Novel spectrophotometric methods for determination of desloratidine in pharmaceutical formulations based on charge transfer reaction

2.1 Apparatus

All the absorption spectral measurements were made using Varian double beam UV–Vis spectrophotometer (Tokyo, Japan) equipped with 10 mm matched quartz cells.

2.2 Materials and reagents

All solvents (methanol, dimethyl sulfoxide, ethanol, acetone and acetonitrile) used in this work were of HPLC grade.

Desloratadine reference standard and bulk powder was supplied from Delta Pharma, Egypt. Its purity was found to be 99.78% according to the manufacturer's method. The commercial formulations used included Aerius tablets labeled to contain (5.0 mg/tablet, Schering-Plough Cooperation, USA) and Desa tablets labeled to contain (5.0 mg/tablet, Delta Pharma, Egypt).

2.3 Construction of calibration curves

Aliquots of methanolic solutions containing (0.05–2.0 mL) of a standard solution of DES (100 μg mL⁻¹) were transferred to separated 10 mL calibrated flasks. To each flask was added 2.0 mL of (3.0 × 10⁻³ M) chromogenic reagent (I–III) solution. Afterward, the obtained mixture was shaken in order to promote the reaction and the volume was completed to the mark with methanol. The absorbance of this final solution was measured at 528, 505 and 560 nm for I, II and III, respectively against a reagent blank. Perform a linear regression analysis using absorbance data vs. concentration of the drug. Use the slope and intercept data obtained from linear regression analysis of the calibration graph to calculate the concentration of an unknown sample. $$\text{Absorbance}=\text{intercept}+(\text{slope X concentration})$$ Alternatively, the concentration of the unknown may be directly obtained by calibration graphing.

2.4 Applications to pharmaceutical formulations (tablets)

The contents of ten tablets were removed and finely powdered using an agate mortar. The combined contents were mixed and weighed accurately. A portion of the powder equivalent to 50 mg of DES was accurately weighed and exactly 25 mL of methanol was added, sonicated for about 20 min, left for a time in a refrigerator to allow any insoluble matter to settle down and then filtered into a 50 mL volumetric flask. The solution was then completed to volume with methanol and the procedure was completed as described for preparing the calibration graph. The nominal contents of the tablets were determined either from the calibration graph or using the corresponding regression equation.

2.5 Stoichiometric relationship

The stoichiometric ratios of the charge transfer complexes formed between DES and I–III reagents were determined by applying the continuous variation method attributable to Job (Job, 1939) and modified by Vosburgh and Coober (1941) at the optimum wavelengths of maximum absorbance. Job's method of continuous variation was employed, a 3.0 × 10⁻³ M standard solution of DES and 3.0 × 10⁻³ M solution of reagent (I–III) were used. A series of solution were prepared in which the total volume of drug and reagent was kept at 2.0 mL. The reagents were mixed in various proportions with drug and diluted to volume in a 10 mL calibrated flask with methanol following the above mentioned procedures.

3.1 Evaluation of the effect of the solvent nature

The first parameter evaluated in the optimization of the experimental conditions was the nature of the solvent employed. The solvent plays an important role in some charge transfer reactions, since it must be able to facilitate the total charge transfer and then allow the complex dissociation and stabilization of the radical anion formed, which is the absorbing species. According to the literature, solvents with high dielectric constant are more effective to execute this task (Kelani et al., 1997; Huang et al., 2006). Taking this fact into account, water would be an excellent solvent for the procedure. However, the poor solubility of the reagents in water did not allow its use in the present case. So, the reaction was tested in ethanol, methanol, acetone, DMSO and acetonitrile media. Although the highest dielectric constant of DMSO and acetonitrile and best sensitivity were achieved with methanol, probably because of the capacity of this solvent to form stable hydrogen bonds with the radical anion. Then, methanol was chosen for further experiments (Fig. 3).

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Figure 3

Effect of different solvents on the charge transfer complex of DES-III complex obtained against III reagent solution also prepared in each solvent. DES concentration = 10 μg L⁻¹ and reagent III concentration = (3.0 × 10⁻³ M).

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3.2 Effect of the reagents concentration

In spectrophotometric analytical methods where maximum sensitivity is desired, the reagent concentration in solution is an important parameter to be studied, since the maximum conversion of the analyte into absorbing species depends on the amount of the reagent available in the solution for reaction and the equilibrium involved. In order to achieve this objective, an experiment was performed when various concentrations of reagents (I–III) solution (3.0 × 10⁻³ M) in the range of 0.25–4.0 mL were added to a fixed drug concentration (10 μg mL⁻¹) (Fig. 4). The results showed that 2.0 mL of (3.0 × 10⁻³ M) reagents solution (I–III) was enough to develop the color to its full intensity. As it can be seen, remarkable increase of the absorbance was verified up to respectively, after this point, it only suffered a slight increase or constant absorbance.

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Figure 4

Effect of (3.0 × 10⁻³ M) reagent concentration on the absorbance of charge transfer complexes formed between DES (10 μg mL⁻¹) and the reagents at the optimum wavelengths.

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3.3 Effect of the reaction time

The optimum reaction time was determined by continuous monitoring of the absorbance at optimum wavelengths of a solution containing 10 μg mL⁻¹ DES plus 2.0 mL of (3.0 × 10⁻³ M) (I–III) reagent, respectively at laboratory ambient temperature (25 ± 2 °C). On raising the temperature, the absorbance of the charge transfer complex was decreased with a hypochromic shift, until decayed at 60 °C. Stable absorbance values were observed from the beginning of the experiment up to 12 h. After this time, absorbance suffered a slight decrease. In view of these results, all spectral measurements were carried out after 3.0 min of mixing of the reagents and 25 ± 2 °C in order to make the method faster.

3.4 Sequence of additions

Drug–reagent–solvent was the favorable sequence of addition for complete color development and highest absorbance at the recommended wavelength.

3.5 Stoichiometry of the reaction

Job's method of the continuous variation (Job, 1939) was employed to determine the stoichiometry of the charge transfer reaction in methanol medium. Keeping the sum of the molar concentrations of the investigated drug (DES) and (I–III) reagents fixed, the ratio of the concentrations of the two substances in the mixture was varied and the absorbances of the mixtures were recorded at optimum wavelengths against a convenient blank solution prepared for each point of the experiment. As shown in Fig. 5, the molar ratio which gave maximum absorbance was 0.33, indicating that they react with (I–III) in a proportion of (1:2) (Drug:reagent) and confirming the assumption raised before when the effect of reagent concentration was studied. In view of this result a reaction mechanism was proposed considering the transfer of free electron of the two nitrogen atoms present in one molecule of drug to the charge-deficient center of the reagent molecule.

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Figure 5

Job's method of continuous variation between DES and Alizarines reagents (I–III).

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3.6 Mechanism of the reaction

Solutions of reagents in methanol exhibit an absorption band with a well defined maximum at 428, 421 and 491 nm, respectively, while the drug solution in methanol showed no absorption in the 400–700 nm range. The addition of drug to the reagent solution in methanol caused an immediate change in the absorption spectrum with the appearance of a new characteristic band with maximum absorption at optimum wavelengths recorded in Table 2.

According to (Ayad et al., 1984) molecular charge-transfer complexes are formed in non-polar solvents while radical anion species are predominant in polar solvents. Also, it is believed that the addition of basic compounds that contain a lone pair of electrons, such as DES, results in the formation of charge-transfer complexes of n–π type. This kind of complexes can be considered as an intermediate molecular-association compound that forms a corresponding radical anion in polar solvents. In this case, radical anions result from the total transfer of charge (Fig. 6).

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Figure 6

Possible mechanism of radical anion formation from III with DES reaction.

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3.7 Validation of the proposed methods

The validity of the methods was tested regarding linearity, specificity, accuracy, repeatability and precision according to International Conference on Harmonization (ICH, 1996) and United States Pharmacopeia (USP, 1999) guidelines.

Linearity – By using the above procedures, linear regression equations were obtained. The regression plots showed that there was a linear dependence of the analytical response in the three methods to the concentration of the drug over the ranges cited in Table 2. Linear regression analysis of the data gave the following equations. For Method I, A = 0.0007 + 0.0521C, r = 0.9998, for method II, A = 0.0072 + 0.0617C, r = 0.9993 and for method III, A = −0.0084 + 0.0509C, r = 0.9998. Where A is the absorbance, C is the concentration of the drug (μg mL⁻¹), and r is the correlation coefficient.

The limit of quantification (LOQ) was determined by establishing the lowest concentration that can be measured according to ICH, 1996. The results are shown in Table 2. The limits of detection (LOD) were determined by establishing the minimum level at which the analyte can be reliably detected, and the results are also summarized in Table 1. LOQ and LOD were calculated according to the following equation (USP, 1999): $$\begin{array}{cc}\hfill & \text{LOQ}=10s/b\hfill \\ \hfill & \text{LOD}=3.3s/b\hfill \end{array}$$ where s is the standard deviation of the intercept of the regression line. b: is the slope of the calibration curve.

The proposed methods were evaluated for the accuracy as percent relative error (% Er) and the precision as percent relative standard deviation (% RSD) (Tables 3).

3.8 Analysis of the pharmaceutical preparation

The proposed methods were applied to the determination of DES in commercial tablets (Aerius and Desa tablets). The methods were tested for linearity, specificity, accuracy, repeatability, and precision according to ICH recommendations. The results of the proposed methods were statistically compared with those obtained using the reference method (EL-Enany et al., 2007). Recovery ± SD values were obtained. Statistical analysis of the results, using Student's t-test and the variance ratio F-test revealed no significant difference between the performance of the proposed and reference methods regarding the accuracy and precision, respectively (Table 6) (Miller and Miller, 2005).