Sensitive and selective spectrophotometric assay of rizatriptan benzoate in pharmaceuticals using three sulphonphthalein dyes

2.5.1 BPB method

Varying aliquots of standard RTB solution in chloroform equivalent to 0.8–16.0 μg ml⁻¹ (0.2–4.0 ml of 20 μg ml⁻¹) were accurately transferred into a series of 5 ml calibrated flasks and the total volume in each flask was brought to 4 ml by adding chloroform. After the addition of 1 ml of 0.1% BPB solution in dichloromethane, the content was mixed well and the absorbance of the ion–pair complex was measured at 425 nm against a reagent blank similarly prepared without adding RTB solution.

2.5.2 BCP method

Aliquots of 0.2–4.0 ml RTB standard solution in chloroform (25 μg ml⁻¹) equivalent to 1.0–20.0 μg ml⁻¹ were measured accurately and transferred into a series of 5 ml calibrated flasks. The total volume in each flask was brought to 4 ml by adding chloroform. To each flask, 1 ml of 0.1% BCP solution in dichloromethane was added and mixed well. The absorbance of the resulting yellow colored chromogen was measured at 425 nm against reagent blank.

2.5.3 BTB method

Into a series of 5 ml calibration flasks, aliquots (0.2–4.0 ml) of standard RTB solution (30 μg ml⁻¹) equivalent to 1.2–24.0 μg ml⁻¹ RTB were accurately transferred and the total volume in each flask was brought to 4 ml by adding chloroform. To each flask 1 ml of 0.025% BTB solution in chloroform was added and mixed well. The absorbance of the yellow colored ion–pair complex was measured at 420 nm against the reagent blank.

In all the methods, a calibration graph was prepared by plotting the increasing absorbance values versus concentration of RTB. The concentration of RTB was read from the calibration graph or computed from the respective regression equation derived using Beer’s law data.

2.5.4 Procedure for tablets

Twenty tablets containing RTB were weighed and pulverized into a fine powder. An amount of tablet powder equivalent to 10 mg of RTB was weighed into a 100 ml calibrated flask, 50 ml of chloroform added and the mixture was shaken for 20 min, then the volume was made up to the mark with the same solvent, mixed well and filtered using Whatman No. 42 filter paper. The first 10 ml portion of the filtrate was discarded and the subsequent portion was diluted appropriately with chloroform to get the working concentrations (20, 25 and 30 μg ml⁻¹ RTB) and subjected to analysis following the procedures described above.

2.5.5 Placebo blank analysis

A placebo blank of the composition: talc (35 mg), starch (25 mg), acacia (20 mg), methyl cellulose (30 mg), sodium citrate (20 mg), magnesium stearate (25 mg) and sodium alginate (25 mg) was transferred to a 50 ml calibration flask, 30 ml of chloroform was added and the mixture was shaken for 20 min. Then the volume was made up to the mark with the same solvent, mixed well and filtered using Whatman No. 42 filter paper and subjected to analysis using the procedures described above.

2.5.6 Synthetic mixture analysis

To the placebo blank of the composition described above, 10 mg of RTB was added and homogenized, transferred to a 100 ml calibrated flask and the solution was prepared as described under “Procedure for tablets”, and then subjected to analysis by the procedures described above.

2.5.7 Procedure for stoichiometric relationship

Job’s method of continuous variations of equimolar solutions was employed: 2.55 × 10⁻⁵ M each of RTB in chloroform and BPB in dichloromethane (Method A) solutions, 3.19 × 10⁻⁵ M each of RTB in chloroform and BCP in dichloromethane (Method B) solutions and 5.1 × 10⁻⁵ M each of RTB and BTB in chloroform (Method C) solutions were prepared separately. A series of solutions was prepared in which the total volume of RTB and reagent was kept at 5 ml. The drug and reagent were mixed in various complementary proportions (0:5, 1:4, 2:3,…, 5:0, inclusive) and completed as directed under the recommended procedures. The absorbance of the resultant ion–pair complex was measured at 425 nm in methods A and B, and 420 nm in method C, respectively.

3.1.1 Effect of solvent

In order to select a suitable solvent for preparation of the reagent solutions used in the study, the reagents were prepared separately in different solvents such as 1,4-dioxane, chloroform, acetonitrile, acetone and dichloromethane, and the reaction of RTB with BPB, BCP or BTB was followed. In methods A and B as shown in Fig. 4, dichloromethane was best suited for the preparation of BPB and BCP solutions, respectively. The chloroform solvent was found to be the ideal solvent for preparation of BTB for method C (Fig. 4). Similarly, the effect of the diluting solvent was studied for all methods and the results showed that none of the solvents except chloroform formed sensitive and stable colored species in methods A, B and C. Therefore, chloroform was used for dilution throughout the investigation.

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Figure 4

Effect of solvents on the formation of RTB–BPB complex (8 μg ml⁻¹ RTB), RTB–BCP complex (10 μg ml⁻¹ RTB) and RTB–BTB complex (12 μg ml⁻¹ RTB).

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3.1.2 Effect of dye concentration

The influence of the concentration of BPB, BCP and BTB on the intensity of the color developed at the selected wavelength was studied (Fig. 5). One milliliter each of 0.1% BPB and BCP or 0.025% BTB was sufficient to produce maximum and reproducible color with minimum blank absorbance.

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Figure 5

Effect of reagent concentration on the formation RTB–BPB complex (8 μg ml⁻¹ RTB), RTB–BCP complex (10 μg ml⁻¹ RTB) and RTB–BTB complex (12 μg ml⁻¹ RTB).

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3.1.3 Effect of reaction time and stability

The optimum reaction time for the development of color at ambient temperature (30 ± 2 °C) was studied and it was found that a 5 min standing time was sufficient for the complete formation of ion–pair complexes in all the methods. The formed color was stable for 1 h in methods A and B and it was stable more than 12 h in method C.

3.1.4 Study of composition of ion–pair complex and its conditional stability constant

The composition and conditional stability constant of the RTB–BPB or RTB–BCP or RTB–BTB complex formed were evaluated by applying Job’s method of continuous variations (Douglas and Donald, 1971) using equimolar concentrations of RTB (prepared by following the general procedure) and the dye. The concentration of dye used in method A was 2.55 × 10⁻⁵ M, method B was 3.19 × 10⁻⁵ M, whereas concentration of RTB and dye is 5.10 × 10⁻⁵ M in method C. The experiments were performed by mixing equimolar solutions of drug and reagent by maintaining the total volume at 5.0 ml. The plots of the mole ratio between drug and reagent versus the absorbance values were prepared (Fig. 6), and the results revealed that the formation of ion–pair complex between drug and reagent followed a 1:1 reaction stoichiometry.

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Figure 6

Job’s plots obtained for (a) 2.55 × 10⁻⁵ M RTB and BPB ion–pair complex (b) 3.19 × 10⁻⁵ M RTB and BCP ion–pair complex and (c) 5.1 × 10⁻⁵ M RTB and BTB ion–pair complex.

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The conditional stability constant (K_f) of the ion–association complex was calculated from the continuous variation data using the following equation (Erk, 2003): $$K_f=\frac{A/A_m}{{[1-A/A_m]}^{n+2}{C}_M{(n)}^n}$$ where A and A_m are the observed maximum absorbance and the absorbance value when all the drug present is associated, respectively. C_M is the mole concentration of drug at the maximum absorbance and n is the stoichiometry which BPB/BCP/BTB ion associates with RTB. The log K_f values were found to be 7.75, 6.47 and 7.19 for BPB method, BCP method, and BTB method, respectively.

3.2.1 Analytical parameters

A linear relation was found to exist between absorbance and the concentration of RTB in the ranges given in Table 2. The calibration graph in each case is described by the equation: $$Y=a+\mathit{bX}$$ where Y = absorbance, a = intercept, b = slope and X = concentration in μg ml⁻¹ obtained by the method of least squares. Correlation coefficient, intercept and slope for the calibration data are summarized in Table 2. Sensitivity parameters such as apparent molar absorptivity and Sandell’s sensitivity values, the limits of detection and quantification calculated as per the current ICH guidelines (ICH, 2005) are compiled in Table 2 and are indicative of the sensitivity of the methods. The limits of detection (LOD) and quantification (LOQ) were calculated according to the same guidelines using the formulae: $$\text{LOD}=3.3\sigma /s\text{and}\text{LOQ}=10\sigma /s$$ where σ is the standard deviation of five reagent blank determinations and s is the slope of the calibration curve.

3.2.2 Accuracy and precision

The accuracy and precision of the methods were evaluated by performing seven replicate analyses on pure drug solution at three different concentration levels (within the working range). The relative error (RE %), an indicator of accuracy (Table 3) was within 1.33 and within day precision, also called the repeatability, expressed as relative standard deviation (RSD %) was less than 1.25 indicating high accuracy and repeatability of the methods. The reproducibility of the methods also known as the day-to-day precision was evaluated by performing replicate analyses on pure drug solution at three levels over a period of five days, preparing all solutions afresh. The day-to-day RSD values (Table 3) were less than 2% reflecting the usefulness of the methods in routine analysis.

3.2.3 Robustness and ruggedness

The robustness of the methods was evaluated by making small incremental changes in the volume of dye (1 ± 0.2 ml) and contact time (5 ± 1 min), and the effect of the changes were studied on the absorbance of the ion–pair complex. The changes had negligible influence on the results as revealed by small intermediate precision values expressed as %RSD (⩽1.44%). Method ruggedness was demonstrated having the analysis done by four analysts, and also by a single analyst performing analysis using four different cuvettes. Intermediate precision values (%RSD) in both instances were in the range 0.96–1.76 indicating acceptable ruggedness. These results are presented in Table 4.

3.2.4 Effect of co-formulated substances

The effect of co-formulated substances was tested by placebo blank and synthetic mixture analyses. A convenient aliquot of the placebo blank solution was subjected to analysis according to the recommended procedures. There was no interference by the inactive ingredients as indicated by the near blank absorbance in all the methods.

The analyses of synthetic mixture solution yielded percent recoveries which ranged between 98.68–101.31 and with standard deviation of 0.57–1.21. The results of this study indicated that the inactive ingredients did not interfere in the assay.

3.2.5 Application to the analysis of tablets

In order to evaluate the analytical applicability of the proposed methods to the quantification of RTB in commercial tablets, the results obtained by the proposed methods were compared with those of the reference method (Altinoz et al., 2002) by applying Student’s t-test for accuracy and F-test for precision. The reference method consisted of the measurement of the absorbance of the tablet extract in water at 225 nm. The results (Table 5) show that the Student’s t- and F-values at 95% confidence level are less than the theoretical values, indicating that there is a good agreement between the results obtained by the proposed methods and the reference method with respect to accuracy and precision.

3.2.6 Recovery studies

The accuracy and validity of the proposed methods were further ascertained by performing recovery studies. Pre-analyzed tablet powder was spiked with pure RTB at three levels (50%, 100% and 150% of that found in tablet powder) and the total was determined by the proposed methods. The percent recovery of pure RTB added was in the range 100.3–103.5% with standard deviation of 0.30–2.12 (Table 6) indicating that the recovery was good, and that the co- formulated substance did not interfere in the determination.