detection Wavelength

Solutions containing FTC and TDF were injected at different wavelengths (240, 254, 260, and 280 nm) under the optimized electrophoretic conditions. Both analytes had sufficient UV absorption at wavelength 254 nm, as shown by their absorption spectra (Figures 2b and c), measured by DAD. Therefore, 254 nm was selected as the optimum detection wavelength for the simultaneous determination of the drugs.

From these optimization studies, the following electrophoretic conditions were selected as optima for final analysis: a fused silica capillary of ID 75 μm, total length of 70 cm, and effective length of 62 cm, 50 mM phosphate solution as BGE at pH 9.50, temperature of 21 °C, applied voltage at 25 kV, injection time of 40 s at 30 mbr injection pressure, and 254 nm as detection wavelength. PAR was used as the IS to guarantee a high level of quantitative performance and significantly reduce the injection-related imprecision and provide a greater control over the sample amount injected. A typical electropherogram obtained from a standard solution of combination drugs under these optimized conditions is shown in Figure 2a. The migration times of FTC, IS, and TDF were 8.55, 12.25, and 14.58 min., respectively. The total time of analysis was <15 min.

Method Validation

IS was important to ensure a high level of quantitative performance. The method was validated according to ICH guidelines [45], in terms of its linearity, limit of detection (LOD) and limit of quantitation (LOQ), precision and accuracy, and solution stability.

Linearity

The linearity of detector responses for both FTC and TDF was determined by plotting peak area ratio of each drug to the IS versus concentration. The analytical data for the calibration graphs are listed in Table 1. The calibration curves were linear in the range of 5–200 μg/mL for FTC and TDF, with high (r) values (>0.999) and small Sy/x values, indicating the closeness of the points to the linear regression line. A typical calibration curve has the following regression equation of y = 0.0061x + 0.0182 for FTC and Y = 0.0098x + 0.0283 for TDF. The high values of the correlation coefficients (r) with negligible intercepts (a) indicate the acceptable linearity of the calibration graphs. Sy/x is a measure of the extent of deviation of the found (measured) y-values from the calculated values. The smaller the Sy/x, the closer the points are to the linear regression line. Standard deviation (SD) of the intercept (Sa) and the slope (Sb) were also calculated [35].

Table 1.

Validation parameters for the determination of emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) by the proposed method

ParameterFTCTDF
Concentration range (μg/mL)5–2005–200
Intercept (a)0.01820.0283
Slope (b)0.00610.0098
Correlation coefficient (r)0.99920.9993
Sa0.011120.01208
Sb0.11 × 10−30.13 × 10−3
Sy/x0.01920.0209
LOD (μg/mL)0.400.36
LOQ (μg/mL)1.251.00

S a, standard deviation of intercept; Sb, standard deviation of slope; Sy/x, standard deviation of the residual; LOD, limit of detection; LOQ, limit of quantitation.

Limits of Detection and Quantitation

The concentrations of the analyte showing signal-to-noise ratios of 3:1 and 10:1 were considered to be the LOD and LOQ, respectively [35]. The LOD for FTC and TDF, at a signal-to-noise ratio of three, and the LOQ at a signal-to-noise ratio of ten were calculated for both drugs as shown in Table 1.

Accuracy

The accuracy of the developed CE method was evaluated by analyzing laboratory-prepared mixtures at three concentration levels of both FTC and TDF (20, 80, and 150 μg/mL), prepared as described under the experimental section. Low percentage error (−1.44 to 1.60) obtained for both drugs indicated high degree of accuracy of the proposed method. The analytical data are shown in Table 2.

Table 2.

Accuracy and precision data for the determination of emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) by the proposed method

AnalyteConc. added (μg/mL)Conc. found mean ± SD (μg/mL)Error %RSD%
Intra-dayFTC2019.68 ± 0.251.601.27
8049.83 ± 0.670.341.34
15078.98 ± 1.321.281.64
TDF2019.92 ± 0.360.401.81
8049.74 ± 0.420.520.84
15079.33 ± 1.110.801.40
Inter-dayFTC2019.85± 0.260.751.31
8049.20 ± 0.851.601.73
15081.15 ± 1.08−1.441.33
TDF2019.95 ± 0.320.251.60
8049.76 ± 0.560.481.13
15080.18 ± 1.15-0.231.43

Precision

Precision was assessed by analyzing standard mixtures at the same concentration levels of both FTC and TDF as those used for accuracy. This was achieved by repeating the assay three times on the same day (intra-day precision) or on three different days (inter-day precision). In both cases, low RSD % values (0.84–1.81%) were achieved, indicating a high degree of method precision (Table 2).

Selectivity

Selectivity is described by the ability of the method to discriminate the analytes from all potential interfering substances. Selectivity of the method was investigated by analyzing standard solutions of FTC and TDF at their LOQ (1.25 and 1 μg/mL for FTC and TDF, respectively). This was assessed by both peak purity and spiking experiments with pure standard compounds. Peak purity was evaluated by the use of a diode array detector (DAD). The ChemStation software allows calculations of the purity factor and similarity curves compared with specified target spectra by comparing the average spectrum with that of the front, apex, and tail of a peak. In order to ascertain the peak purity, the calculated peak purity value should be within a certain pre-specified limit, the so-called threshold value. The calculated peak purity did not exceed the threshold values, indicating that there were no impurity peaks detected at the retention times of each individual drug and internal standard at the level of LOQ or less. Excipients commonly co-formulated with the studied drug such as magnesium stearate, cellulose, starch, calcium hydrogen phosphate, colloidal silicon dioxide, and coloring agents did not interfere in the determination of FTC and TDF, indicating the high selectivity of the proposed method (Figure 4).

Figure 4.
Figure 4.

A typical electropherogram of a prepared tablet solution containing 100 μg/mL FTC (1), 25 μg/mL PAR, IS (2), and 150 μg/mL TDF (3)

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2017.00057

Application to commercial tablets

The applicability of the proposed method was examined by analyzing commercially available Truvada® tablets. The obtained results of the test solutions are shown in Figure 4. The results of the proposed method were statistically compared with those obtained using the previously published HPLC method [15]. Statistical analysis of the results, using Student's t test and variance ratio (F test) [35], revealed no significant difference between the performance of the proposed and reference methods regarding the accuracy and precision, respectively (Table 3). The reference method depends on RP-HPLC determination of both FTC and TDF using Hypersil C-18 analytical column with a mobile phase consisting of buffer system–acetonitrile, 60:40 (v/v). The method was linear in the concentration range of 50–300 (FTC) and 75–450 μg/mL (TDF) with LOD and LOQ of 0.58 and 1.77 μg/mL, respectively, for both drugs. Thus, compared to the reference method, our proposed method is considered more sensitive with lower linearity range for both drugs. Moreover, it has the general advantages of CE separations compared to the HPLC methods of analysis, as mentioned under Introduction section.

Table 3.

Statistical analysis of assay results and recovery experiments in commercial Truvada® tablets

FTCTDF
Recovery% ± RSD99.66 ± 0.7399.47 ± 0.33
Recovery% ± RSD100.22 ± 0.65100.78 ± 0.64
ta1.311.61
Fb1.181.40

Recovery% ± RSD: mean ± RSD for five and six determinations using the proposed CE method and reference method, respectively.

Theoretical values for t and F at P = 0.05 are 2.3 and 5.19, respectively.

Stability Indicating Property (Accelerated Degradation)

This study was performed to determine the ability of the proposed method to separate FTC and TDF from their degradation products generated during forced degradation studies and to assess the stability profile and characteristics of FTC and TDF. Both drugs were subjected to different stress conditions: acid-base hydrolysis, oxidation, dry heat, neutral hydrolysis, and photochemical degradation. This was based on the comparison of the absorption spectra of stressed samples with those of standard solutions using DAD.

In addition, to check and ensure the homogeneity (peak purity) of all peaks in the stressed sample solutions, DAD was employed. Peak purity tests performed using the DAD were useful to show that the analyte peak did not contain more than one substance since all calculated peak purity values did not exceed the threshold limits.

In all cases, recovery based on the amount of drug remaining as well as percentage degradation was calculated and presented in Table 4.

Table 4.

Summary of forced degradation study results of FTC and TDF

Stress conditionsFTC
TDF
% Recovery% Degradation% Recovery% Degradation
Acid hydrolysis (0.1 N HCl , 100 °C, 1 h)49.08%50.92%16.58%83.41%
Base hydrolysis (0.1 N NaOH, 100 °C, 1 h)4.62%95.37%48.06%51.93%
Oxidative degradation (3% H2o2, 80 °C, 3 h)58.06%41.94%78.39%21.61%
Dry heat (100 °C, 2 h)75.33%24.66%20.97%79.03%
Neutral hydrolysis (100 °C, 1 h)99.80%ND41.31%58.68%
Photochemical degradation Day light (3 days)98.90%ND99.32%ND
UV 254 nm (2 h)99.21%ND98.99%ND
UV 366 nm (2 h)99.45%ND99.51%ND

ND, not detected.

Acid and Base Hydrolysis

Drug recovery of 49.08% (FTC) and 16.58% (TDF), and of 4.62% (FTC) and 48.06% (TDF) from the acid- and base-stressed samples, respectively, suggested the significant degradation of both drugs. However, more relative degradation of TDF in acid conditions and of FTC in basic conditions was noticed, as shown in Table 4. Electropherograms of acid and base degraded samples are shown in Figure 5a and b, respectively.

Figure 5.
Figure 5.

Typical electropherograms of a standard mixture of 100 μg/mL FTC (1), 25 μg/mL PAR, IS (2), and 100 μg/mL TDF (3), a); and its corresponding acid hydrolysis, b); base hydrolysis, c); oxidative degradation, d); neutral hydrolysis, e); and dry heat degradation, f)

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2017.00057

Oxidative Degradation

The electropherogram of the FTC and TDF samples treated with 3% (v/v) H2O2 showed a significant degradation of both drugs, with greater effect on the FTC (Figure 5c). Oxidative degradation resulted in 41.94% degradation of FTC compared with 21.61% for TDF, as shown in Table 4.

Neutral Hydrolysis

Studies under neutral conditions were performed by heating drug substance in distilled water at 100 °C, resulting in no degradation of FTC while significant degradation was recorded (58.68%) for TDF (Table 4, Figure 5e).

Dry Heat Testing

Heating the drug powders in a thermostated oven at 100 °C for 2 h produced degradation of both FTC and TDF (Figure 5f). However, more significant degradation of TDF (79.03%) was recorded compared with FTC (24.66%), as shown in Table 4.

Photochemical Degradation

Practically, no degradation of FTC or TDF was indicated after exposure of drug solutions to direct daylight for 3 days. Moreover, the stability of both drugs was also investigated by UV irradiation of FTC and TDF mixture solution at short wavelength at 254 nm and long wavelength at 366 nm (for 2 h). The results indicated that both drugs were almost stable under these UV conditions. A summary of forced degradation results is given in Table 4.

Conclusion

A stability-indicating CE–DAD method was developed for the separation of two antiviral drugs (FTC and TDF) in a single run. All parameters affecting the electrophoretic analysis including buffer pH, buffer strength, organic modifiers, voltage, applied injection pressure, injection time, cartridge temperature, and wavelength were optimized. The final separation was performed with BGE consisting of phosphate solution (50 mM, pH 9.5), 25 kV applied voltage, and 30 mbr for 40 s, 21 °C at detection wavelength of 254 nm.

Forced degradation studies were performed on bulk samples of the two drugs under different stress conditions. These included acid and base hydrolysis, oxidative degradation, dry heat, neutral hydrolysis, and photochemical degradation studies. The proposed method proved to be stability indicating by the separation of the two analytes from the forced-degradation products. Purity assessment was achieved using DAD. This method can be used for the quality assurance of these drugs in bulk and dosage forms.

Acknowledgment

The authors would like to extend their appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the research group project no. RGP-VPP-331.

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If the inline PDF is not rendering correctly, you can download the PDF file here.

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    Through internet communication, World Health Organization. http://www.who.int/hiv/en/. Accessed February 2015.

  • 2.

    Through internet communication, http://aidsinfo.nih.gov/education-materials/fact-sheets/19/73/the-hiv-life-cycle/. Accessed February 2015.

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    Cihlar T. ; Ray S. A. Antiviral Res. 2010, 85, 39.

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    Orsega S. ; J. Nurse Pract. 2015, 11.

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    • Export Citation
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    Patil S. V. ; Pishvikar S. S.; More S. A. Spectrophotometric Method for Degradation Study of Tenofovir Disopoxil Fumarates, Int. J. Pharm. Sci. Res. 2012, 3, 4363.

    • Search Google Scholar
    • Export Citation
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    Ashour H. K. ; Belal T. S. Arabian J. Chem. http://dx.doi.org/10.1016/j.arabjc.2013.06.024, 2013.

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    Behera A. ; Parida A.; Meher A. K.; Sankar D. G.; Moitra S. K.; Sil S. C. Int. J. Pharmtech Res. 2011, 3, 1874.

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    ICH Guidelines Q2 (R1) Validation of Analytical Procedures: Text and Methodology. In: Proceeding of the International Conference on Harmonization, Geneva, Switzerland 2005.

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