# Method Development and Validation for Estimation of Eperisone Hydrochloride as API and in Tablet Dosage Form by Two Spectroscopic Methods

Authors: Joytosh Banerjee; Renu Solanki; Badri Prakash Nagori
Journal: ISRN Analytical Chemistry
 (2013)

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## Abstract

Two simple and sensitive spectrophotometric methods have been developed for the determination of eperisone hydrochloride based on its ability to be detected in UV region (Normal UV) and its oxidation using potassium permanganate in alkaline medium (kinetic spectroscopic). The detection was done at 261.40 nm and 603.5 nm. The different experimental parameters affecting the method development were studied and optimized. The initial rate and fixed time method were utilized to construct calibration graph, and 5 minutes and 3 minutes, respectively, were found suitable for the determination of the concentration of drug. Linearity was found over the concentration range of 2–20μg/mL, 15–30 μg/mL, and 15–35 μg/mL by UV, initial rate, and fixed time methods, respectively. The results were validated as per the ICH guidelines. RSD values were found to be less than 2%. The methods were applied for estimation of eperisone hydrochloride in RAPISONE (Abbott, Maharashtra). The assay results were found to be 100.4% ± 0.08, 99.93% ± 0.05, and 99.41% ± 0.04 by UV, initial rate, and fixed time method, respectively. Statistical comparison of the proposed methods showed a good agreement indicating no significant difference in accuracy and precision, thus confirming the suitability of UV and kinetic method for the estimation of eperisone hydrochloride in bulk as well as in tablet dosage forms.


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## Body

## 1. Introduction

Eperisone hydrochloride is an antispasmodic drug sold in Japan, India, Philippines, and Bangladesh [1]. It belongs to piperidinopropiophenone analogues, and its structure is shown in Figure 1. It acts by relaxing both skeletal muscles and vascular smooth muscles and demonstrates reduction of myotonia, improvement of circulation, and suppression of pain reflex [2]. Kinetic spectrophotometric methods have regained interest in the recent past due to its specific advantages as well as improved capabilities of the modern instrument [3–5]. LC-ESI-MS [6], GC-MS [7, 8], and HPLC [9, 10] methods were proposed for the determination of eperisone hydrochloride in biological fluids. Various methods were proposed for the estimation of degradation products [11]. Japanese Pharmacopeia reports potentiometric method for its estimation [12]. Kinetic spectrophotometric method has not been reported so far in any literature. The objective of the research is to develop and validate UV and kinetic spectrophotometric method for the estimation of eperisone hydrochloride for routine analysis in bulk and tablet dosage forms. The methods would be statistically compared for their suitability for estimation of the drug.Figure 1
Chemical structure of eperisone hydrochloride.


## 2. Material and Method

Double distilled water was used as solvent to prepare solutions. Potassium permanganate (KMnO4) (LOBA CHEMIE, India) was accurately weighed and dissolved in distilled water to obtain a solution of 6 × 10−3M as given in the Indian Pharmacopoeia (I.P.). Sodium hydroxide (NaOH) (LOBA CHEMIE, India) was also accurately weighed and dissolved in distilled water to obtain a solution of 1.0 M in distilled water. Shimadzu UV-1800 model with spectral band width one nm and wavelength accuracy of 0.3 nm was used for spectral measurements. One cm matched quartz cells were used for carrying the analyte solution. Weighing was done on electronic balance (GR-200, A & D Company). Glassware used in each procedure were washed and dried at the end of each day’s work. Pure drug of eperisone hydrochloride was obtained as gift sample from Sun Pharma Industries, Mumbai. Tablets of brand name RAPISONE (Batch no. AME-008B), marketed by Abbott, Maharashtra, were procured from local pharmacy.


### 2.1. Preparation of Standard Stock Solution

#### 2.1.1. For Normal UV Spectrophotometric Method

Standard stock solution of 1000μg/mL was prepared by dissolving 100 mg of pure active pharmaceutical ingredient in 100 mL of double distilled water in a volumetric flask.


#### 2.1.2. For Kinetic Spectrophotometric Method

Stock solution A (for method development): 100 mg of eperisone hydrochloride was accurately weighed and transferred to 100 mL volumetric flask. It was dissolved in 25 mL of doubly distilled water, and the volume was made up to 100 mL with the same. The final solution contained 1000μg/mL of eperisone hydrochloride solution.Stock solution B (for determination of stoichiometry): 177.5 mg of eperisone hydrochloride was accurately weighed and transferred to 10 mL volumetric flask. It was dissolved in doubly distilled water, and the volume was made up to 10 mL with the same. The final solution contained 17750μg/mL of eperisone hydrochloride solution.


### 2.2. Preparation of Sample Solution of Pharmaceutical Tablets

#### 2.2.1. For Normal UV Method

Twenty tablets were taken, and the I.P. method was followed to determine the average weight. Weighed tablets were triturated well. A quantity of powder equivalent to 10 mg of drug was transferred to 100 mL volumetric flask and mixed with 25 mL of doubly distilled water. The solution was sonicated for 10 minutes, and the volume was made up to the mark with doubly distilled water solvent. The solution was then filtered through Whatman filter paper no. 42.


#### 2.2.2. For Kinetic Spectrophotometric Method

20 tablets were weighed and triturated well. A quantity of powder equivalent to 50 mg of drug was transferred to 100 mL volumetric flask and mixed with 25 mL of double distilled water, and the solution was sonicated for 15 minutes. The volume was finally made up to 100 mL with double distilled water. The solution was filtered through Whatman filter paper no. 42. The filtrate was diluted with water to obtain working concentration in the range of 15–25μg/mL for the drug.


### 2.3. Procedure

#### 2.3.1. Normal UV Method

Ten mL of stock solution A was taken and further diluted to 100 mL to obtain working stock solution of 100μg/mL. From the solution, 2 mL was transferred to a ten mL volumetric flask, and the volume was made up to ten mL with double distilled water to get the final solution of 20 μg/mL. The solution was scanned between 400 nm and 200 nm. Wavelength of maximum absorbance (λmax) was found to be 261.40 nm. Different aliquots (0.2, 0.4,…,2.0 mL) of working stock solution were accurately measured and diluted in a series of ten mL volumetric flask with doubly distilled water to obtain concentrations of 1, 2, 3, 4,…, and 20 μg/mL.


#### 2.3.2. Initial Rate Method

A series of dilutions (150–350μg/mL) was prepared by pipetting out different aliquots of stock solution A into a series of 10 mL volumetric flask. At the time of observation, 1.6 mL of sodium hydroxide solution (1 M) was added, followed by 2.0 mL of potassium permanganate solution (0.006 M) to each flask and then diluted to the volume with double distilled water. The content of mixture of each flask was mixed well, and the increase in absorbance was recorded (in Kinetics mode) at 603.5 nm as a function of time for 15 minutes against reagent blank treated similarly. The initial rate of reaction at different concentrations was obtained from the slope of tangent to the absorbance time curve (at 5 minutes).


#### 2.3.3. Fixed Time Method

In this method, the absorbance was recorded accurately at preselected time of 3, 6, 9, 12, and 15 minutes against reagent blank treated similarly. The calibration graphs were constructed by plotting the absorbances against various concentrations of the series of drug solution. The concentration of the drug was determined from either calibration curve or regression equation.


## 2.1. Preparation of Standard Stock Solution

### 2.1.1. For Normal UV Spectrophotometric Method

Standard stock solution of 1000μg/mL was prepared by dissolving 100 mg of pure active pharmaceutical ingredient in 100 mL of double distilled water in a volumetric flask.


### 2.1.2. For Kinetic Spectrophotometric Method

Stock solution A (for method development): 100 mg of eperisone hydrochloride was accurately weighed and transferred to 100 mL volumetric flask. It was dissolved in 25 mL of doubly distilled water, and the volume was made up to 100 mL with the same. The final solution contained 1000μg/mL of eperisone hydrochloride solution.Stock solution B (for determination of stoichiometry): 177.5 mg of eperisone hydrochloride was accurately weighed and transferred to 10 mL volumetric flask. It was dissolved in doubly distilled water, and the volume was made up to 10 mL with the same. The final solution contained 17750μg/mL of eperisone hydrochloride solution.


## 2.1.1. For Normal UV Spectrophotometric Method

Standard stock solution of 1000μg/mL was prepared by dissolving 100 mg of pure active pharmaceutical ingredient in 100 mL of double distilled water in a volumetric flask.


## 2.1.2. For Kinetic Spectrophotometric Method

Stock solution A (for method development): 100 mg of eperisone hydrochloride was accurately weighed and transferred to 100 mL volumetric flask. It was dissolved in 25 mL of doubly distilled water, and the volume was made up to 100 mL with the same. The final solution contained 1000μg/mL of eperisone hydrochloride solution.Stock solution B (for determination of stoichiometry): 177.5 mg of eperisone hydrochloride was accurately weighed and transferred to 10 mL volumetric flask. It was dissolved in doubly distilled water, and the volume was made up to 10 mL with the same. The final solution contained 17750μg/mL of eperisone hydrochloride solution.


## 2.2. Preparation of Sample Solution of Pharmaceutical Tablets

### 2.2.1. For Normal UV Method

Twenty tablets were taken, and the I.P. method was followed to determine the average weight. Weighed tablets were triturated well. A quantity of powder equivalent to 10 mg of drug was transferred to 100 mL volumetric flask and mixed with 25 mL of doubly distilled water. The solution was sonicated for 10 minutes, and the volume was made up to the mark with doubly distilled water solvent. The solution was then filtered through Whatman filter paper no. 42.


### 2.2.2. For Kinetic Spectrophotometric Method

20 tablets were weighed and triturated well. A quantity of powder equivalent to 50 mg of drug was transferred to 100 mL volumetric flask and mixed with 25 mL of double distilled water, and the solution was sonicated for 15 minutes. The volume was finally made up to 100 mL with double distilled water. The solution was filtered through Whatman filter paper no. 42. The filtrate was diluted with water to obtain working concentration in the range of 15–25μg/mL for the drug.


## 2.2.1. For Normal UV Method

Twenty tablets were taken, and the I.P. method was followed to determine the average weight. Weighed tablets were triturated well. A quantity of powder equivalent to 10 mg of drug was transferred to 100 mL volumetric flask and mixed with 25 mL of doubly distilled water. The solution was sonicated for 10 minutes, and the volume was made up to the mark with doubly distilled water solvent. The solution was then filtered through Whatman filter paper no. 42.


## 2.2.2. For Kinetic Spectrophotometric Method

20 tablets were weighed and triturated well. A quantity of powder equivalent to 50 mg of drug was transferred to 100 mL volumetric flask and mixed with 25 mL of double distilled water, and the solution was sonicated for 15 minutes. The volume was finally made up to 100 mL with double distilled water. The solution was filtered through Whatman filter paper no. 42. The filtrate was diluted with water to obtain working concentration in the range of 15–25μg/mL for the drug.


## 2.3. Procedure

### 2.3.1. Normal UV Method

Ten mL of stock solution A was taken and further diluted to 100 mL to obtain working stock solution of 100μg/mL. From the solution, 2 mL was transferred to a ten mL volumetric flask, and the volume was made up to ten mL with double distilled water to get the final solution of 20 μg/mL. The solution was scanned between 400 nm and 200 nm. Wavelength of maximum absorbance (λmax) was found to be 261.40 nm. Different aliquots (0.2, 0.4,…,2.0 mL) of working stock solution were accurately measured and diluted in a series of ten mL volumetric flask with doubly distilled water to obtain concentrations of 1, 2, 3, 4,…, and 20 μg/mL.


### 2.3.2. Initial Rate Method

A series of dilutions (150–350μg/mL) was prepared by pipetting out different aliquots of stock solution A into a series of 10 mL volumetric flask. At the time of observation, 1.6 mL of sodium hydroxide solution (1 M) was added, followed by 2.0 mL of potassium permanganate solution (0.006 M) to each flask and then diluted to the volume with double distilled water. The content of mixture of each flask was mixed well, and the increase in absorbance was recorded (in Kinetics mode) at 603.5 nm as a function of time for 15 minutes against reagent blank treated similarly. The initial rate of reaction at different concentrations was obtained from the slope of tangent to the absorbance time curve (at 5 minutes).


### 2.3.3. Fixed Time Method

In this method, the absorbance was recorded accurately at preselected time of 3, 6, 9, 12, and 15 minutes against reagent blank treated similarly. The calibration graphs were constructed by plotting the absorbances against various concentrations of the series of drug solution. The concentration of the drug was determined from either calibration curve or regression equation.


## 2.3.1. Normal UV Method

Ten mL of stock solution A was taken and further diluted to 100 mL to obtain working stock solution of 100μg/mL. From the solution, 2 mL was transferred to a ten mL volumetric flask, and the volume was made up to ten mL with double distilled water to get the final solution of 20 μg/mL. The solution was scanned between 400 nm and 200 nm. Wavelength of maximum absorbance (λmax) was found to be 261.40 nm. Different aliquots (0.2, 0.4,…,2.0 mL) of working stock solution were accurately measured and diluted in a series of ten mL volumetric flask with doubly distilled water to obtain concentrations of 1, 2, 3, 4,…, and 20 μg/mL.


## 2.3.2. Initial Rate Method

A series of dilutions (150–350μg/mL) was prepared by pipetting out different aliquots of stock solution A into a series of 10 mL volumetric flask. At the time of observation, 1.6 mL of sodium hydroxide solution (1 M) was added, followed by 2.0 mL of potassium permanganate solution (0.006 M) to each flask and then diluted to the volume with double distilled water. The content of mixture of each flask was mixed well, and the increase in absorbance was recorded (in Kinetics mode) at 603.5 nm as a function of time for 15 minutes against reagent blank treated similarly. The initial rate of reaction at different concentrations was obtained from the slope of tangent to the absorbance time curve (at 5 minutes).


## 2.3.3. Fixed Time Method

In this method, the absorbance was recorded accurately at preselected time of 3, 6, 9, 12, and 15 minutes against reagent blank treated similarly. The calibration graphs were constructed by plotting the absorbances against various concentrations of the series of drug solution. The concentration of the drug was determined from either calibration curve or regression equation.


## 3. Results and Discussion

### 3.1. For Normal UV Method

Calibration curve was plotted atλmax of 261.40 nm as shown in Figure 2. Linearity was found in the range of 2–22 μg/mL as shown in Figure 3. The various optical parameters are reported in Table 1.Table 1
Optical parameters and  regression characteristic for eperisone hydrochloride.
Sr. no.
Parameter
Result
(
1
)
Absorption maxima (nm)
261.40
(
2
)
Linearity range (μg/mL)
2–20
(
3
)
Standard regression equation
Y
=
0.04867
X
-
0.00058
(
4
)
Correlation coefficient (r2)
0.99936
(
5
)
Molar absorptivity (L/mol/cm)
14718.93
(
6
)
A (1%, A cm)
497.5
(
7
)
Sandell’s sensitivity (μg/cm2)
0.02
(
8
)
Intraday precision (% RSD)
0.14
(
9
)
Interday precision (% RSD)
0.28
(
10
)
LOD (μg/mL)
0.33
(
11
)
LOQ (μg/mL)
1.0Figure 2
Absorption scan of eperisone hydrochloride in double distilled water (λmax=261.40 nm).Figure 3
Calibration curve of eperisone hydrochloride in doubly distilled water (2–22μg/mL).


### 3.2. Optimization of Parameters for Kinetic Spectrophotometric Method

Potassium permanganate, as strong oxidizing agent, has been used in oxidimetric analytical method for the determination of many compounds. The valency of manganese changes during the course of the reaction. The heptavalent manganese ion changes to the green color (Mn VI), while, in neutral and acidic medium, the permanganate is further reduced to colorless (Mn II). The behavior of permanganate was the basis for its use in the development of spectrophotometric method. The absorption spectrum of aqueous potassium permanganate solution in alkaline medium exhibited an absorption band at 526 nm. The addition of eperisone hydrochloride to this solution produces a new characteristic band at 603.5 nm. This is shown in Figure4. The band is due to the formation of manganate ion, which resulted from the oxidation of eperisone hydrochloride by potassium permanganate in alkaline medium. The intensity of the color increases with time; therefore, a kinetically based method was successfully developed for the determination of eperisone hydrochloride in their pharmaceutical dosage formulations.Figure 4
Absorption spectra of (A) eperisone hydrochloride (20μg/mL); (B) alkaline potassium permanganate (0.006 M), and (C) the reaction product.


### 3.3. Effect of Potassium Permanganate Concentration

The absorbance increases substantially with increasing the concentration of potassium permanganate as shown in Figure5. Maximum absorbance was obtained when 2.0 mL of 0.006 M of potassium permanganate was used. Thus, the adoption of 2 mL of potassium permanganate in the final solution proved to be adequate for the determination of eperisone hydrochloride (final concentration was 0.0012 M).Figure 5
Effect of potassium permanganate on the reaction between the investigated eperisone hydrochloride (20μg/mL) and alkaline potassium permanganate.


#### 3.3.1. Effect of Sodium Hydroxide Concentration

Maximum absorption was obtained when 1.6 mL of 1 M NaOH was used. Over this volume, no deliberate change in absorbance could be detected. So, 1.6 mL of 1 M of NaOH was used as an optimum value as shown in Figure6.Figure 6
Effect of sodium hydroxide concentration (1 M) on the reaction between eperisone hydrochloride (20μg/mL) and alkaline potassium permanganate.


#### 3.3.2. Effect of Temperature

At room temperature, the reaction rate increases substantially with time. Moreover, heating the solution was found to increase the rate of the reaction but caused precipitation of MnO2 also. Therefore, room temperature was selected as the optimum temperature.


#### 3.3.3. Stoichiometry and Reaction Mechanism [13, 14]

The stoichiometric ratio between potassium permanganate and eperisone hydrochloride was determined by Job’s method and was found to be 4 : 1. Corrected Job’s plot is shown in Figure7. Eperisone hydrochloride was found to be susceptible for oxidation with alkaline potassium permanganate producing a green color at λmax of 603.5 nm.Figure 7
Job’s plot of continuous variation between potassium permanganate and eperisone hydrochloride.


#### 3.3.4. Kinetics of the Reaction

Under the optimum conditions, the absorbance time curves of eperisone hydrochloride with potassium permanganate reagent were constructed (as shown in Figures8 and 9). The initial rate of the reaction was determined from the slope of tangents of the absorption time curves. The order of the reaction with respect to permanganate was determined by studying the reaction at different concentrations of permanganate with fixed concentration of investigated eperisone hydrochloride. The plot of initial rate (ΔA/Δt) against initial absorbance was linear passing through origin indicating that the initial order of the reaction with respect to permanganate was 1 as shown in Figure 10.Figure 8
Representing change in absorbance with respect to time as recorded by UV in Kinetics mode.Figure 9
Absorption versus time for the reaction between eperisone hydrochloride (6.76 × 10−5M) and KMnO4 (0.3 to 1.8 × 10−3M).Figure 10
Plot of initial rate (dA/dt, where t=300 s) versus initial absorbance A.The order with respect to investigated eperisone hydrochloride was evaluated by measuring the rate of the reaction at several concentrations of eperisone hydrochloride at a fixed concentration of permanganate reagent as shown in Figure11. This was done by plotting the logarithm of initial rate of the reaction versus logarithm of molar concentration of investigated eperisone hydrochloride and was found to be approximately 1 as shown in Figure 12. However, under the optimized experimental conditions, the concentrations of eperisone hydrochloride were determined using relative excess amount of potassium permanganate and sodium hydroxide solutions. Therefore, pseudo-zero-order conditions were obtained with respect to their concentrations.Figure 11
Absorption versus time for the reaction between eperisone hydrochloride (5.07 to 11.8 × 10−5 M) and KMnO4 (1.2 × 10−4 M).Figure 12
Plot of log initial rate (dA/dt, where t=300 s) versus Log concentration.


#### 3.3.5. Quantitation by Kinetic Methods

(1)  Initial Rate Method. Initial rate of reaction would follow pseudo-first-order and were found to obey (1):
(1)V=dAdt=K′Cn,
where V is the reaction rate, A is absorbance, t is the measuring time, K′ is the pseudo-first-order rate constant, C is the molar concentration, and n is the order of the reaction. The logarithmic form of the reaction is shown in (2):
(2)logv=logΔAΔt=logK′+nlogC.
Regression analysis was performed using the method of least square to evaluate the slopes, intercepts, and correlation coefficients. The analytical parameters and results of regression analysis are given in Table 2. The value of n(≈1) in the regression equation confirmed that the reaction of eperisone hydrochloride with the potassium permanganate was a pseudo-first-order with respect to eperisone hydrochloride concentration. The limits of detection (LOD) were calculated, and results obtained confirmed good sensitivity of the proposed method and consequently their capabilities to determine low amount of eperisone hydrochloride.Table 2
Data representing analytical parameters for the initial rate method for determination of eperisone hydrochloride with alkaline potassium permanganate (Δt=300 sec).
Method
Linearity (μg/mL)
LogK
Slope(n)
r
2
LOD (μg/mL)
LOQ (μg/mL)
Initial rate
15–30
0.998
0.808
0.998
0.46
1.41(2)  Fixed Time Method. In this method, the absorbance of the reaction solution containing varying amounts of eperisone HCl was measured at preselected fixed time. Calibration curve of absorbance versus the concentration of the drug at fixed time was plotted (shown in Figure 13). The regression equation, correlation coefficients, and detection limits were calculated and reported in Table 3. The lowest detection limit was obtained at fixed time of 6 minutes. However the fixed time of 3 minutes showed a wider concentration range for quantification.Table 3
Analytical parameters for the fixed time method for determination of eperisone hydrochloride with alkaline potassium permanganate.
Reaction time (sec)
Linearity range (μg/mL)
Intercept
S.D.
Slope
S.D.
r
2
LOD (μg/mL)
LOQ (μg/mL)
180
15–35
0.035
0.01
0.029
0.00
0.998
0.65
1.99
360
15–30
0.105
0.00
0.033
0.00
0.999
0.15
0.46
540
15–25
0.11
0.02
0.035
0.00
0.998
1.81
5.49
720
15–20
0.07
0.02
0.087
0.01
1
0.79
2.40
900
15–20
0.1
0.01
0.033
0.00
1
1.15
3.49Figure 13
Calibration plot of absorbance versus the concentration of eperisone hydrochloride at preselected fixed time of 3 minutes.


### 3.4. Validation of Proposed Methods [15, 16]

#### 3.4.1. Specificity

Specificity of the UV method was evaluated by addition of common tablet excipients available in the laboratory and recording the UV scan. No interference was detected from the added excipients at 261.40 nm, and the variations in absorbance were negligible.In case of kinetic spectroscopic method, both the proposed methods were performed in visible range away from the UV-absorption region. A very minor change in the absorbance values was seen during the time duration.


#### 3.4.2. Linearity

Linearity data are studied for both methods and reported in Tables1, 2, and 3.


#### 3.4.3. Accuracy

Accuracy studies in terms of percentage recovery (percentage) were studied for normal UV and kinetic spectroscopic method and reported in Tables4, 5, and 6. The results of mean % recovery were within the range of 98–102%, and % RSD was less than 2% so the proposed method was found to be accurate.Table 4
Result of accuracy studies by normal UV method.
Formulation
Amount taken (μg/mL)
Amount added (μg/mL)
Amount found± S.D.
% recovery ± S.D.
Mean % recovery± S.D.
RAPISONE
5
3
8.14
±
0.0
101.8
±
0.0
100.62
±
1.02
5
5
9.99
±
0.0
99.95
±
0.0
5
7
12.01
±
.
00
100.11
±
0.0Table 5
Data representing evaluation of accuracy of the analytical procedure using initial rate method.
S. no.
Amount of tablet soln added (μg/mL)
Amount of std. soln added (μg/mL)
LogdA/dt  (mean)
Amount found(μg/mL)
% recovery
Mean %recovery ± S.D.
% RSD
(
1
)
10
10
−2.851
19.98
99.92
100.54
±
0.77
0.76
(
2
)
10
15
−2.872
25.07
100.29
(
3
)
10
20
−2.775
30.42
101.41Table 6
Data representing evaluation of accuracy of the analytical procedure using fixed time method.
S. no.
Amount of tablet soln added (μg/mL)
Amount of std. soln added (μg/mL)
Mean Abs.
Amount found (μg/mL)
% recovery
Mean % recovery ± S.D.
% RSD
(
1
)
10
10
0.628
19.88
99.41
99.22
±
0.16
0.16
(
2
)
10
15
0.753
24.78
99.12
(
3
)
10
20
0.892
29.74
99.14


#### 3.4.4. Precision

The developed methods were subjected to precision studies. The analytical techniques demonstrated good precision, and the data is given in Tables7, 8, and 9.Table 7
Intraday and Interday precision for normal UV method (n=6).
Amount taken(μg/mL)
Intra-day precision
Inter-day precision
% assay ± S.D.
% assay ± S.D.
8
100.82
±
0.77
99.96
±
1.06
10
100.63
±
0.77
100.2
±
0.91
12
100.55
±
1.71
100.52
±
0.98
Mean % assay ± S.D.
100.67
±
0.14
100.22
±
0.28
% RSD
0.140
0.285Table 8
Data showing intraday precision analysis for initial rate method and fixed time method.
Conc. (μg/mL)
Initial rate method
Fixed time method
Mean± S.D.
% RSD
Mean± S.D.
% RSD
20
20.024
±
0.04
0.211
19.89
±
0.18
0.92
25
25.0
±
0
0
24.78
±
0.19
0.76
30
30.01
±
0.01
0.033
29.89
±
0.19
0.64
Mean % RSD
0.081
0.777Table 9
Data showing interday precision analysis for initial rate method and fixed time method.
Conc. (μg/mL)
Initial rate method
Fixed time method
Mean± S.D.
% RSD
Mean± S.D.
% RSD
20
20.02
±
0.015
0.076
19.83
±
0
.
16
0.805
25
25.06
±
0.06
0.246
24.76
±
0
.
20
0.829
30
30.07
±
0.06
0.220
29.65
±
0.35
1.180
Mean % RSD
0.180
0.938


#### 3.4.5. Detection and Quantification Limits

The values are reported in Tables1, 2, and 3.


## 3.1. For Normal UV Method

Calibration curve was plotted atλmax of 261.40 nm as shown in Figure 2. Linearity was found in the range of 2–22 μg/mL as shown in Figure 3. The various optical parameters are reported in Table 1.Table 1
Optical parameters and  regression characteristic for eperisone hydrochloride.
Sr. no.
Parameter
Result
(
1
)
Absorption maxima (nm)
261.40
(
2
)
Linearity range (μg/mL)
2–20
(
3
)
Standard regression equation
Y
=
0.04867
X
-
0.00058
(
4
)
Correlation coefficient (r2)
0.99936
(
5
)
Molar absorptivity (L/mol/cm)
14718.93
(
6
)
A (1%, A cm)
497.5
(
7
)
Sandell’s sensitivity (μg/cm2)
0.02
(
8
)
Intraday precision (% RSD)
0.14
(
9
)
Interday precision (% RSD)
0.28
(
10
)
LOD (μg/mL)
0.33
(
11
)
LOQ (μg/mL)
1.0Figure 2
Absorption scan of eperisone hydrochloride in double distilled water (λmax=261.40 nm).Figure 3
Calibration curve of eperisone hydrochloride in doubly distilled water (2–22μg/mL).


## 3.2. Optimization of Parameters for Kinetic Spectrophotometric Method

Potassium permanganate, as strong oxidizing agent, has been used in oxidimetric analytical method for the determination of many compounds. The valency of manganese changes during the course of the reaction. The heptavalent manganese ion changes to the green color (Mn VI), while, in neutral and acidic medium, the permanganate is further reduced to colorless (Mn II). The behavior of permanganate was the basis for its use in the development of spectrophotometric method. The absorption spectrum of aqueous potassium permanganate solution in alkaline medium exhibited an absorption band at 526 nm. The addition of eperisone hydrochloride to this solution produces a new characteristic band at 603.5 nm. This is shown in Figure4. The band is due to the formation of manganate ion, which resulted from the oxidation of eperisone hydrochloride by potassium permanganate in alkaline medium. The intensity of the color increases with time; therefore, a kinetically based method was successfully developed for the determination of eperisone hydrochloride in their pharmaceutical dosage formulations.Figure 4
Absorption spectra of (A) eperisone hydrochloride (20μg/mL); (B) alkaline potassium permanganate (0.006 M), and (C) the reaction product.


## 3.3. Effect of Potassium Permanganate Concentration

The absorbance increases substantially with increasing the concentration of potassium permanganate as shown in Figure5. Maximum absorbance was obtained when 2.0 mL of 0.006 M of potassium permanganate was used. Thus, the adoption of 2 mL of potassium permanganate in the final solution proved to be adequate for the determination of eperisone hydrochloride (final concentration was 0.0012 M).Figure 5
Effect of potassium permanganate on the reaction between the investigated eperisone hydrochloride (20μg/mL) and alkaline potassium permanganate.


### 3.3.1. Effect of Sodium Hydroxide Concentration

Maximum absorption was obtained when 1.6 mL of 1 M NaOH was used. Over this volume, no deliberate change in absorbance could be detected. So, 1.6 mL of 1 M of NaOH was used as an optimum value as shown in Figure6.Figure 6
Effect of sodium hydroxide concentration (1 M) on the reaction between eperisone hydrochloride (20μg/mL) and alkaline potassium permanganate.


### 3.3.2. Effect of Temperature

At room temperature, the reaction rate increases substantially with time. Moreover, heating the solution was found to increase the rate of the reaction but caused precipitation of MnO2 also. Therefore, room temperature was selected as the optimum temperature.


### 3.3.3. Stoichiometry and Reaction Mechanism [13, 14]

The stoichiometric ratio between potassium permanganate and eperisone hydrochloride was determined by Job’s method and was found to be 4 : 1. Corrected Job’s plot is shown in Figure7. Eperisone hydrochloride was found to be susceptible for oxidation with alkaline potassium permanganate producing a green color at λmax of 603.5 nm.Figure 7
Job’s plot of continuous variation between potassium permanganate and eperisone hydrochloride.


### 3.3.4. Kinetics of the Reaction

Under the optimum conditions, the absorbance time curves of eperisone hydrochloride with potassium permanganate reagent were constructed (as shown in Figures8 and 9). The initial rate of the reaction was determined from the slope of tangents of the absorption time curves. The order of the reaction with respect to permanganate was determined by studying the reaction at different concentrations of permanganate with fixed concentration of investigated eperisone hydrochloride. The plot of initial rate (ΔA/Δt) against initial absorbance was linear passing through origin indicating that the initial order of the reaction with respect to permanganate was 1 as shown in Figure 10.Figure 8
Representing change in absorbance with respect to time as recorded by UV in Kinetics mode.Figure 9
Absorption versus time for the reaction between eperisone hydrochloride (6.76 × 10−5M) and KMnO4 (0.3 to 1.8 × 10−3M).Figure 10
Plot of initial rate (dA/dt, where t=300 s) versus initial absorbance A.The order with respect to investigated eperisone hydrochloride was evaluated by measuring the rate of the reaction at several concentrations of eperisone hydrochloride at a fixed concentration of permanganate reagent as shown in Figure11. This was done by plotting the logarithm of initial rate of the reaction versus logarithm of molar concentration of investigated eperisone hydrochloride and was found to be approximately 1 as shown in Figure 12. However, under the optimized experimental conditions, the concentrations of eperisone hydrochloride were determined using relative excess amount of potassium permanganate and sodium hydroxide solutions. Therefore, pseudo-zero-order conditions were obtained with respect to their concentrations.Figure 11
Absorption versus time for the reaction between eperisone hydrochloride (5.07 to 11.8 × 10−5 M) and KMnO4 (1.2 × 10−4 M).Figure 12
Plot of log initial rate (dA/dt, where t=300 s) versus Log concentration.


### 3.3.5. Quantitation by Kinetic Methods

(1)  Initial Rate Method. Initial rate of reaction would follow pseudo-first-order and were found to obey (1):
(1)V=dAdt=K′Cn,
where V is the reaction rate, A is absorbance, t is the measuring time, K′ is the pseudo-first-order rate constant, C is the molar concentration, and n is the order of the reaction. The logarithmic form of the reaction is shown in (2):
(2)logv=logΔAΔt=logK′+nlogC.
Regression analysis was performed using the method of least square to evaluate the slopes, intercepts, and correlation coefficients. The analytical parameters and results of regression analysis are given in Table 2. The value of n(≈1) in the regression equation confirmed that the reaction of eperisone hydrochloride with the potassium permanganate was a pseudo-first-order with respect to eperisone hydrochloride concentration. The limits of detection (LOD) were calculated, and results obtained confirmed good sensitivity of the proposed method and consequently their capabilities to determine low amount of eperisone hydrochloride.Table 2
Data representing analytical parameters for the initial rate method for determination of eperisone hydrochloride with alkaline potassium permanganate (Δt=300 sec).
Method
Linearity (μg/mL)
LogK
Slope(n)
r
2
LOD (μg/mL)
LOQ (μg/mL)
Initial rate
15–30
0.998
0.808
0.998
0.46
1.41(2)  Fixed Time Method. In this method, the absorbance of the reaction solution containing varying amounts of eperisone HCl was measured at preselected fixed time. Calibration curve of absorbance versus the concentration of the drug at fixed time was plotted (shown in Figure 13). The regression equation, correlation coefficients, and detection limits were calculated and reported in Table 3. The lowest detection limit was obtained at fixed time of 6 minutes. However the fixed time of 3 minutes showed a wider concentration range for quantification.Table 3
Analytical parameters for the fixed time method for determination of eperisone hydrochloride with alkaline potassium permanganate.
Reaction time (sec)
Linearity range (μg/mL)
Intercept
S.D.
Slope
S.D.
r
2
LOD (μg/mL)
LOQ (μg/mL)
180
15–35
0.035
0.01
0.029
0.00
0.998
0.65
1.99
360
15–30
0.105
0.00
0.033
0.00
0.999
0.15
0.46
540
15–25
0.11
0.02
0.035
0.00
0.998
1.81
5.49
720
15–20
0.07
0.02
0.087
0.01
1
0.79
2.40
900
15–20
0.1
0.01
0.033
0.00
1
1.15
3.49Figure 13
Calibration plot of absorbance versus the concentration of eperisone hydrochloride at preselected fixed time of 3 minutes.


## 3.3.1. Effect of Sodium Hydroxide Concentration

Maximum absorption was obtained when 1.6 mL of 1 M NaOH was used. Over this volume, no deliberate change in absorbance could be detected. So, 1.6 mL of 1 M of NaOH was used as an optimum value as shown in Figure6.Figure 6
Effect of sodium hydroxide concentration (1 M) on the reaction between eperisone hydrochloride (20μg/mL) and alkaline potassium permanganate.


## 3.3.2. Effect of Temperature

At room temperature, the reaction rate increases substantially with time. Moreover, heating the solution was found to increase the rate of the reaction but caused precipitation of MnO2 also. Therefore, room temperature was selected as the optimum temperature.


## 3.3.3. Stoichiometry and Reaction Mechanism [13, 14]

The stoichiometric ratio between potassium permanganate and eperisone hydrochloride was determined by Job’s method and was found to be 4 : 1. Corrected Job’s plot is shown in Figure7. Eperisone hydrochloride was found to be susceptible for oxidation with alkaline potassium permanganate producing a green color at λmax of 603.5 nm.Figure 7
Job’s plot of continuous variation between potassium permanganate and eperisone hydrochloride.


## 3.3.4. Kinetics of the Reaction

Under the optimum conditions, the absorbance time curves of eperisone hydrochloride with potassium permanganate reagent were constructed (as shown in Figures8 and 9). The initial rate of the reaction was determined from the slope of tangents of the absorption time curves. The order of the reaction with respect to permanganate was determined by studying the reaction at different concentrations of permanganate with fixed concentration of investigated eperisone hydrochloride. The plot of initial rate (ΔA/Δt) against initial absorbance was linear passing through origin indicating that the initial order of the reaction with respect to permanganate was 1 as shown in Figure 10.Figure 8
Representing change in absorbance with respect to time as recorded by UV in Kinetics mode.Figure 9
Absorption versus time for the reaction between eperisone hydrochloride (6.76 × 10−5M) and KMnO4 (0.3 to 1.8 × 10−3M).Figure 10
Plot of initial rate (dA/dt, where t=300 s) versus initial absorbance A.The order with respect to investigated eperisone hydrochloride was evaluated by measuring the rate of the reaction at several concentrations of eperisone hydrochloride at a fixed concentration of permanganate reagent as shown in Figure11. This was done by plotting the logarithm of initial rate of the reaction versus logarithm of molar concentration of investigated eperisone hydrochloride and was found to be approximately 1 as shown in Figure 12. However, under the optimized experimental conditions, the concentrations of eperisone hydrochloride were determined using relative excess amount of potassium permanganate and sodium hydroxide solutions. Therefore, pseudo-zero-order conditions were obtained with respect to their concentrations.Figure 11
Absorption versus time for the reaction between eperisone hydrochloride (5.07 to 11.8 × 10−5 M) and KMnO4 (1.2 × 10−4 M).Figure 12
Plot of log initial rate (dA/dt, where t=300 s) versus Log concentration.


## 3.3.5. Quantitation by Kinetic Methods

(1)  Initial Rate Method. Initial rate of reaction would follow pseudo-first-order and were found to obey (1):
(1)V=dAdt=K′Cn,
where V is the reaction rate, A is absorbance, t is the measuring time, K′ is the pseudo-first-order rate constant, C is the molar concentration, and n is the order of the reaction. The logarithmic form of the reaction is shown in (2):
(2)logv=logΔAΔt=logK′+nlogC.
Regression analysis was performed using the method of least square to evaluate the slopes, intercepts, and correlation coefficients. The analytical parameters and results of regression analysis are given in Table 2. The value of n(≈1) in the regression equation confirmed that the reaction of eperisone hydrochloride with the potassium permanganate was a pseudo-first-order with respect to eperisone hydrochloride concentration. The limits of detection (LOD) were calculated, and results obtained confirmed good sensitivity of the proposed method and consequently their capabilities to determine low amount of eperisone hydrochloride.Table 2
Data representing analytical parameters for the initial rate method for determination of eperisone hydrochloride with alkaline potassium permanganate (Δt=300 sec).
Method
Linearity (μg/mL)
LogK
Slope(n)
r
2
LOD (μg/mL)
LOQ (μg/mL)
Initial rate
15–30
0.998
0.808
0.998
0.46
1.41(2)  Fixed Time Method. In this method, the absorbance of the reaction solution containing varying amounts of eperisone HCl was measured at preselected fixed time. Calibration curve of absorbance versus the concentration of the drug at fixed time was plotted (shown in Figure 13). The regression equation, correlation coefficients, and detection limits were calculated and reported in Table 3. The lowest detection limit was obtained at fixed time of 6 minutes. However the fixed time of 3 minutes showed a wider concentration range for quantification.Table 3
Analytical parameters for the fixed time method for determination of eperisone hydrochloride with alkaline potassium permanganate.
Reaction time (sec)
Linearity range (μg/mL)
Intercept
S.D.
Slope
S.D.
r
2
LOD (μg/mL)
LOQ (μg/mL)
180
15–35
0.035
0.01
0.029
0.00
0.998
0.65
1.99
360
15–30
0.105
0.00
0.033
0.00
0.999
0.15
0.46
540
15–25
0.11
0.02
0.035
0.00
0.998
1.81
5.49
720
15–20
0.07
0.02
0.087
0.01
1
0.79
2.40
900
15–20
0.1
0.01
0.033
0.00
1
1.15
3.49Figure 13
Calibration plot of absorbance versus the concentration of eperisone hydrochloride at preselected fixed time of 3 minutes.


## 3.4. Validation of Proposed Methods [15, 16]

### 3.4.1. Specificity

Specificity of the UV method was evaluated by addition of common tablet excipients available in the laboratory and recording the UV scan. No interference was detected from the added excipients at 261.40 nm, and the variations in absorbance were negligible.In case of kinetic spectroscopic method, both the proposed methods were performed in visible range away from the UV-absorption region. A very minor change in the absorbance values was seen during the time duration.


### 3.4.2. Linearity

Linearity data are studied for both methods and reported in Tables1, 2, and 3.


### 3.4.3. Accuracy

Accuracy studies in terms of percentage recovery (percentage) were studied for normal UV and kinetic spectroscopic method and reported in Tables4, 5, and 6. The results of mean % recovery were within the range of 98–102%, and % RSD was less than 2% so the proposed method was found to be accurate.Table 4
Result of accuracy studies by normal UV method.
Formulation
Amount taken (μg/mL)
Amount added (μg/mL)
Amount found± S.D.
% recovery ± S.D.
Mean % recovery± S.D.
RAPISONE
5
3
8.14
±
0.0
101.8
±
0.0
100.62
±
1.02
5
5
9.99
±
0.0
99.95
±
0.0
5
7
12.01
±
.
00
100.11
±
0.0Table 5
Data representing evaluation of accuracy of the analytical procedure using initial rate method.
S. no.
Amount of tablet soln added (μg/mL)
Amount of std. soln added (μg/mL)
LogdA/dt  (mean)
Amount found(μg/mL)
% recovery
Mean %recovery ± S.D.
% RSD
(
1
)
10
10
−2.851
19.98
99.92
100.54
±
0.77
0.76
(
2
)
10
15
−2.872
25.07
100.29
(
3
)
10
20
−2.775
30.42
101.41Table 6
Data representing evaluation of accuracy of the analytical procedure using fixed time method.
S. no.
Amount of tablet soln added (μg/mL)
Amount of std. soln added (μg/mL)
Mean Abs.
Amount found (μg/mL)
% recovery
Mean % recovery ± S.D.
% RSD
(
1
)
10
10
0.628
19.88
99.41
99.22
±
0.16
0.16
(
2
)
10
15
0.753
24.78
99.12
(
3
)
10
20
0.892
29.74
99.14


### 3.4.4. Precision

The developed methods were subjected to precision studies. The analytical techniques demonstrated good precision, and the data is given in Tables7, 8, and 9.Table 7
Intraday and Interday precision for normal UV method (n=6).
Amount taken(μg/mL)
Intra-day precision
Inter-day precision
% assay ± S.D.
% assay ± S.D.
8
100.82
±
0.77
99.96
±
1.06
10
100.63
±
0.77
100.2
±
0.91
12
100.55
±
1.71
100.52
±
0.98
Mean % assay ± S.D.
100.67
±
0.14
100.22
±
0.28
% RSD
0.140
0.285Table 8
Data showing intraday precision analysis for initial rate method and fixed time method.
Conc. (μg/mL)
Initial rate method
Fixed time method
Mean± S.D.
% RSD
Mean± S.D.
% RSD
20
20.024
±
0.04
0.211
19.89
±
0.18
0.92
25
25.0
±
0
0
24.78
±
0.19
0.76
30
30.01
±
0.01
0.033
29.89
±
0.19
0.64
Mean % RSD
0.081
0.777Table 9
Data showing interday precision analysis for initial rate method and fixed time method.
Conc. (μg/mL)
Initial rate method
Fixed time method
Mean± S.D.
% RSD
Mean± S.D.
% RSD
20
20.02
±
0.015
0.076
19.83
±
0
.
16
0.805
25
25.06
±
0.06
0.246
24.76
±
0
.
20
0.829
30
30.07
±
0.06
0.220
29.65
±
0.35
1.180
Mean % RSD
0.180
0.938


### 3.4.5. Detection and Quantification Limits

The values are reported in Tables1, 2, and 3.


## 3.4.1. Specificity

Specificity of the UV method was evaluated by addition of common tablet excipients available in the laboratory and recording the UV scan. No interference was detected from the added excipients at 261.40 nm, and the variations in absorbance were negligible.In case of kinetic spectroscopic method, both the proposed methods were performed in visible range away from the UV-absorption region. A very minor change in the absorbance values was seen during the time duration.


## 3.4.2. Linearity

Linearity data are studied for both methods and reported in Tables1, 2, and 3.


## 3.4.3. Accuracy

Accuracy studies in terms of percentage recovery (percentage) were studied for normal UV and kinetic spectroscopic method and reported in Tables4, 5, and 6. The results of mean % recovery were within the range of 98–102%, and % RSD was less than 2% so the proposed method was found to be accurate.Table 4
Result of accuracy studies by normal UV method.
Formulation
Amount taken (μg/mL)
Amount added (μg/mL)
Amount found± S.D.
% recovery ± S.D.
Mean % recovery± S.D.
RAPISONE
5
3
8.14
±
0.0
101.8
±
0.0
100.62
±
1.02
5
5
9.99
±
0.0
99.95
±
0.0
5
7
12.01
±
.
00
100.11
±
0.0Table 5
Data representing evaluation of accuracy of the analytical procedure using initial rate method.
S. no.
Amount of tablet soln added (μg/mL)
Amount of std. soln added (μg/mL)
LogdA/dt  (mean)
Amount found(μg/mL)
% recovery
Mean %recovery ± S.D.
% RSD
(
1
)
10
10
−2.851
19.98
99.92
100.54
±
0.77
0.76
(
2
)
10
15
−2.872
25.07
100.29
(
3
)
10
20
−2.775
30.42
101.41Table 6
Data representing evaluation of accuracy of the analytical procedure using fixed time method.
S. no.
Amount of tablet soln added (μg/mL)
Amount of std. soln added (μg/mL)
Mean Abs.
Amount found (μg/mL)
% recovery
Mean % recovery ± S.D.
% RSD
(
1
)
10
10
0.628
19.88
99.41
99.22
±
0.16
0.16
(
2
)
10
15
0.753
24.78
99.12
(
3
)
10
20
0.892
29.74
99.14


## 3.4.4. Precision

The developed methods were subjected to precision studies. The analytical techniques demonstrated good precision, and the data is given in Tables7, 8, and 9.Table 7
Intraday and Interday precision for normal UV method (n=6).
Amount taken(μg/mL)
Intra-day precision
Inter-day precision
% assay ± S.D.
% assay ± S.D.
8
100.82
±
0.77
99.96
±
1.06
10
100.63
±
0.77
100.2
±
0.91
12
100.55
±
1.71
100.52
±
0.98
Mean % assay ± S.D.
100.67
±
0.14
100.22
±
0.28
% RSD
0.140
0.285Table 8
Data showing intraday precision analysis for initial rate method and fixed time method.
Conc. (μg/mL)
Initial rate method
Fixed time method
Mean± S.D.
% RSD
Mean± S.D.
% RSD
20
20.024
±
0.04
0.211
19.89
±
0.18
0.92
25
25.0
±
0
0
24.78
±
0.19
0.76
30
30.01
±
0.01
0.033
29.89
±
0.19
0.64
Mean % RSD
0.081
0.777Table 9
Data showing interday precision analysis for initial rate method and fixed time method.
Conc. (μg/mL)
Initial rate method
Fixed time method
Mean± S.D.
% RSD
Mean± S.D.
% RSD
20
20.02
±
0.015
0.076
19.83
±
0
.
16
0.805
25
25.06
±
0.06
0.246
24.76
±
0
.
20
0.829
30
30.07
±
0.06
0.220
29.65
±
0.35
1.180
Mean % RSD
0.180
0.938


## 3.4.5. Detection and Quantification Limits

The values are reported in Tables1, 2, and 3.


## 4. Application of the Proposed Method

The developed methods were applied for the determination of eperisone HCl on pharmaceutical tablet dosage form. The tablet content was computed from its corresponding regression equations. The obtained mean recovery values of the labeled amounts were 100.4% ± 0.07, 99.93% ± 0.05, and 99.41% ± 0.04 as reported in Table10.Table 10
Estimation of eperisone hydrochloride in tablet dosage form.
Brand name
Label claim (mg)
Conc. found (mg)
Conc. found (mg)(initial rate)
Conc. found (mg)(fixed time)
RAPISONE
50 mg
10.04
20.03
19.94
9.94
19.93
19.84
10.14
19.98
19.87
Mean
10.04
19.98
19.88
% assay
100.4
99.93
99.42


## 5. Statistical Treatment of Data [17, 18]

The accuracy and precision of the kinetic methods (initial rate and fixed time) were statistically compared with that of UV spectrophotometric method. Statistical comparison was done by applying Student’st-test and ANOVA. There was no significant difference found between the calculated and theoretical values at 95% confidence level as reported in Tables 11, 12, and 13. This indicated the similarities between the proposed and the reference method for the determination of eperisone HCl in the tablet dosage form.Table 11
Results oft-test applied for recovery of drugs to compare initial rate method  and fixed time method with normal UV method.
S. no.
Tablet name
Initial rate method
UV method
Fixed time method
UV method
(
1
)
RAPISONE
99.93
101.8
99.42
101.8
(
2
)
100.29
99.95
99.12
99.95
(
3
)
101.41
100.11
99.15
100.11
Mean
100.54
100.62
99.22
100.62
Df
2
2
t
Stat
0.081
2.79
t
Critical two-tail
4.302
4.302Table 12
Results oft-test applied for precision data of drugs to compare both the methods.
S. no.
Tablet name
Initial rate method
UV method
Fixed time method
UV method
(
1
)
RAPISONE
100.08
101.2
100
101.2
(
2
)
100.22
100.3
98.41
100.3
(
3
)
100.23
98.33
100
98.33
Mean
100.18
99.94
99.47
99.94
Df
2
2
t
Stat
−0.268
0.434
t
Critical two-tail
4.302
4.302Table 13
Data representing analytical treatment of assay results.
Tablet name
Statistical tool
Method
F (ANOVA)
F (Crit)
Initial rate
Fixed time
Reference
RAPISONE
% Assay
99.93
99.42
100.04
0.108
5.143
t
stat
0.878
0.750
t
crit
4.302
4.302
F stat
0.003
0.002
F crit
0.052
0.052


## 6. Conclusion

In the present study, two spectroscopic methods (UV and kinetic spectroscopy) were developed, validated, and statistically compared for determining eperisone HCl in API as well as in tablet dosage forms. Normal UV method proved to be a simple and cost-effective method for the estimation of the drug. Kinetic spectroscopic method has been used in the past for the elemental analysis and detection of biomolecules in biological fluids (like enzymes). The applicability of this method has been successfully studied and extended in this research. The developed method involves measurements in the visible region and is more selective than the reported UV based methods. Further, the developed methods utilized doubly distilled water as solvent which is easily available, safe, and economical as well. The initial rate and fixed time methods can be easily applied as they do not require elaborate samples and/or tedious procedures for the analysis. Besides, both methods are sensitive enough for the analysis of lower amount of the drug. Furthermore, the developed methods do not require expensive instruments and/or critical analytical reagents. Statistical comparison of the two developed methods proved that there was no significant difference in the accuracy, precision, and quantitative capabilities. These advantages give the proposed methods a great value to the spectroscopic analysis of eperisone HCl in quality control laboratories.



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