Spectrophotometric determination and thermodynamic studies of the charge transfer complexation of emedastine difumarate with some π-acceptors

2.1.1 Materials and reagents

All solvents used were of spectroscopic grade and bidistilled water was used throughout the work. Emedastine difumarate was kindly supplied from Chem Swiss, SIGMA Co., Egypt. Its purity was found to be 99.00% according to The United States Pharmacopeia (2013). Emedastine 0.05% ophthalmic solution labeled to contain 0.5 mg emedastine difumarate per 1 mL (Batch No., 190409-F₁, manufactured by SIGMA Co. Egypt) was purchased from the local market. DDQ and CA (Aldrich, Germany) 0.2% (w/v), were prepared in acetonitrile and found to be stable for at least one week when stored in refrigerator. TCNQ (Aldrich, Germany) 0.1% (w/v), was freshly prepared in acetonitrile. Acetone, acetonitrile, 1,4-dioxane, ethanol, methanol (Merck Co., Germany), chloroform (Fischer Scientific, UK), anhydrous Na₂SO₄, ammonia solution 33% (Adwic Co., Egypt) were used.

2.1.2 Emedastine difumarate standard solutions

Emedastine difumarate standard solution, 1 mg mL⁻¹ was prepared in methanol. This stock solution was subsequently used for preparing working standard solutions, 0.5 mg mL⁻¹ for DDQ and, 0.08 mg mL⁻¹ for TCNQ using acetonitrile. Emedastine 1 × 10⁻³ M solution was prepared by dissolving an accurately weighed amount of 53.457 mg in 10 mL methanol. The volume was completed to 100 mL with acetonitrile.

2.2.1 Construction of calibration curves

Aliquots equivalent to 50.0–1000.0 μg or 200.0–2000.0 μg of emedastine difumarate working standard solutions (0.5 or 1 mg mL⁻¹) were transferred into two separate series of 10 mL volumetric flasks. To each series, 2.5 mL of each; 0.1% DDQ or 0.1% CA was added separately. Then the volume was completed with acetonitrile. The absorbance was measured after about 20 and 5 min at 457 and 519 nm of DDQ and CA, respectively, against the appropriate reagent blank. Plots of absorbance against drug concentration were plotted and regression parameters were computed.

For TCNQ, into a series of stopper test tubes, aliquots of working drug solution (0.08 mg mL⁻¹) equivalent to 8.0–160.0 μg of emedastine were transferred, followed by 2.5 mL of 0.1% of TCNQ solution and 2 mL acetonitrile. The tubes were heated in a thermostatic water bath at 60 °C for 30 min then cooled and the content of each tube was transferred quantitatively into a 10 mL volumetric flask and completed to the mark with acetonitrile. The absorbance was measured at 841 nm against a reagent blank treated similarly. The absorbance was plotted against corresponding drug concentration and the regression equations were calculated.

2.2.2 Determination of stoichiometry of the charge transfer complex by Job’s method of continuous variation

Three series of 2 mL quantities of mixtures of equimolar solutions of emedastine difumarate-DDQ, emedastine difumarate-CA and emedastine difumarate-TCNQ were made. The first and third series were made from 1 × 10⁻³ M of each DDQ and TCNQ and acceptor by compromising complementary proportions of the two solutions (0.2:1.8, 0.4:1.6,.., 1.8:0.2) in 10 mL volumetric flasks and stopper test tubes for the first and third series, respectively. The second series was made by the same procedure, using solutions of emedastine difumarate and CA each of, 5 × 10⁻³ M in 10 mL volumetric flasks. Then the detailed procedures described under, “Construction of calibration curves”, were followed. The absorbance was measured, at 457, 520 and 481 nm for the charge transfer complexes of DDQ-emedastine, CA-emedastine and TCNQ-emedastine, respectively each against its appropriate blank.

2.2.3 Determination of stability constant, molar absorptivity and standard free energy change

Different volumes; 0.1–0.5 mL of 1 × 10⁻³ M solution of emedastine in acetonitrile were transferred into a series of 10 mL volumetric flasks. To each flask, 5 mL of 10⁻⁴ M DDQ or CA in acetonitrile was added separately. For TCNQ, different volumes; 0.1–0.5 mL of 1 × 10⁻³ M drug solution in acetonitrile were transferred into a series of 10 mL stopper test tubes. Then 2 mL of 10⁻⁴ M TCNQ in acetonitrile was added. The detailed procedures described under, “Construction of calibration curves”, were followed for each method.

2.2.4 Determination of thermodynamic parameters of emedastine-TCNQ complex

Into a series of stopper test tubes, aliquots of working solutions of emedastine equivalent to 80 μg of emedastine were transferred, followed by 2.5 mL of 0.1% TCNQ and 2 mL of acetonitrile. The tubes were heated in a thermostatic water bath at different temperatures (40, 50, 60 °C) for different time intervals. At the specified time intervals, the tubes were cooled and the content of each tube was transferred quantitatively to 10 mL volumetric flasks. Then the detailed procedures described under “Construction of calibration curves”, were followed.

2.2.5 Application to pharmaceutical formulations

An accurately measured volume of the mixed emedastine eye drop solution (five bottles) equivalent to 10 mg of emedastine difumarate was quantitatively transferred into a beaker and evaporated under vacuum, then dissolved in 5 mL methanol and transferred quantitatively to 10 mL volumetric flask and completed with acetonitrile, then filtered and completed to 10 mL with acetonitrile. The procedure described under, “Construction of calibration curves”, was followed.

3.2.1 Effect of reagent volume

Different volumes; 0.5–3.0 mL of each DDQ and CA (0.2% w/v) and TCNQ (0.1% w/v) were studied. Highest absorbance at the relevant maxima was obtained upon using 2.5 mL of each.

3.2.2 Effect of solvents

Different solvents were tried for the complexation reaction. Acetonitrile afford maximum color intensity, which may be attributed to its high dielectric constant that promotes maximum yield of radical anions, and its high solvating power for π acceptors (Geffken and Salem, 2006).

3.2.3 Effect of time and temperature

Complete color development was attained instantaneously at room temperature between drug and CA, which remained stable for further 1 h. For DDQ maximum absorbance was developed after 20 min and remained stable for further 15 min.

However, with TCNQ the complex formation was slow over a period of two hr, which suggests heating at different temperatures (40–60 °C). The highest color intensity was attained, after heating at 60 °C for 30 min and remained stable for further 25 min.

3.2.4 Stoichiometry of the reaction

On studying the molar ratio between drug and each of, DDQ, CA and TCNQ, using Job’s method of continuous variation for; 1 × 10⁻³ M solutions for DDQ, CA or TCNQ it was found to be 1:1 (Rose, 1964), this indicates that only one site is responsible for the formation of the complex and a univalent charged species, is the possible site of the charge transfer process.

3.2.5 Association constants and standard free energy

The association constants were calculated for the interaction of emedastine with either DDQ, CA or TCNQ, using the Benesi–Hildebrand equation (Benesi and Hidelbrand, 1949) which depends on the experimental conditions that one of the two component species should be present in large excess, so that its concentration is virtually unaltered on formation of the complex. $$\frac{[A_o]}{A^{\text{AD}}}=\frac{1}{{\epsilon }^{\text{AD}}}+\frac{1}{{\epsilon }^{\text{AD}}{K}_C^{\text{AD}}}X\frac{1}{[D^o]}$$ where [A_O] and [D_O] are the total concentrations of the interacting species (acceptor and donor, respectively), A^AD is the absorbance of the complex, ε^AD is the molar absorptivity of the complex and $K_C^{\text{AD}}$ is the association constant of the complex.

On plotting the values of [A_O]/A^AD versus 1/[D_O] straight line was obtained (Fig. 2) whose intercept and the slope were calculated and the following equations were obtained: $$\frac{[\mathit{Ao}]}{A_{\lambda }^{\mathit{AD}}}=19.95\times {10}^{-5}+7\times {10}^{-8}\times \frac{1}{[D_0]}\text{DDQ complex}$$ $$\frac{[\mathit{Ao}]}{A_{\lambda }^{\mathit{AD}}}=39.79\times {10}^{-5}+1.6\times {10}^{-8}\times \frac{1}{[D_0]}\text{CA complex}$$ $$\frac{[\mathit{Ao}]}{A_{\lambda }^{\mathit{AD}}}=3.221\times {10}^{-5}+6\times {10}^{-8}\times \frac{1}{[D_0]}\text{TCNQ complex}$$ The standard free energy change of complexation (ΔG^o) is related to the association constant by the following equation (Vogel’s, 1989; Martin and Swarbrick, 1969). $$\mathrm{\Delta }{G}^{°}=-2.303\mathit{RT}\log K_C^{\mathit{AD}}$$ where ΔG^o is the standard free energy of the complex (kcal/mol), R is the gas constant (1.987 cal mol⁻¹ deg⁻¹), T is the temp in Kelvin (273 + °C) and $K_C^{\mathit{AD}}$ is the association constant of complex (L/mol), ( $K_C^{\mathit{AD}}$ ), (ε^AD) and (ΔG^o) were calculated and the results are represented in Table 1.

Close

Figure 2

Benesi–Hildebrand plot for DDQ, CA and TCNQ complexes.

Export to PPT

3.3.1 Linearity and sensitivity

Under the specified optimum reaction conditions, the calibration curves for emedastine difumarate with the different Pi acceptors employed in the present work were constructed.

The regression equations for the results were derived using the least-squares method. In all cases, Beer’s law plots were linear in the concentration range of 5.0–100.0, 20.0–200.0, and 0.8–16.0 μg mL⁻¹, for emedastine difumarate with DDQ, CA and TCNQ, respectively with very small intercepts and good correlation coefficients in the specified concentration range (Table 2). For accurate determination, Sandell sensitivities were calculated which reflected the sensitivity and accuracy of the proposed methods. The limits of detection (LOD) and quantification (LOQ) were determined, using the formula: LOD = 3.3 σ/S and LOQ = 10 σ/S, where σ = the standard deviation of the intercepts of regression lines, S = the slope of the calibration curve.

3.3.2 Accuracy and precision

The accuracy was evaluated by performing analysis of quality control samples with known concentration level of emedastine difumarate. The concentrations of the drug were calculated from slope and intercept obtained from the corresponding regression equation of the standard calibration curve. The recovery percentages and the mean recovery were then calculated. Statistical comparison between the proposed spectrophotometric and the official or manufacturer’s methods using student’s t-test and F-ratio at the 95% confidence level reveals an insignificant difference (Table 3). The accuracy was also assessed by applying the standard addition technique. Satisfactory results were obtained in good agreement with the labeled claim (Table 3).

The precisions of the assays (repeatability and intermediate precision) were determined for emedastine difumarate concentrations. Repeatability was assessed by analyzing five concentration levels in triplicates, using the proposed procedures, where RSD were 0.427–0.82% (intra-assay precision). Intermediate precision was evaluated by assaying the same concentrations in triplicate on three successive days. The assays gave satisfactory results; the relative standard deviations (RSD) were less than 2% (Table 2). This level of precision of the proposed methods was adequate for the quality control analysis of emedastine.

3.3.3 Ruggedness and robustness

The ruggedness of the proposed methods was assessed by applying the procedures using two different instruments in two different laboratories at different elapsed time. Results obtained from lab-to-lab and day-to-day variations were found to be reproducible as RSD did not exceed 2%. Robustness of the procedures was assessed by evaluating the influence of small variation of experimental variables: concentrations of reagents, and reaction time, on the analytical performance of the method. In these experiments, one experimental parameter was changed while the other parameters were kept unchanged, and the recovery percentage was calculated each time. The small variations in any of the variables did not significantly affect the results; recovery percentages were 99.78 ± 0.746–100.21 ± 0.468%. This provided an indication for the reliability of the proposed methods during routine work.

3.3.4 Application to pharmaceutical formulations

The obtained satisfactory validation results made the proposed procedures suitable for the routine quality control analysis of emedastine. The proposed method was applied for the determination of emedastine in its ophthalmic solution. The obtained mean recoveries ± RSD% ranged from 100.04 ± 0.68 to 100.22 ± 0.59. The methods were also assured by applying the standard addition technique (Table 3). The results were statistically compared with those obtained by the reported method, proving no significant difference with respect to accuracy and precision (Table 3).