A double beam UV-visible spectrophotometer (UV-160, Shimadzu, Japan) in the wavelength range of 200–800 was used for spectrophotometric measurements.
A Waters HPLC system consisting of a Model 515 pump, a Model 717 plus Autosampler and a Model 486 UV-visible detector (All from Waters, Milford, USA) were used. Multi-channel Chrom&Spec software for chromatography (version 1.5×) was used for data processing.
Rizatriptan benzoate (purity 99.8%) was from Merck (Batch No: 000705126 0048038) and was kindly provided by Food and Drug Laboratory Research Center, Ministry of Health, Tehran, Iran. Bromocresol green (BCG) and the other analytical grade chemicals were purchased from Merck (Darmstadt, Germany).
A standard solution of rizatriptan benzoate (5 × 10-4 M) was prepared by dissolving 13.5 mg of the drug in 100 ml of distilled water.
A 5 × 10-4 M solution of BCG was prepared by dissolving 349 mg of BCG in 1000 ml of distilled water by adding 2 ml of 0.1 M NaOH for better solubility.
The phosphate buffer (0.1 M) was prepared by dissolving 1.78 g of NaH2PO4 in 1000 ml of distilled water. The pH value was adjusted to 3.0.
The Britton- Robinson buffers in the pH range of 2–7 were prepared from an equal mixture of 0.1 M acetic acid, 0.1 M boric acid and 0.1 M phosphoric acid. The pH value was adjusted using a 1 M NaOH solution.
General procedure for sample preparation
An aliquot of 2 ml of rizatriptan solution (5 × 10-4 M), 1 ml of buffer solution (pH 3), 6 ml of BCG reagent (5 × 10-4 M), and 1 ml of distilled water were transferred to a 100 ml separating funnel. The mixture was extracted three times using 5, 3 and 2 ml portions of chloroform. The separating funnel was vigorously shacked for 30 seconds for extraction. The organic phase was separated and dehydrated by passing over anhydrous sodium sulfate and was transferred to a 10 ml volumetric flask. The volume was completed with chloroform and the absorbance was measured at 416 nm against a reagent blank.
Optimization of the reaction conditions
Selection of the optimum pH
The effect of pH on ion-pair complex formation was studied by using the general procedure and Britton-Robinson buffers in the range of 2–7. Also, different kinds of buffers (phthalate, acetate, phosphate) at the same pH value were studied to find out the effect of buffer type.
Selection of the reagent amount
Using 2 ml of rizatriptan solution (5 × 10-4 M) and 1 ml of phosphate buffer (pH 3.0), different amounts of BCG reagent (0.5–7 ml) were added and after extraction, the absorbance of the resulting solution was measured at 416 nm.
Selection of the extracting solvent
To find out the best extracting solvent, chloroform; dichloromethane; ethyl acetate; and diethyl ether were used as extracting solvents and the absorbance of the resulting ion-pair complexes were compared.
The effect of the reaction time
The effect of time on the ion-pair complex formation was studied at various time intervals in the range of 0–60 min.
The Job’s method of continuous variations was applied to find out the stoichiometric ratio of the ion-pair complexation. Different volumes of rizatriptan and BCG, at equal molar concentrations (4 × 10-4 M), were mixed with a fixed total volume and the absorbance of the resulting ion-pair complex was measured according to the general procedure. The resulting absorbance was plotted over the mole ratio of rizatriptan.
Determination of rizatriptan in dosage forms
Twenty Rizatriptan tablets (5 mg) (Farabi Pharmaceutical Co, Iran) were finely powdered using a mortar and pestle. Next, an amount of the resulted power equivalent to one tablet (5 mg of rizatriptan) was accurately weighed and transferred to a 100 ml volumetric flask and about 70 ml of distilled water was added. The mixture was sonicated for 15 min and made up to volume by the same solvent. After filtration through a 0.45 μm syringe filter, the concentration of rizatriptan was measured according to the general procedure. The amount of rizatriptan was calculated by comparing the obtained absorbance with a standard solution at the same concentration. The same procedure was also performed for determination of rizatriptan by HPLC using a previously published method.
The relative recovery of rizatriptan from dosage form was evaluated by standard addition method. Rizatriptan standard solution at concentration level of 40 μg/ml was added to a solution obtained from tablet powder equal to one tablet. After ion-pair complexation and according to the general procedure, the absorbance of this solution was compared with a standard solution at 40 μg/ml after subtraction of the absorbance of a tablet powder. The relative recovery was then calculated.
Six series of rizatriptan solutions in the range of 0.5–50 μg/ml and also 10–100 μg/ml were prepared and the absorbance was measured according to the general procedure. The calibration curves were constructed and the statistical data were calculated. For calibration curves in the range of 0.5–50 μg/ml, 5 ml (instead of 2 ml) of rizatriptan solution were used.
Precision and accuracy
To find out the precision and accuracy, three sets of different solutions of rizatriptan at three different concentration levels for each calibration range were prepared in triplicate and measured for 3 days. The within-day and between-day precision and accuracy were calculated.