The optimum conditions for color development for each method were established by varying the parameters one at a time, keeping the others fixed and observing the effect produced on the absorbance of the colored species.
The spectrophotometric method for the determination of PiCl and DEX is based on their oxidation with a known excess of KMnO4 in acidic medium and subsequent determination of residual oxidant by reacting it with fixed amount of amaranth dye (AM), acid orange II (AO), indigocarmine (indigo) and methylene blue (MB) show characteristics λmax values at 521, 485, 610 and 664 nm for AM, AO, indigo and MB methods, respectively (Fig. ).
Absorption spectra of the oxidation product between 8.0 μg mL-1 PiCl, KMnO4 and, (a) AM, (b) AO, (c) Indigo and (d) MB by heating at 60 ± 2°C for 5.0 min.
Effect of heating time
In order to obtain the highest and most stable absorbance, the effect of heating time on the oxidation reaction of PiCl and DEX was catalyzed by heating in a water bath at 60 ± 2°C for the periods ranging for 2.5-20 min. the time required to complete the reaction and maximum absorbance was obtained after 5.0 min for PiCl and 10 min for DEX. After oxidation process, the solution must be cooled at least for 3.0 min before addition of dye (Fig. ).
Effect of heating time on the oxidation of 8.0 μg mL-1 PiCl-dye at the optimum wavelengths λmax of each dye.
Effect of oxidant concentration
When a study on the effect of KMnO4 on color development was performed, it was observed that in both cases the absorbance increased with increase in the volume of KMnO4 (5.0 × 10-4 M). It reached maximum when 2.0 ml and 1.5 ml of KMnO4 solution was added to a total volume of 10 ml for PiCl and DEX, respectively. The color intensity decreased above the upper limits. Therefore, 2.0 ml and 1.5 ml of KMnO4 were used for all measurements (Fig. ).
Effect of volume of 5.0 × 10-4 M KMnO4 on the development of the reaction product: 8.0 μg mL-1 DEX with MB and 10 μg mL-1 PiCl with AO.
Effect of acid concentration
To study the effect of acid concentration, different types of acids were examined (H2SO4, H3PO4 and CH3COOH) to achieve maximum yield of redox reaction. The results indicated that the sulphuric acid was the preferable acid with KMnO4 as oxidant. The reaction was performed in a series of 10 mL volumetric flask containing 8.0 μg mL-1 of the cited drugs, different volumes (0.1–2.5 mL) of 2.0 M H2SO4 and 2.0 and 1.5 mL of KMnO4 (5.0 × 10-4 M) with PiCl and DEX, respectively were added. After 5.0 min for PiCl and 10 min for DEX heating time at 60 ± 2°C in a water bath, the solution was cooled for about 3.0 min; the dyes (1.0, 1.5, 1.2 and 2.0 mL of AM, AO, indigo and MB, respectively) were added, then complete to 10 mL total volume with water. It was found that the maximum absorbance was obtained at 0.5 mL of 2.0 M H2SO4. Above this volume, the absorbance decreased for PiCl, where as for DEX the absorbance remained constant. Therefore, a volume of 0.5 ml of 2.0 M H2SO4, was used for all measurements (Fig. ).
Effect of mL added of Sulfuric acid (2.0 M) on absorbance of PiCl with (5.0 × 10-4 M) KMnO4 and dyes (5.0 × 10-4 M).
Effect of dye concentration
In order to ascertain the linear relationship between the volume of added KMnO4 and the decrease in absorbance of AM, AO, Indigo and MB, experiments were performed using 0.5 mL of 2.0 M H2SO4 with varying volumes of KMnO4. The decrease in absorbance was found to be linear up to 2.0 and 1.5 mL of 5.0 × 10-4 M KMnO4 with optimum volumes (1.0, 1.5, 1.2 and 2.0 mL of AM, AO, indigo and MB, respectively) for 8.0 μg mL-1 of PiCl and DEX (Fig. ). The color was found to be stable up to 24 h.
Effect of added dyes (5.0 × 10-4 M) on absorbance of 10 μg mL-1 of PiCl with KMnO4 (5.0 × 10-4 M).
Job’s method of continuous variation and the molar ratio method described by Yoe and Jones (25
), was employed to determined the stoichiometry of drug, oxidant and dyes. Keeping the sum of the molar concentration of both fixed, the ratio of the concentrations of each two in the mixture was varied and the absorbances of the mixture were recorded at the suitable wavelength against reagent blank. The maximum absorbance corresponds to the stoichiometric ratio. Stoichiometric ratio was found to be 1:1 for drug to oxidant; drug to dyes and oxidant to dyes as shown in (Fig. ) (Table ).
Continuous variations graph for the reaction between 5.0 × 10-4 M PiCl and 5.0 × 10-4 M KMnO4 with dyes (5.0 × 10-4 M).
Analytical parameters and optical characteristics of the proposed methods with PiCl and DEX
Validation of the proposed methods
Linearity. At described experimental conditions for PiCl and DEX determination, standard calibration curves for PiCl and DEX with KMnO4 and dyes, were constructed by plotting absorbance’s vs. concentrations. The statistical parameters were given in the regression equation calculated from the calibration graphs, along with the standard deviations of the slope (Sb) and the intercept (Sa) on the ordinate and the standard deviation residuals (Sy/x).
The linearity of calibration graphs was proved by the high values of the correlation coefficient (r) and the small values of the y-intercepts of the regression equations. The apparent molar absorptivities of the resulting colored ion-pair complexes and relative standard deviation of response factors for each proposed spectrophotometric method were also calculated and recorded in Table . The molar absorptivity of D>C>B>A method for PiCl, while for DEX the molar absorptivity of D>A>C>B method.
The detection limits (LOD) for the proposed methods were calculated using the following equation (27
LOD = 3s / k
where s is the standard deviation of replicate determination values under the same conditions as for the sample analysis in the absence of the analyte and k is the sensitivity, namely the slope of the calibration graph. In accordance with the formula, the detection limits were found to be 0.30, 0.16, 0.21 and 0.18 μg mL-1 for A, B, C and D methods, respectively. Whereas; for DEX the detection limits were found to be 0.09, 0.26, 0.37 and 0.21 μg mL-1 for A, B, C and D methods, respectively.
The limits of quantitation, LOQ, defined as (27
LOQ = 10 s / k
According to this equation, the limit of quantitation were found to be 0.99, 0.53, 0.69 and 0.60 μg mL-1 for A, B, C and D methods, respectively. Whereas; for DEX the detection limits were found to be 0.30, 0.87, 1.23 and 0.70 μg mL-1 for A, B, C and D methods, respectively.
Specificity, Precision, and Accuracy. Specificity of Oxidation-reduction reaction and selective determination of PiCl and DEX with KMnO4 and dyes could be possible. Percentage relative standard deviation (RSD %) as precision and percentage relative error (Er %) as accuracy of the suggested method were calculated. Precision was carried out by six determinations at four different concentrations in these spectrophotometric methods. The percentage relative error calculated using the following equation:
Er % = [(founded – added) / added] × 100
The inter-day precision and accuracy results are shown in (Table ). These results of accuracy and precision show that the proposed methods have good repeatability and reproducibility.
Evaluation of accuracy and precision data for PiCl and DEX obtained by the proposed methods
Robustness and Ruggedness. For the evaluation of method robustness, some parameters were interchanged; KMnO4 concentration, dye concentration, wavelength range, and heating time. The capacity remained unaffected by small deliberate variations. Method ruggedness was expressed as RSD % of the same procedure applied by two analysts and in two different instruments on different days. The results showed no statistical differences between different analysts and instruments suggesting that the developed methods were robust and rugged.
In pharmaceutical analysis, it is important to test the selectivity towards excipients and additives added to the pharmaceutical preparations of PiCl and DEX. It is clear from the results obtained for the pharmaceutical preparations that the commonly encountered excipients such as starch, talc, glucose, alginate and stearate did not interfere indicating a high selectivity for determining the studied PiCl and DEX in its dosage forms.
The proposed methods were successfully applied to determine the drugs studied PiCl and DEX in tablets and drops. Six replicate determinations were made. Moreover, to check the validity of the proposed methods, dosage forms were tested for possible interference with standard addition method. There was no significant difference between slopes of calibration curves and standard addition methods at four methods. Therefore it is concluded that the excipients in pharmaceutical dosage forms of PiCl and DEX such as starch, lactose, glucose, sugar, talc, sodium chloride, titanium dioxide, and magnesium stearate were not found any interference in the analysis of PiCl and DEX. The results were compared statistically by student’s t- test (for accuracy) and variance ratio F- test (for precision) with official methods at 95% confidence level with five degrees of freedom (Table , ). The results showed that the t
- values were less than the critical value (27
) indicating that there were no significant differences between the proposed and official methods. Because the proposed methods were more reproducible with high recoveries they can be recommended for routine analysis in majority of drug quality control laboratories.
Application of the standard addition technique for the determination of PiCl in dosage forms using the proposed methods
Application of the standard addition technique for the determination of DEX in dosage forms using the proposed methods
Chemistry of colored species
The proposed methods are based on the oxidation of the cited drugs by excess of KMnO4 to form oxidation products besides unreacted KMnO4 (step 1), and followed by the determination of unreacted KMnO4 by measuring the decrease in the absorbance of AM, AO, Indigo and MB dyes at their λmax (step 2). The possible sequences of reactions are presented in Fig. .
The possible sequences of Oxidation-reduction reaction.