A new simple, reliable, inexpensive, and accurate method was developed for the quantification of Frovatriptan Succinate Monohydrate in different physiological media at 244 nm in bulk and in tablet dosage forms. The developed method is an attempt to surpass the disadvantages associated with the reported methods, namely, less sensitive and tedious in usage for routine purposes. Beer’s law was followed over the range of 1.0 µg/mL to 4.5 µg/mL. Stability indicating assay method was developed and validated as per the ICH guidelines using various parameters, for example, accuracy, precision, limit of quantification, limit of detection, robustness, ruggedness, solution stability, recovery, forced degradation (hydrolysis, photo degradation, thermal degradation, and oxidation), and so forth. Percent relative standard deviation associated with all the parameters was less than 2, showing compliance with the acceptance criteria of ICH guidelines. The developed method was very sensitive as limit of quantification and limit of detection were found to be 0.025 µg/mL and 0.00625 µg/mL, respectively. Forced degradation studies of drug reveal good stability under the chosen experimental conditions.
Chemically, the Frovatriptan Succinate Monohydrate (FSM) is 3-methylamino-6-carboxamido-1,2,3,4-tetrahydrocarbazole succinate monohydrate (Figure
Structure of Frovatriptan Succinate Monohydrate (FSM).
Despite tremendous utility of FSM in migraine, there was no simple, sensitive, and reliable stability indicating that UV spectroscopic method has been reported for its analysis in the vast reviewed literature. Only two UV spectroscopic methods have been mentioned in the literature for the analysis of FSM in 0.1
As in pharmaceutical industry, time and expense play a very crucial role. Pharmaceutical industries always admire simple and more sensitive methods for routine usage. Therefore, a need of simple, reliable, inexpensive, and accurate stability indicating method for analysis of FSM as bulk or as pharmaceutical dosage forms has always been felt.
The present study was aimed to develop and validate analytical methods for the analysis of FSM in distilled water, phosphate buffer (pH 6.7), and acetate buffer (pH 5.5) at two different storage conditions, that is, room temperature (
FSM was procured as a gift sample from Azakem Labs Pvt. Ltd., India. Potassium dihydrogen orthophosphate, sodium hydroxide, and sodium acetate trihydrate were obtained from Himedia Ltd., India. Hydrogen peroxide (30% w/v) was obtained from Moly chem., India. All other chemicals used were of analytical grade.
Double beam UV-visible spectrophotometer (Shimadzu, UV-1800, Japan) in connection with computer having UV Probe 2.34 software and equipped with matched quartz cells of 1.0 cm each was used.
Different stock solutions were prepared in distilled water, phosphate buffer (pH 6.7), and acetate buffer (pH 5.5) by accurately weighing about 156.4 mg of FSM (equivalent to 100 mg of frovatriptan) in a volumetric flask of 100 mL capacity. Volume was made up to the mark using suitable media. Different dilutions were made in the range of 1
Accuracy of the developed method was determined with the aid of three different concentration levels, namely, low concentration (LC) 2.0
Repeatability and intermediate precision studies were determined for developed methods using same concentration levels as were taken in accuracy studies. Percent RSD and recovery were taken as precision measure.
Linearity was determined by preparing a series of dilutions in three media and then measuring the absorbance at 244 nm (
LOD and LOQ measurements were determined by subjecting minimum concentration of calibration plot to series of dilutions. Absorbance was measured in replicate of six for each case, till RSD (%) was came as
Robustness was determined by taking similar concentration levels as were taken in accuracy studies and storing them at two different temperatures (
Test solution (2.5
Least square regression equation of FSM in different media at two different temperatures had shown
Accuracy data associated with LC, IC, and HC is mentioned in Table
Accuracy data of developed method for analysis of FSM in various media (
Media | Levels | Storage conditions | |||||
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Freeze (5°C ± 0.5°C) | Room (25°C ± 0.5°C) | ||||||
Absorbance (±SD) | RSD (%) | Recovery (%) | Absorbance (±SD) | RSD (%) | Recovery (%) | ||
Distilled water | LC |
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Phosphate buffer (pH 6.7) | LC |
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Acetate buffer (pH 5.5) | LC |
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IC |
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HC |
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The RSD (%) data associated with repeatability studies ranges from 0.432 to 1.139 and 0.332 to 1.392 for distilled water at freeze temperature and room temperature, respectively. The % RSD data associated with repeatability studies in phosphate buffer (pH 6.7) ranges from 0.460–1.283 and 0.666–1.119 at freeze temperature and room temperature, respectively. The % RSD data associated with repeatability studies in acetate buffer (pH 5.5) ranges from 0.706 to 1.722 and 0.275 to 1.004 at freeze temperature and room temperature, respectively. In intermediate precision, values of RSD (%) varies from 0.563 to 1.254 and 0.578 to 1.164 for distilled water at freeze temperature and room temperature, respectively. The phosphate buffer (pH 6.7) values of RSD (%) vary from 1.020 to 1.397 and 1.089 to 1.371 at freeze temperature and room temperature, respectively. The acetate buffer (pH 5.5) values of RSD (%) vary from 0.629 to 1.344 and 0.684 to 1.100 at freeze temperature and room temperature, respectively. Recovery ranges from 96% to 104.9% in both types of precision study.
Beer’s law follows over the range of 1.0
Linearity data for developed method of FSM.
Media | Storage conditions | |||||
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Freeze (5°C ± 0.5°C) | Room (25°C ± 0.5°C) | |||||
Slope | Intercept |
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Slope | Intercept |
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Distilled water | 0.1716 | −0.0058 | 0.9984 | 0.1716 | −0.0058 | 0.9984 |
Phosphate buffer (pH 6.7) | 0.1889 | +0.0138 | 0.9983 | 0.1885 | +0.0009 | 0.9995 |
Acetate buffer (pH 5.5) | 0.1914 | +0.005 | 0.9999 | 0.1901 | +0.0013 | 0.9996 |
Linearity plots of FSM in different media. (a) Linearity curve of FSM in distilled water at freeze temperature, (b) linearity curve of FSM in distilled water at room temperature, (c) linearity curve of FSM in phosphate buffer (pH = 6.7) at freeze temperature, (d) linearity curve of FSM in phosphate buffer (pH = 6.7) at room temperature, (e) linearity curve of FSM in acetate buffer (pH = 5.5) at freeze temperature, and (f) linearity curve of FSM in acetate buffer (pH = 5.5) at freeze temperature.
LOQ and LOD for FSM were found to be 0.025
LOQ and LOD data for developed method of FSM (
Concentration ( |
Absorbance (±SD) | RSD (%) |
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0.025 |
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11.208 |
0.0125 |
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27.200 |
0.006 |
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30.051 |
Variation in pH of media by
Effect of change in pH of media by ±0.1 unit on the sensitivity of developed method of FSM (
Concentration ( |
Absorbance (±SD) | RSD (%) |
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2 |
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0.371 |
2.5 |
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0.479 |
3 |
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0.289 |
When paired Student’s
Test solution was found to undergo photolysis after 12 hours of exposure to radiations ranging from 300 to 800 nm. Extent of degradation after 12 hours was found to be 31.34%. Plot constructed between amount remaining to degrade (
Photolytic degradation of FSM (
Time (hours) | Concentration remaining |
RSD (%) | Degradation (%) |
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0 |
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0.554 | 3.532 |
0.5 |
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0.338 | 4.498 |
1 |
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0.220 | 4.424 |
2 |
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0.000 | 4.572 |
3 |
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0.136 | 11.636 |
6 |
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0.697 | 14.089 |
9 |
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0.396 | 18.253 |
12 |
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0.126 | 31.338 |
Storage of test solution at 70°C for 7 days yields no significant degradation (Table
Thermal stability data of FSM (
Time (days) | Concentration remaining |
RSD (%) |
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1 |
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0.449 |
2 |
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0.330 |
3 |
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0.331 |
4 |
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0.217 |
5 |
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0.331 |
6 |
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0.330 |
7 |
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0.125 |
When constant volume (1.0 mL) of various strengths HCl from (0.1 N to 1.0 N) was added individually to 10 mL of test solution, it was found to be stable in 0.1 N, 0.2 N, 0.3 N, and 0.4 N HCl for 6 hours without heating. Addition of other strengths of HCl yields significant degradation of more than 31% within 6 hours of exposure (Table
Acid catalysed hydrolysis of FSM (
Strength (N) | Amount remaining to degrade (µg/mL) | ||
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Time ( |
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0 | 3 | 6 | |
0.1 | 2.437 | 2.437 | 2.383 |
0.2 | 2.344 | 2.153 | 2.114 |
0.3 | 2.129 | 2.011 | 2.015 |
0.4 | 2.108 | 1.966 | 1.959 |
0.5 | 1.957 | 1.645 | 1.613 |
0.6 | 1.862 | 1.568 | 1.508 |
0.7 | 1.839 | 1.469 | 1.428 |
0.8 | 1.791 | 1.378 | 1.325 |
0.9 | 1.563 | 1.282 | 1.114 |
1 | 1.417 | 1.271 | 0.817 |
Forced degradation (acid catalysed hydrolysis) plots of FSM.
Exposure of test solution to various strength of NaOH from (0.1 N to 1.0 N) without heating over 6 hours showed its stability in 0.1 N only (Table
Base catalysed hydrolysis of FSM (
Strength (N) | Amount remaining to degrade ( |
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Time ( |
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0 | 3 | 6 | |
0.1 | 2.486 | 2.461 | 2.451 |
0.2 | 1.727 | 1.693 | 1.535 |
0.3 | 1.697 | 1.669 | 1.472 |
0.4 | 1.644 | 1.588 | 1.423 |
0.5 | 1.558 | 1.549 | 1.388 |
0.6 | 1.443 | 1.434 | 1.362 |
0.7 | 1.414 | 1.389 | 1.346 |
0.8 | 1.347 | 1.339 | 1.294 |
0.9 | 1.283 | 1.240 | 1.160 |
1 | 1.153 | 1.056 | 0.955 |
Forced degradation (base catalysed hydrolysis) plots of FSM.
In oxidative degradation studies, FSM was found to be stable on the exposure of
Oxidative stability data of FSM.
Time (minutes) | Volume of 3% w/v solution added per 10 mL of drug solution | |||||
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0.1 mL | 0.15 mL. | 0.2 mL | 0.1 mL | 0.15 mL | 0.2 mL | |
Concentration ( |
Concentration ( |
Concentration (µg/mL) | Degradation (%) | Degradation (%) | Degradation (%) | |
0 | 2.390 | 2.390 | 2.390 | 4.385 | 4.385 | 4.385 |
15 | 2.389 | 2.381 | 2.381 | 4.456 | 4.743 | 4.743 |
90 | 2.381 | 2.376 | 2.372 | 4.743 | 4.959 | 5.102 |
180 | 2.372 | 2.358 | 2.358 | 5.102 | 5.676 | 5.676 |
360 | 2.360 | 2.331 | 2.329 | 5.605 | 6.753 | 6.825 |
Oxidative degradation plots of FSM using hydrogen peroxide as oxidizing agent.
Test solution was found to be stable for 90 days on storage at room temperature (
Different media for analytical method development were selected based on the solubility of drug, economy, and applicability of the developed method. Values of
FSM is a potent and selective agonist of 5-HT1B and 5-HT1D receptors. Among all the triptans, it has the longest elimination half-life of 26 hours, even after 11 years of its existence in pharmaceutical market, till now no simple, sensitive, and economical UV-spectroscopy method for the analysis of FSM has been reported. Hence, an attempt had been taken to the developed method in distilled water, phosphate buffer (pH 6.7), and acetate buffer (pH 5.5). The developed method was found to be reliable and accurate. Irrespective to media, FSM shows the maximum absorbance at the wavelength of 244 nm. Forced degradation studies show stability of FSM test solution under the chosen experimental conditions.