Spectrophotometric determination of diphenhydramine hydrochloride in pharmaceutical preparations and biological fluids via ion-pair formation

2.3.1 Procedure for calibration curve

Aliquots of DPH in the concentration range stated in Table 1, were transferred into a series of 50 ml separating funnels. A 2.5 ml of 5 × 10⁻⁴ M dye solution and 2.0 ml of buffer solutions of (pH 3.6, pH 5.0, or pH 4, or pH 4.6) were placed. The volume of the aqueous phase was adjusted to 10 ml with distilled water and mixed well. The funnels were shaken vigorously with 10 ml of dichloromethane for 2 min. The two phases were allowed to stand for clear separation and the dichloromethane layer was dried by running through anhydrous sodium sulfate. The absorbance of the organic phase was measured at 632, 514, 428 and 414 nm for PB, EBT, MO and BCP, respectively, against dichloromethane. The calibration graphs are linear over the concentration ranges and are given in Table 1.

2.3.2 Stoichiometric relationship

Job’s method of continuous variations was employed; 2 × 10⁻⁴ M solution of PB and DPH, 5 × 10⁻⁴ M of EBT, MO, BCP and DPH solution were used. A series of solutions were prepared in which the total volume of drug and reagent was kept at 1.8 ml. The reagents were mixed in various proportions and completed to 10 ml of distilled water, following the above-mentioned procedure.

2.3.3 Procedure for the assay of tablets

Ten tablets were weighed and powdered. A portion of the powder equivalent to 20 mg of DPH was weighed and dissolved in distilled water, filtered into a 100 ml calibrated flask to remove the insoluble matter and diluted to volume with water. Solutions of working range concentration were prepared by proper dilution of this stock solution with water and followed the above procedure for the analysis.

2.3.4 Procedure for the assay of urine samples

The proposed method was applied to the determination of DPH in human urine collected from six normal people and transferred into well-cleaned sample tubes. Human urine was 50-fold diluted with distilled water. For the determination of DPH in urine, 1.0 ml of diluted urine was put in a 50 ml separating funnel. The solution was prepared and then, absorbance was measured following the same procedure as that for standard solutions. The absolute recovery was determined for DPH by comparing the representative absorbance of the treated urine with the absorbance of the standard drug at the same concentration.

3.1.1 Absorption spectra

Anionic dyes such as patent blue (PB), eriochrome black T (EBT), methyl orange (MO) and bromocresol purple (BCP) form ion-association complexes in acidic buffer solution with the positively charged drug and these complexes are quantitatively extracted into dichloromethane. Absorption spectra of the ion-pair complexes extracted into dichloromethane show a maximum at 632, 514, 428 and 414 nm for BP, EBT, MO and BCP, respectively, as shown in Fig. 1. The reagent blank under similar conditions showed no absorption.

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Figure 1

Absorption spectra of 3 μg ml⁻¹ of DPH–PB complex, 9 μg ml⁻¹ of DPH with EBT, MO and BCP complex against dichloromethane as blank.

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3.1.2 Effect of pH and buffer media

It was observed that effective extraction for the complexes depends on the type of buffer used and its pH. The effect of buffer solution was studied by extracting the coloured complexes in the presence of various buffers, such as KCl–HCl (1.0–2.2), NaOAc–AcOH (3.0–6.0), potassium hydrogen phthalate–NaOH (4.1–5.9) and NaOAc–HCl (7.0–9.0). The experiments showed that the maximum colour intensity and constant absorbance were observed in NaOAc–AcOH for PB, MO and BCP. For EBT reagent, phthalate buffer was the buffer of choice, which did not interfere and gave the highest sensitivity. The results are shown in Fig. 2 and it is evident that absorbance of the complexes with PB, EBT, MO and BCP was found to be constant within the pH ranges 3.0–3.8, 5.0–5.6, 3.0–4.2 and 4.0–5.0, respectively. Thus, all the absorbance measurements were made at pH 3.6, 5.2, 4.0 and 4.6 with PB, EBT, MO and BCP, respectively. The optimum amount of buffer solution used to give constant absorbance was also studied and found to be 2.0 ml.

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Figure 2

Effect of pH on the absorbance of ion-pair complexes of PB with 3 μg ml⁻¹ DPH and EBT, MO and BCP with 9 μg ml⁻¹ DPH.

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3.1.3 Selecting of the extracting solvents

Several water-immiscible organic solvents, including benzene, toluene, carbon tetrachloride, chloroform and dichloromethane were investigated. Dichloromethane was selected because of its slightly higher efficiency on colour intensity and the shortest time to reach the equilibrium between both phases.

3.1.4 Sequence of addition

From the experiment, we can see that when the addition sequence was dye–buffer–drug, the absorbance was the highest. When the addition sequence was drug–dye–buffer, the absorbance was less.

3.1.5 Effect of time and temperature

The effect of time on the formation and stability of the ion-association complexes was studied by measuring the absorbance’s of the extracted ion-association complexes at increasing time intervals. At the beginning, the absorbance increased gradually along with the time. After 10 min it achieved stability and remained basically unaltered for more than 3 h.

The effect of temperature on the absorbance was also, examined in the range of 30–100 °C. The results showed that the ion-association complexes were performed almost instantaneously in all cases at room temperature (25 ± 2 °C) and the influence of temperature was little. So, the absorbance was measured at room temperature (25 ± 2 °C).

3.1.6 Effect of dye concentration

The effect of the dye concentration on the intensity of the colour developed at selected wavelengths was tested using different milliliters of 5 × 10⁻⁴ M of the reagents. It is apparent from Fig. 3, that the maximum absorbance in each case, was found with 2.0 ml of dyestuff, beyond which absorbance was constant. Thus 2.5 ml of each dyestuff was used for ion-pair complex formation throughout the experiment.

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Figure 3

Effect of 5 × 10⁻⁴ M dye on the absorbance of ion-pair complexes of: PB with 3.0 μg ml⁻¹ of DPH, EBT, MO and BCP with 9.0 μg ml⁻¹ of DPH.

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3.1.7 Stoichiometric ratio of the ion-association complex

The stoichiometry of the ion-association complex formed by electrostatic attraction between positive protonated drug (DPH⁺) and the anion of bromocresol purple (BCP⁻) (as a model example of the used dyes) was investigated by applying the continuous variation method (Job, 1928; Vosburgh and Cooper, 1941) at the wavelength of maximum absorbance. The results obtained showed that the stoichiometric ratio of the ion-association complex is 1:1 (drug:dye) in all cases (Fig. 4). The suggested mechanism of DPH⁺–BCP⁻ ion-pair complex formation is described in Scheme 2. The extraction equilibrium can be represented as follows: $${\text{DPH}}_{\text{aq}.}^{+}+{\text{D}}_{\text{aq}.}^{-}\leftrightarrow {\text{DPH}}^{+}{\text{D}}_{\text{aq}.}^{-}\leftrightarrow {\text{DPH}}^{+}{\text{D}}_{\text{org}.}^{-}$$

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Figure 4

Continuous-variations study of drug–dye systems: DPH–PB (2.0 × 10⁻⁴ M); DPH–EBT; DPH–MO and BCP–DPH (5.0 × 10⁻⁴ M) ion-pair complexes.

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Scheme 2

Probable mechanism for the formation of 1:1 DPH–BCP.

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3.1.8 Conditional stability constants (K_f) of ion-pair complexes

The conditional stability constants (K_f) of the ion-pair associates for DPH under the experimental conditions described above were calculated from the continuous variation data using the following equation (Inczedy, 1976): $$K_f=\frac{A/A_m}{[1-A/A_m]{\hspace{0.17em}}^{n+1}{C}_D^n}(n){\hspace{0.17em}}^n$$ where A and A_m are the observed maximum absorbance and the absorbance value of the drug present is associated, respectively. C_D is the molar concentration of drug at the maximum and n is the stoichiometric constant with which dye ion associates with drug. In accordance with the formula, the conditional stability constants were found to be 4.974, 3.985, 4.181 and 4.309 for PB, EBT, MO and BCP, respectively.

The standard free energy change (ΔG°) was related to the association constant and calculated by the following equation (Martin et al., 1969): $$\mathrm{\Delta }G°=-2.303\mathit{RT}\log K_f$$ where (ΔG°) is the free energy change of the complex (kJ mol⁻¹), R the gas constant (1.987 cal mol⁻¹ deg⁻¹), T is the temperature in kelvin (273+ °C), and K_f is the association constant of drug–reagent ion-pair complexes (l mol⁻¹).

3.1.9 Specificity and effect of excipients

Formation of the ion-pair complex with anionic dyes needs a basic drug; therefore, no possible interference is expected from other co-formulated drugs lacking any basic center. A systematic study of the effect of excipients was performed, following the proposed procedures for a 10-ml sample system, by adding a known amount of excipients to the fixed DPH concentration (3.0 μg ml⁻¹). The results revealed the fact that no significant interference was observed from the excipients, such as glucose, fructose, sucrose, lactose and starch commonly present in pharmaceutical formulations. However, the drug content from the powdered tablets was extracted into methylene chloride, which completely eliminates the common excipients found in drug formulations.

3.2.1 Conformity to Beer’s law

Beer’s law is obeyed in the concentration (C, μg ml⁻¹) range of 0.5–3.0 μg ml⁻¹ with PB, 2.0–16 μg ml⁻¹ with EBT, 2.0–10 μg ml⁻¹ with MO and 1.0–10 μg ml⁻¹ with BCP. The molar absorptivity (ɛ) calculated and found to be 1.32 × 10⁵, 2.36 × 10⁴, 3.68 × 10⁴ and 3.07 × 10⁴ l mol⁻¹ cm⁻¹, for PB, EBT, MO and BCP, respectively, indicating high sensitivity of the reagents under investigations for the determination of the cited drug.

The optical characteristics such as Beer’s law limits, correlation coefficient (r) and Sandell’s sensitivities (Sandell, 1950) are recorded in Table 1. The regression analysis using the method of least square was made for the slope (b), intercept (a) and standard deviation of the residuals (S_y/x) obtained from different concentrations are given in Table 1. The intercept of the lines were very small indicating that there is no systematic difference between determined and expected concentration within the investigated range using the proposed method.

3.2.2 Precision and accuracy

In order to determine the accuracy and precision of the proposed method, solution containing four different concentrations of DPH were prepared and five replicates determinations, covering the usable concentration range, were carried out for the pure form and the pharmaceutical preparation of the drug under investigation. The analytical results obtained for this investigation are summarized in Table 2. The low values of percent relative standard deviation (RSD%) indicate good precision and reproducibility of the proposed method. The average percent recoveries obtained were quantitative (99.85–100.6), indicating good accuracy of the method. The mean values obtained and the calculated standard deviations are compared with those obtained by the pharmacopoeia method of DPH (United States Pharmacopoeia, 2000) by applying the t- and F-tests (Table 3). Such comparison showed that there is no significant difference, at 95% confidence level, between the mean values or variances obtained by the proposed and the pharmacopoeia method. This indicates the high accuracy and precision of the present method.

3.2.3 Analytical application

The validity of the proposed method for the analysis of DPH in tablets and urine was examined by analyzing the sample using the proposed method.

3.2.4 Determination of DPH in tablets and urine

The proposed method has been successfully applied to the determination of DPH in commercial tablets purchased locally and urine samples. The results are shown in Table 3. The mean percent recovery obtained from five replicate measurements of tablets and urine containing DPH were 99.86–101.02% (Table 3) and indicate that the proposed method was effective for the determination of DPH in tablets and urine samples. The calculated Student’s t-values and F-values did not exceed the theoretical ones at 95% confidence level. Therefore, there is no significant difference between the proposed method and official method (United States Pharmacopoeia, 2000).