# A Stability Indicating Assay Method Development and Validation for the Frovatriptan Succinate Monohydrate by Using UV: A Spectrophotometric Technique

Authors: Hitesh Verma; Surajpal Verma; Harmanpreet Singh
Journal: ISRN Spectroscopy
 (2013)

---

## Abstract

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.


---

## Body

## 1. Introduction

Chemically, the Frovatriptan Succinate Monohydrate (FSM) is 3-methylamino-6-carboxamido-1,2,3,4-tetrahydrocarbazole succinate monohydrate (Figure1). It is a potent and selective agonist of 5-HT1B and 5-HT1D receptors. By acting on 5-HT1B receptor, it causes constriction of dilated arteriovenous anastomoses. The action on 5-HT1D causes the inhibition of Substance P and calcitonin gene-related peptide (CGRP) release. Among all the triptans, it has the longest elimination half-life (t1/2) of 26 hours. This carabzole derivative was approved by FDA in 2001. It has all the ideal characteristics to be used as a first line drug for menstruation migraine [1–3].Figure 1
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 0.1N NaOH and 0.1 N HCl at 280.2 nm and 279 nm, but these methods are less sensitive and tedious to be used for routine purposes because they involve first-order derivatization, second-order derivatization, and area-under the-curve (AUC) analysis in order to make their method sensitive [4, 5]. HPLC methods were reported in the literature for their analysis in dosage forms and in blood samples [6–8]. Till now, no forced degradation data is available in the literature for FSM.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 (25°C) and freeze temperature (5°C) at 244 nm. Forced degradation studies were carried out using aqueous solution of FSM (2.5 μg/mL). Test solution was exposed to various types of ICH prescribed stress conditions, like hydrolysis (neutral, acid catalyzed, and base catalysed), photolysis, thermal degradation, and oxidation, so as to check its stability. Developed methods were validated as per ICH guidelines and validation data was evaluated with the aid of various statistical parameters [9].


## 2. Materials and Methods

### 2.1. Chemicals and Reagents

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.


### 2.2. Instruments

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.


### 2.3. Construction of Calibration Plot

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 1μg/mL–3.5 μg/mL and analysed.


### 2.4. Analytical Method Validation

#### 2.4.1. Accuracy

Accuracy of the developed method was determined with the aid of three different concentration levels, namely, low concentration (LC) 2.0μg/mL, intermediate concentration (IC) 2.5 μg/mL, and a higher concentration (HC) 3.0 μg/mL. LC, IC, and HC were prepared in all the three media from independent stock solutions, in duplicate, and were analysed (n=3). Accuracy of the method was determined with the help of standard deviation (SD), percent relative standard deviation (RSD), and recovery at each level.


#### 2.4.2. Precision

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.


#### 2.4.3. Linearity

Linearity was determined by preparing a series of dilutions in three media and then measuring the absorbance at 244 nm (n=3), so as to find out the range over which Beer’s law followed. Linear regression coefficient value (R2) was taken as linearity measure.


#### 2.4.4. Limit of Quantification (LOQ) and Limit of Detection (LOD)

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≥10% and ≥30%, which signify LOQ and LOD of the developed method.


#### 2.4.5. Robustness

Robustness was determined by taking similar concentration levels as were taken in accuracy studies and storing them at two different temperatures (5∘C and 25∘C) and by varying the pH of the media by ±0.1 units.


#### 2.4.6. Forced Degradation Studies

Test solution (2.5μg/mL) was exposed to different strengths of hydrochloric acid (0.1 N to 1.0 N), sodium hydroxide (0.1 N to 1.0 N), and hydrogen peroxide solutions (from 3% w/v to 30% w/v), and the extent of degradation was analysed with respect to time and volume of exposure. Test solution was also exposed separately to the radiations of 300–800 nm and to 70∘C temperature to evaluate the extent of photolytic and thermal degradation, respectively [10, 11].


## 2.1. Chemicals and Reagents

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.


## 2.2. Instruments

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.


## 2.3. Construction of Calibration Plot

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 1μg/mL–3.5 μg/mL and analysed.


## 2.4. Analytical Method Validation

### 2.4.1. Accuracy

Accuracy of the developed method was determined with the aid of three different concentration levels, namely, low concentration (LC) 2.0μg/mL, intermediate concentration (IC) 2.5 μg/mL, and a higher concentration (HC) 3.0 μg/mL. LC, IC, and HC were prepared in all the three media from independent stock solutions, in duplicate, and were analysed (n=3). Accuracy of the method was determined with the help of standard deviation (SD), percent relative standard deviation (RSD), and recovery at each level.


### 2.4.2. Precision

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.


### 2.4.3. Linearity

Linearity was determined by preparing a series of dilutions in three media and then measuring the absorbance at 244 nm (n=3), so as to find out the range over which Beer’s law followed. Linear regression coefficient value (R2) was taken as linearity measure.


### 2.4.4. Limit of Quantification (LOQ) and Limit of Detection (LOD)

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≥10% and ≥30%, which signify LOQ and LOD of the developed method.


### 2.4.5. Robustness

Robustness was determined by taking similar concentration levels as were taken in accuracy studies and storing them at two different temperatures (5∘C and 25∘C) and by varying the pH of the media by ±0.1 units.


### 2.4.6. Forced Degradation Studies

Test solution (2.5μg/mL) was exposed to different strengths of hydrochloric acid (0.1 N to 1.0 N), sodium hydroxide (0.1 N to 1.0 N), and hydrogen peroxide solutions (from 3% w/v to 30% w/v), and the extent of degradation was analysed with respect to time and volume of exposure. Test solution was also exposed separately to the radiations of 300–800 nm and to 70∘C temperature to evaluate the extent of photolytic and thermal degradation, respectively [10, 11].


## 2.4.1. Accuracy

Accuracy of the developed method was determined with the aid of three different concentration levels, namely, low concentration (LC) 2.0μg/mL, intermediate concentration (IC) 2.5 μg/mL, and a higher concentration (HC) 3.0 μg/mL. LC, IC, and HC were prepared in all the three media from independent stock solutions, in duplicate, and were analysed (n=3). Accuracy of the method was determined with the help of standard deviation (SD), percent relative standard deviation (RSD), and recovery at each level.


## 2.4.2. Precision

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.


## 2.4.3. Linearity

Linearity was determined by preparing a series of dilutions in three media and then measuring the absorbance at 244 nm (n=3), so as to find out the range over which Beer’s law followed. Linear regression coefficient value (R2) was taken as linearity measure.


## 2.4.4. Limit of Quantification (LOQ) and Limit of Detection (LOD)

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≥10% and ≥30%, which signify LOQ and LOD of the developed method.


## 2.4.5. Robustness

Robustness was determined by taking similar concentration levels as were taken in accuracy studies and storing them at two different temperatures (5∘C and 25∘C) and by varying the pH of the media by ±0.1 units.


## 2.4.6. Forced Degradation Studies

Test solution (2.5μg/mL) was exposed to different strengths of hydrochloric acid (0.1 N to 1.0 N), sodium hydroxide (0.1 N to 1.0 N), and hydrogen peroxide solutions (from 3% w/v to 30% w/v), and the extent of degradation was analysed with respect to time and volume of exposure. Test solution was also exposed separately to the radiations of 300–800 nm and to 70∘C temperature to evaluate the extent of photolytic and thermal degradation, respectively [10, 11].


## 3. Results

### 3.1. Calibration Curve

Least square regression equation of FSM in different media at two different temperatures had shownR2 value close to 1 which ensured high degree of positive correlation between the two variables, namely, absorbance and concentration.


### 3.2. Analytical Method Validation

#### 3.2.1. Accuracy

Accuracy data associated with LC, IC, and HC is mentioned in Table1. In each case, RSD (%) was less than 2, which shows compliance with ICH guidelines. Recovery as in accuracy studies were found to be 100%–104% for distilled water, 97%–100% for phosphate buffer (pH 6.7), and 96%–100% for acetate buffer (pH 5.5), respectively.Table 1
Accuracy data of developed method for analysis of FSM in various media (n=3).
Media
Levels
Storage conditions
Freeze (5°C ± 0.5°C)
Room (25°C ± 0.5°C)
Absorbance (±SD)
RSD (%)
Recovery (%)
Absorbance (±SD)
RSD (%)
Recovery (%)
Distilled water
LC
0.351
±
0.001
0.285
103.931
0.352
±
0.003
0.714
104.320
IC
0.441
±
0.003
0.600
104.112
0.437
±
0.004
0.804
103.102
HC
0.514
±
0.003
0.625
100.997
0.520
±
0.003
0.484
102.161
Phosphate buffer (pH 6.7)
LC
0.388
±
0.002
0.536
100.139
0.382
±
0.004
0.944
97.460
IC
0.475
±
0.004
0.740
98.154
0.475
±
0.004
0.740
97.580
HC
0.578
±
0.003
0.556
99.849
0.578
±
0.003
0.612
97.425
Acetate buffer (pH 5.5)
LC
0.374
±
0.003
0.706
98.289
0.364
±
0.001
0.275
96.313
IC
0.480
±
0.006
1.163
99.404
0.471
±
0.004
0.925
99.756
HC
0.554
±
0.007
1.212
98.275
0.567
±
0.006
1.003
100.047


#### 3.2.2. Precision

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.


#### 3.2.3. Linearity

Beer’s law follows over the range of 1.0μg/mL–4.5 μg/mL in all the media at both storage conditions. The values of slope, intercept, and regression coefficient (R2) associated with linearity plots are listed in Table 2. Values of regression coefficients are close to 1 which shows that there is a directed positive correlation between the two variables, namely, absorbance and concentration (Figure 2).Table 2
Linearity data for developed method of FSM.
Media
Storage conditions
Freeze (5°C ± 0.5°C)
Room (25°C ± 0.5°C)
Slope
Intercept
R
2
Slope
Intercept
R
2
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
0.9996Linearity 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.
(a)
(b)
(c)
(d)
(e)
(f)


#### 3.2.4. LOQ and LOD

LOQ and LOD for FSM were found to be 0.025μg/mL and 0.00625 μg/mL, respectively. Data associated with LOD and LOQ analysis is mentioned in Table 3.Table 3
LOQ and LOD data for developed method of FSM (n=6).
Concentration (μg/mL)
Absorbance (±SD)
RSD (%)
0.025
0.028
±
0.003
11.208
0.0125
0.013
±
0.003
27.200
0.006
0.009
±
0.003
30.051


#### 3.2.5. Robustness

Variation in pH of media by±0.1 unit and storage conditions do not seem to have effect on the absorption values and λmax (Table 4).Table 4
Effect of change in pH of media by ±0.1 unit on the sensitivity of developed method of FSM (n=6).
Concentration (μg/mL)
Absorbance (±SD)
RSD (%)
2
0.346
±
0.001
0.371
2.5
0.436
±
0.002
0.479
3
0.520
±
0.003
0.289When paired Student’st-test (two tailed) was applied on the accessed data, calculated value (0.9073) was much less than that of the table value at 95% confidence level, which ensures the robustness of the experiment. Similar results were obtained when prepared dilutions were subjected to two different storage conditions (freeze and room) for 24 hours.


#### 3.2.6. Forced Degradation Studies

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 (y-axis) and time (x-axis) yields a straight line curve with a negative slope R2 value of 0.9421 (Table 5).Table 5
Photolytic degradation of FSM (n=6).
Time (hours)
Concentration remaining(μg/mL ± SD)
RSD   (%)
Degradation (%)
0
2.415
±
0.003
0.554
3.532
0.5
2.391
±
0.002
0.338
4.498
1
2.393
±
0.001
0.220
4.424
2
2.390
±
0.000
0.000
4.572
3
2.219
±
0.001
0.136
11.636
6
2.160
±
0.003
0.697
14.089
9
2.060
±
0.002
0.396
18.253
12
1.744
±
0.001
0.126
31.338Storage of test solution at 70°C for 7 days yields no significant degradation (Table6).Table 6
Thermal stability data of FSM (n=6).
Time (days)
Concentration remaining(μg/mL ± SD)
RSD (%)
1
2.498
±
0.002
0.449
2
2.493
±
0.002
0.330
3
2.489
±
0.002
0.331
4
2.485
±
0.001
0.217
5
2.489
±
0.002
0.331
6
2.493
±
0.002
0.330
7
2.489
±
0.001
0.125When 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 (Table7 and Figure 3).Table 7
Acid catalysed hydrolysis of FSM (n=6).
Strength (N)
Amount remaining to degrade (µg/mL)
Time (t) (hours)
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
0.817Figure 3
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 (Table8 and Figure 4). In the rest of the sample solutions, significant degradation occurs within 6 hours of exposure (>30%).Table 8
Base catalysed hydrolysis of FSM (n=6).
Strength (N)
Amount remaining to degrade (μg/mL)
Time (t) (hours)
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
0.955Figure 4
Forced degradation (base catalysed hydrolysis) plots of FSM.In oxidative degradation studies, FSM was found to be stable on the exposure of≤0.2 mL volume of 3% w/v solution of hydrogen peroxide per 10 mL of test solution without heating. Addition of more volume (>0.2 mL) or solution containing higher concentration of hydrogen peroxide causes immediate degradation of test samples (Table 9 and Figure 5).Table 9
Oxidative stability data of FSM.
Time (minutes)
Volume of 3% w/v solution added per 10 mL of drug solution
0.1 mL
0.15 mL.
0.2 mL
0.1 mL
0.15 mL
0.2 mL
Concentration (μg/mL)
Concentration (μg/mL)
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
6.825Figure 5
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 (25∘C±0.5∘C).


## 3.1. Calibration Curve

Least square regression equation of FSM in different media at two different temperatures had shownR2 value close to 1 which ensured high degree of positive correlation between the two variables, namely, absorbance and concentration.


## 3.2. Analytical Method Validation

### 3.2.1. Accuracy

Accuracy data associated with LC, IC, and HC is mentioned in Table1. In each case, RSD (%) was less than 2, which shows compliance with ICH guidelines. Recovery as in accuracy studies were found to be 100%–104% for distilled water, 97%–100% for phosphate buffer (pH 6.7), and 96%–100% for acetate buffer (pH 5.5), respectively.Table 1
Accuracy data of developed method for analysis of FSM in various media (n=3).
Media
Levels
Storage conditions
Freeze (5°C ± 0.5°C)
Room (25°C ± 0.5°C)
Absorbance (±SD)
RSD (%)
Recovery (%)
Absorbance (±SD)
RSD (%)
Recovery (%)
Distilled water
LC
0.351
±
0.001
0.285
103.931
0.352
±
0.003
0.714
104.320
IC
0.441
±
0.003
0.600
104.112
0.437
±
0.004
0.804
103.102
HC
0.514
±
0.003
0.625
100.997
0.520
±
0.003
0.484
102.161
Phosphate buffer (pH 6.7)
LC
0.388
±
0.002
0.536
100.139
0.382
±
0.004
0.944
97.460
IC
0.475
±
0.004
0.740
98.154
0.475
±
0.004
0.740
97.580
HC
0.578
±
0.003
0.556
99.849
0.578
±
0.003
0.612
97.425
Acetate buffer (pH 5.5)
LC
0.374
±
0.003
0.706
98.289
0.364
±
0.001
0.275
96.313
IC
0.480
±
0.006
1.163
99.404
0.471
±
0.004
0.925
99.756
HC
0.554
±
0.007
1.212
98.275
0.567
±
0.006
1.003
100.047


### 3.2.2. Precision

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.


### 3.2.3. Linearity

Beer’s law follows over the range of 1.0μg/mL–4.5 μg/mL in all the media at both storage conditions. The values of slope, intercept, and regression coefficient (R2) associated with linearity plots are listed in Table 2. Values of regression coefficients are close to 1 which shows that there is a directed positive correlation between the two variables, namely, absorbance and concentration (Figure 2).Table 2
Linearity data for developed method of FSM.
Media
Storage conditions
Freeze (5°C ± 0.5°C)
Room (25°C ± 0.5°C)
Slope
Intercept
R
2
Slope
Intercept
R
2
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
0.9996Linearity 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.
(a)
(b)
(c)
(d)
(e)
(f)


### 3.2.4. LOQ and LOD

LOQ and LOD for FSM were found to be 0.025μg/mL and 0.00625 μg/mL, respectively. Data associated with LOD and LOQ analysis is mentioned in Table 3.Table 3
LOQ and LOD data for developed method of FSM (n=6).
Concentration (μg/mL)
Absorbance (±SD)
RSD (%)
0.025
0.028
±
0.003
11.208
0.0125
0.013
±
0.003
27.200
0.006
0.009
±
0.003
30.051


### 3.2.5. Robustness

Variation in pH of media by±0.1 unit and storage conditions do not seem to have effect on the absorption values and λmax (Table 4).Table 4
Effect of change in pH of media by ±0.1 unit on the sensitivity of developed method of FSM (n=6).
Concentration (μg/mL)
Absorbance (±SD)
RSD (%)
2
0.346
±
0.001
0.371
2.5
0.436
±
0.002
0.479
3
0.520
±
0.003
0.289When paired Student’st-test (two tailed) was applied on the accessed data, calculated value (0.9073) was much less than that of the table value at 95% confidence level, which ensures the robustness of the experiment. Similar results were obtained when prepared dilutions were subjected to two different storage conditions (freeze and room) for 24 hours.


### 3.2.6. Forced Degradation Studies

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 (y-axis) and time (x-axis) yields a straight line curve with a negative slope R2 value of 0.9421 (Table 5).Table 5
Photolytic degradation of FSM (n=6).
Time (hours)
Concentration remaining(μg/mL ± SD)
RSD   (%)
Degradation (%)
0
2.415
±
0.003
0.554
3.532
0.5
2.391
±
0.002
0.338
4.498
1
2.393
±
0.001
0.220
4.424
2
2.390
±
0.000
0.000
4.572
3
2.219
±
0.001
0.136
11.636
6
2.160
±
0.003
0.697
14.089
9
2.060
±
0.002
0.396
18.253
12
1.744
±
0.001
0.126
31.338Storage of test solution at 70°C for 7 days yields no significant degradation (Table6).Table 6
Thermal stability data of FSM (n=6).
Time (days)
Concentration remaining(μg/mL ± SD)
RSD (%)
1
2.498
±
0.002
0.449
2
2.493
±
0.002
0.330
3
2.489
±
0.002
0.331
4
2.485
±
0.001
0.217
5
2.489
±
0.002
0.331
6
2.493
±
0.002
0.330
7
2.489
±
0.001
0.125When 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 (Table7 and Figure 3).Table 7
Acid catalysed hydrolysis of FSM (n=6).
Strength (N)
Amount remaining to degrade (µg/mL)
Time (t) (hours)
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
0.817Figure 3
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 (Table8 and Figure 4). In the rest of the sample solutions, significant degradation occurs within 6 hours of exposure (>30%).Table 8
Base catalysed hydrolysis of FSM (n=6).
Strength (N)
Amount remaining to degrade (μg/mL)
Time (t) (hours)
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
0.955Figure 4
Forced degradation (base catalysed hydrolysis) plots of FSM.In oxidative degradation studies, FSM was found to be stable on the exposure of≤0.2 mL volume of 3% w/v solution of hydrogen peroxide per 10 mL of test solution without heating. Addition of more volume (>0.2 mL) or solution containing higher concentration of hydrogen peroxide causes immediate degradation of test samples (Table 9 and Figure 5).Table 9
Oxidative stability data of FSM.
Time (minutes)
Volume of 3% w/v solution added per 10 mL of drug solution
0.1 mL
0.15 mL.
0.2 mL
0.1 mL
0.15 mL
0.2 mL
Concentration (μg/mL)
Concentration (μg/mL)
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
6.825Figure 5
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 (25∘C±0.5∘C).


## 3.2.1. Accuracy

Accuracy data associated with LC, IC, and HC is mentioned in Table1. In each case, RSD (%) was less than 2, which shows compliance with ICH guidelines. Recovery as in accuracy studies were found to be 100%–104% for distilled water, 97%–100% for phosphate buffer (pH 6.7), and 96%–100% for acetate buffer (pH 5.5), respectively.Table 1
Accuracy data of developed method for analysis of FSM in various media (n=3).
Media
Levels
Storage conditions
Freeze (5°C ± 0.5°C)
Room (25°C ± 0.5°C)
Absorbance (±SD)
RSD (%)
Recovery (%)
Absorbance (±SD)
RSD (%)
Recovery (%)
Distilled water
LC
0.351
±
0.001
0.285
103.931
0.352
±
0.003
0.714
104.320
IC
0.441
±
0.003
0.600
104.112
0.437
±
0.004
0.804
103.102
HC
0.514
±
0.003
0.625
100.997
0.520
±
0.003
0.484
102.161
Phosphate buffer (pH 6.7)
LC
0.388
±
0.002
0.536
100.139
0.382
±
0.004
0.944
97.460
IC
0.475
±
0.004
0.740
98.154
0.475
±
0.004
0.740
97.580
HC
0.578
±
0.003
0.556
99.849
0.578
±
0.003
0.612
97.425
Acetate buffer (pH 5.5)
LC
0.374
±
0.003
0.706
98.289
0.364
±
0.001
0.275
96.313
IC
0.480
±
0.006
1.163
99.404
0.471
±
0.004
0.925
99.756
HC
0.554
±
0.007
1.212
98.275
0.567
±
0.006
1.003
100.047


## 3.2.2. Precision

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.


## 3.2.3. Linearity

Beer’s law follows over the range of 1.0μg/mL–4.5 μg/mL in all the media at both storage conditions. The values of slope, intercept, and regression coefficient (R2) associated with linearity plots are listed in Table 2. Values of regression coefficients are close to 1 which shows that there is a directed positive correlation between the two variables, namely, absorbance and concentration (Figure 2).Table 2
Linearity data for developed method of FSM.
Media
Storage conditions
Freeze (5°C ± 0.5°C)
Room (25°C ± 0.5°C)
Slope
Intercept
R
2
Slope
Intercept
R
2
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
0.9996Linearity 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.
(a)
(b)
(c)
(d)
(e)
(f)


## 3.2.4. LOQ and LOD

LOQ and LOD for FSM were found to be 0.025μg/mL and 0.00625 μg/mL, respectively. Data associated with LOD and LOQ analysis is mentioned in Table 3.Table 3
LOQ and LOD data for developed method of FSM (n=6).
Concentration (μg/mL)
Absorbance (±SD)
RSD (%)
0.025
0.028
±
0.003
11.208
0.0125
0.013
±
0.003
27.200
0.006
0.009
±
0.003
30.051


## 3.2.5. Robustness

Variation in pH of media by±0.1 unit and storage conditions do not seem to have effect on the absorption values and λmax (Table 4).Table 4
Effect of change in pH of media by ±0.1 unit on the sensitivity of developed method of FSM (n=6).
Concentration (μg/mL)
Absorbance (±SD)
RSD (%)
2
0.346
±
0.001
0.371
2.5
0.436
±
0.002
0.479
3
0.520
±
0.003
0.289When paired Student’st-test (two tailed) was applied on the accessed data, calculated value (0.9073) was much less than that of the table value at 95% confidence level, which ensures the robustness of the experiment. Similar results were obtained when prepared dilutions were subjected to two different storage conditions (freeze and room) for 24 hours.


## 3.2.6. Forced Degradation Studies

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 (y-axis) and time (x-axis) yields a straight line curve with a negative slope R2 value of 0.9421 (Table 5).Table 5
Photolytic degradation of FSM (n=6).
Time (hours)
Concentration remaining(μg/mL ± SD)
RSD   (%)
Degradation (%)
0
2.415
±
0.003
0.554
3.532
0.5
2.391
±
0.002
0.338
4.498
1
2.393
±
0.001
0.220
4.424
2
2.390
±
0.000
0.000
4.572
3
2.219
±
0.001
0.136
11.636
6
2.160
±
0.003
0.697
14.089
9
2.060
±
0.002
0.396
18.253
12
1.744
±
0.001
0.126
31.338Storage of test solution at 70°C for 7 days yields no significant degradation (Table6).Table 6
Thermal stability data of FSM (n=6).
Time (days)
Concentration remaining(μg/mL ± SD)
RSD (%)
1
2.498
±
0.002
0.449
2
2.493
±
0.002
0.330
3
2.489
±
0.002
0.331
4
2.485
±
0.001
0.217
5
2.489
±
0.002
0.331
6
2.493
±
0.002
0.330
7
2.489
±
0.001
0.125When 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 (Table7 and Figure 3).Table 7
Acid catalysed hydrolysis of FSM (n=6).
Strength (N)
Amount remaining to degrade (µg/mL)
Time (t) (hours)
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
0.817Figure 3
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 (Table8 and Figure 4). In the rest of the sample solutions, significant degradation occurs within 6 hours of exposure (>30%).Table 8
Base catalysed hydrolysis of FSM (n=6).
Strength (N)
Amount remaining to degrade (μg/mL)
Time (t) (hours)
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
0.955Figure 4
Forced degradation (base catalysed hydrolysis) plots of FSM.In oxidative degradation studies, FSM was found to be stable on the exposure of≤0.2 mL volume of 3% w/v solution of hydrogen peroxide per 10 mL of test solution without heating. Addition of more volume (>0.2 mL) or solution containing higher concentration of hydrogen peroxide causes immediate degradation of test samples (Table 9 and Figure 5).Table 9
Oxidative stability data of FSM.
Time (minutes)
Volume of 3% w/v solution added per 10 mL of drug solution
0.1 mL
0.15 mL.
0.2 mL
0.1 mL
0.15 mL
0.2 mL
Concentration (μg/mL)
Concentration (μg/mL)
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
6.825Figure 5
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 (25∘C±0.5∘C).


## 4. Discussion

Different media for analytical method development were selected based on the solubility of drug, economy, and applicability of the developed method. Values ofR2 in calibration plots and during linearity analysis were near to 1 in each case, which show direct positive correlation between the concentration of the solution and the absorption of UV light. Analyses of accuracy and precision data show that the method is accurate and reliable. LOD and LOQ data analysis reflect that the developed method is very sensitive and selective. Forced degradation studies were performed as per ICH guidelines, and it was found that FSM was stable in these stressed conditions.


## 5. Conclusion

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.



---

*Source: 361385-2013-05-02.xml*