Rizatriptan is a new selective 5-HT1B/1D agonist which is used in the treatment of migraine headaches. Two simple, rapid, accurate, and economical spectrophotometric methods are described for the determination of rizatriptan benzoate (RTB) in its pure form and pharmaceutical preparations. These methods are based on the charge-transfer complexation reaction between rizatriptan benzoate as
Drug quality control is a branch of analytical chemistry that has a wide impact on the health of human being, so the development of a reliable, quick, and accurate method for the active ingredient determination is always welcomed. Of the chemical neurotransmitter substances, serotonin (5-hydroxytriptamine or 5-HT) is perhaps the most implicated in the treatment of migraine, one among them is rizatriptan benzoate (RTB). RTB is a selective 5-hydroxytryptamine1B/1D (5-
Rizatriptan (RTB) is not official in any pharmacopoeia. The literature survey revealed several reported analytical approaches for the determination of RTB in dosage forms and biological materials including liquid chromatography-electrospray tandem mass spectrometry, LC-MS/MS [
In the literature, only a few visible spectrophotometric methods have been described for the determination of RTB. Shanmukha Kumar et al. [
However, the reported methods, particularly those based on chromatography are complex, require expensive experimental setup, and skilled personnel and inaccessible to many laboratories in developing and underdeveloped nations. In contrast, visible spectrophotometry is considered as the most convenient analytical technique in most quality control and clinical laboratories. Spectrophotometric methods have several advantages, such as low interference level, good analytical selectivity, and they are easier, less expensive, and less time consuming compared with most of the other methods [
Comparison of the proposed and the existing visible spectrophotometric methods.
Sl. No. | Reagent/s used | Methodology |
|
Linear range, |
Reaction time, min | Remarks | Ref. |
---|---|---|---|---|---|---|---|
(1) | (a) Methyl orange. |
Extracted ion-pair complex was measured. |
420 |
10–50 |
2 |
Involves strict pH control and extraction step. |
[ |
(b) Ferric chloride, 2,2′-bipyridyl | Complex formed between reduced Fe(II) and 2,2′-bipyridyl measured. | 490 | 4–20 |
5 | Heating required, multistep reaction. | ||
| |||||||
(a) 2,6-Dichloro quinone-4-chlorimide (DCQC) |
Oxidative coupling product measured. |
610 |
5–25 |
NA |
Narrow linear range. |
||
(2) | (b)1,2-Napthoquinone-4-sulphonic acid (NQS). |
Color produced by replacement of imino group of RTB by sulfonate group of NQS measured. |
480 |
15–75 |
NA |
Narrow linear range. |
[ |
(c) Brucine, sodium metaperiodate. | Oxidative coupling product measured. | 530 |
8–40 |
NA | Narrow linear range. | ||
| |||||||
(a) Ferric chloride,1,10-phenanthroline. |
Complex formed with 1,10-phenanthroline, ferric chloride measured. |
510 |
2–10 |
10 |
Heating required, multi step reaction, narrow linear range. |
||
(3) | (b) Folin-Ciocalteu reagent, sodium hydroxide. |
Reduced FC-reagent was measured. |
610 |
2–10 |
5 |
Narrow linear range. |
[ |
(c) Alizarin red | Extracted ion-pair complex was measured. | 425 |
4–20 |
2 | Involves strict pH control and extraction step. | ||
| |||||||
(4) | 7,7,8,8- Tetracyanoquinodimethane (TCNQ) | Charge transfer complex measured | 744 |
10–100 |
15 | Heating required, reagent is expensive. |
[ |
| |||||||
(a) Bromophenol blue (BPB) |
425 |
0.8–16 |
5 |
||||
(5) | (b) Bromocresol purple (BCP) |
Extraction free ion-pair complexes measured. | 425 |
1–20 |
5 | Simple, rapid, sensitive, selective, and no heating step. Use of single reagent and no extraction step involved. | |
(c) Bromothymol blue (BTB) | 420 | 1.2–24 |
5 | ||||
| |||||||
(6) | NBS (a) JG |
Resulting color measured | 620 |
0.5–8 |
15 |
Highly sensitive, no heating |
|
(b) CMG | 540 | 1.5–30 |
15 |
||||
| |||||||
(7) | (a) p-Chloranilic acid (p-CA) |
Radical anion was measured. |
530 |
14–245 |
Instantaneous |
Single-step instantaneous reaction, no heating or extraction step, no pH control, wide linear dynamic ranges. | Present methods |
(b) 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) | Radical anion was measured. | 590 | 4–70 |
Instantaneous |
NR: not reported; NA: not available.
The aim of this study was to establish simple, sensitive, precise, and inexpensive procedures for the quantification of RTB in pharmaceutical preparations. The basis of the proposed methods is molecular interactions between electron donors and electron acceptors. These interactions are generally associated with the formation of intensely colored charge transfer (CT) complexes which absorb radiation in the visible region [
All absorption measurements were made using a Systronics model 106 digital spectrophotometer (Systronics Ltd, Ahmedabad, India) with 1 cm path length matched quartz cells.
Pharmaceutical grade RTB certified to be 99.65% pure was obtained as a gift sample from Jubilant life Sciences, Nanjangud, Mysore, India, and used as received. Rizora-10 and Rizora-5 from Intas pharmaceuticals Ltd., Ahmedabad, India, both tablets were purchased from local commercial sources. 1,4-Dioxane and acetonitrile (spectroscopic grade) were purchased from Merck Specialities Pvt Ltd., Mumbai, India.
p-Chloranilic acid (p-CA) and 2,3-dichloro-5,6-dicyanoquinone (DDQ) both 0.1% solutions (both from S.D. Fine Chem Ltd, Mumbai), were prepared freshly in 1,4-dioxane.
For p-CA method, a 350
Varying aliquots of standard RTB solution equivalent to 14.0–245.0
Into a series of 5 mL calibration flasks, aliquots (0.2–3.5 mL) of standard 100
Standard graphs were prepared by plotting the absorbance versus RTB concentrations, and the concentration of the unknown was read from the calibration graph or computed from the respective regression equation derived using the absorbance-concentration data.
Twenty tablets were weighed and pulverized. An amount of tablet powder equivalent to 35 mg RTB was transferred into a 100 mL volumetric flask and about 70 mL of acetonitrile was added to the flask. The content was shaken well for 20 min and diluted to the mark with the same solvent. The resulting solution was filtered through Whatman No. 42 filter paper and used for the assay by following the general procedure described for p-CA method. The resulting tablet extract (350
A placebo blank containing starch (10 mg), acacia (15 mg), hydroxyl cellulose (10 mg), sodium citrate (10 mg), talc (20 mg), magnesium stearate (15 mg), and sodium alginate (10 mg) was prepared and its solution prepared as described under tablets and then subjected to analysis.
A synthetic mixture was separately prepared by adding pure RTB (50 mg) to the above mentioned placebo blank and the mixture was homogenized. The mixture containing 35 mg of RTB was weighed and its extract was prepared as described for tablets. The extracts containing three different concentrations of RTB were subjected to assay according to the general procedures described earlier and the percentage recovery of RTB was computed.
Job’s method of continuous variations of equimolar solutions was employed to establish the stoichiometry of the colored products. The solutions equivalent to 7.66 × 10−4 and 2.55 × 10−4 M RTB were prepared. Further, 7.66 × 10−4 M p-CA (method A) and 2.55 × 10−4 M DDQ (method B) solutions were prepared in 1,4-dioxane. A series of solutions was prepared in which the total volume of RTB and reagent was kept at 5 mL. The drug and reagent solutions were mixed in various complementary proportions (0 : 5, 1 : 4, 2 : 3,…,5 : 0, inclusive) and made up to mark with acetonitrile. The absorbance of the resulting colored species was measured at 530 nm in method A and 590 nm in method B.
The reaction of p-CA as a
Absorption spectra of charge transfer complex of RTB-p-CA (140
Absorption spectra of charge transfer complex of RTB-DDQ (40
The chemistry involved in the proposed methods is based on the reaction of the basic nitrogen (The more basic secondary amine) of RTB as
The dissociation of the
The tentative reaction mechanism for RTB-p-CA complex.
The tentative reaction mechanism for RTB-DDQ complex.
The effect of the reagent concentration on the intensity of the color developed at the selected wavelengths was ascertained by adding different amounts of the reagents p-CA and DDQ to fixed concentrations of 150 and 40
Effect of reagent concentration on color development: (a) RTB (150
To dissolve RTB, acetonitrile was preferred to chloroform, dichloromethane, acetone, 2-propanol, dichloroethane, 1,4-dioxane, methanol or ethanol, because the complex formed in these solvents either had very low absorbance values or precipitated upon dilution. Whereas in the case of reagents, highly intense colored products were formed when 1,4-dioxane medium was used as a solvent to dissolve p-CA and DDQ. Therefore, acetonitrile and 1,4-dioxane were chosen as solvents to dissolve RTB and the reagents, respectively. Similarly, the effect of the diluting solvent was studied and the results showed that none of the solvents except acetonitrile favored sensitive and stable colored species in both methods. Thus, acetonitrile was used for dilution throughout the investigation.
The optimum reaction time was determined by following the color development upon the addition of p-CA and DDQ solutions to the RTB solution at room temperature. Complete color development was attained instantaneously with both the reagents. The absorbance of these radical anions remained stable for at least 3 hrs and 2 hrs for method A and method B, respectively.
Job’s continuous variations graph for the reaction between RTB and p-CA or DDQ (Figure
Job’s Continuous-variation plot: (a) [RTB] and [p-CA] = 7.66 ×10−4 M (b) [RTB] and [DDQ] = 2.55 × 10−4 M.
Under optimum experimental conditions for determination of the drug under study, the absorbance versus concentration plots were found to be linear over the concentration ranges stated in Table
Regression and analytical parameters.
Parameter | p-CA method | DDQ method |
---|---|---|
|
530 | 590 |
Beer’s law limits ( |
14–245 | 4–70 |
Molar absorptivity (l mol−1 cm−1) | 1.30 × 103 | 5.22 × 103 |
Sandell sensitivity* ( |
0.3002 | 0.0751 |
Limit of detection ( |
1.36 | 0.60 |
Limit of quantification ( |
4.11 | 1.83 |
Regression equation, |
||
Intercept,( |
0.0077 | 0.0078 |
Slope,( |
0.0032 | 0.0127 |
Correlation coefficient ( |
0.9999 | 0.9999 |
Standard deviation of intercept ( |
0.00336 | 0.00402 |
Standard deviation of slope ( |
0.00002 | 0.00009 |
*Limit of determination as the weight in
The LOD for the proposed methods were calculated using the equation [
The LOQ, defined as [
In order to determine the precision of the proposed methods, solutions containing three different concentrations of RTB were prepared and analyzed in five replicates and the analytical results are summarized in Table
Evaluation of intraday and interday precision and accuracy.
Method | RTB taken |
Intraday ( |
Interday ( |
||||
---|---|---|---|---|---|---|---|
RTB founda ( |
%RSDb | %REc | RTB founda( |
%RSDb | %REc | ||
Method A | 70.0 | 69.2 | 1.2 | 1.1 | 70.3 | 1.4 | 0.4 |
140.0 | 142.2 | 1.0 | 1.6 | 142.3 | 1.1 | 1.7 | |
210.0 | 212.5 | 0.6 | 1.2 | 212.6 | 1.0 | 1.3 | |
| |||||||
Method B | 20.0 | 20.1 | 0.5 | 0.7 | 20.3 | 0. 9 | 1.6 |
40.0 | 39.9 | 1.7 | 0.4 | 40.2 | 1.4 | 0.6 | |
60.0 | 60.8 | 1.1 | 1.4 | 61.0 | 1.5 | 1.6 |
aMean value of five determinations; bRelative standard deviation (%); cRelative error (%).
The robustness of the methods was evaluated by making small incremental changes in the volume of dye (
Robustness and ruggedness.
Method |
RTB taken, |
Method robustness | Method robustness | |
---|---|---|---|---|
Parameter altered | ||||
Reagent volume, mLa | Interanalysts |
Intercuvettes | ||
RSD, % ( |
RSD, % ( |
RSD, % ( |
||
Method A | 70.0 | 0.9 | 1.7 | 1.5 |
140.0 | 1.3 | 1.3 | 1.2 | |
210.0 | 1.0 | 1.1 | 1.3 | |
| ||||
Method B | 20.0 | 1.0 | 1.0 | 1.6 |
40.0 | 1.1 | 1.5 | 1.6 | |
60.0 | 1.4 | 1.6 | 1.4 |
aVolumes of reagent were 1 ± 0.1 mL.
In order to evaluate the selectivity of the proposed methods for the analysis of pharmaceutical formulations, the effect of the presence of common excipients, such as talc, starch, acacia, hydroxyl cellulose, sodium citrate, magnesium stearate, and sodium alginate was tested for possible interference in the assay by placebo blank and synthetic mixture analyses.
The analysis of synthetic mixture solution prepared as described earlier yielded percent recoveries which ranged between 99.02–102.1 and with standard deviation of 0.79–1.94 (
The proposed methods were successfully applied to the determination of RTB in two representative tablets Rizora-10 and Rizora-5. The results obtained are showed in Table
Results of analysis of tablets by the proposed methods.
Tablet brand name | Label claim mg/tablet | Found (percent of label claim ± SD)a | ||
---|---|---|---|---|
Reference method | Proposed methods | |||
Method A | Method B | |||
Rizora-10 | 10 |
100.17 ± 0.61 | 99.76 ± 0.78 | 101.12 ± 0.93 |
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Rizora-5 | 5 |
99.89 ± 0.77 | 100.89 ± 1.13 | 101.01 ± 0.97 |
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aMean value of five determinations.
Tabulated
Tabulated
To ascertain the accuracy and validity of the proposed methods, recovery experiment was performed
Results of recovery study by standard addition method.
Tablets studied | Method A | Method B | ||||||
---|---|---|---|---|---|---|---|---|
RTB in tablets, |
Pure RTB added, |
Total found, |
Pure RTB recovered |
RTB in |
Pure RTB |
Total found, |
Pure RTB recovered |
|
69.83 | 35.0 | 105.09 | 100.7 ± 1.98 | 20.22 | 10.0 | 30.32 | 101.0 ± 1.75 | |
Rizora 10 | 69.83 | 70.0 | 140.55 | 101.0 ± 1.40 | 20.22 | 20.0 | 40.78 | 102.8 ± 0.57 |
69.83 | 105.0 | 176.81 | 101.9 ± 1.00 | 20.22 | 30.0 | 50.62 | 101.3 ± 0.51 | |
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70.62 | 35.0 | 106.38 | 102.2 ± 1.32 | 20.20 | 10.0 | 30.35 | 101.5 ± 0.49 | |
Rizora 5 | 70.62 | 70.0 | 142.12 | 102.1 ± 0.85 | 20.20 | 20.0 | 40.83 | 103.2 ± 0.87 |
70.62 | 105.0 | 176.75 | 101.1 ± 1.69 | 20.20 | 30.0 | 50.75 | 101.8 ± 0.51 |
*Mean value of three determinations.
The methods are based on well-characterized charge-transfer complexation reaction, and have the advantages of simplicity, speed, accuracy and precision, and use of inexpensive equipment compared to the reported HPLC and LC-MS methods. Other advantage of these methods is wide linear range. The DDQ method is more sensitive than the p-CA method as seen from the higher molar absorptivity value. Moreover, the proposed methods can be performed at room temperature. Thus, the methods are useful for the quality control and routine analysis of RTB in pharmaceuticals since there is no interference from the common excipients that might be found in commercial formulations.
The authors wish to acknowledge Cipla India Ltd., Mumbai, India, for providing the gift sample of atenolol. One of the authors (K. N. Prashanth) also wishes to thank the authorities of the University of Mysore for giving permission and facilities to carry out the research work.