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Research Article CODEN: IJPRNK Impact Factor: 5.567 ISSN: 2277-8713 Satyajit P. Sathe, IJPRBS, 2017; Volume 6(4): 93-118 IJPRBS

Available Online at www.ijprbs.com 93

METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS

ESTIMATION OF PARACETAMOL AND EPERISONE HYDROCHLORIDE IN

PHARMACEUTICAL FORMULATION BY UV SPECTROSCOPIC METHOD”

MR. SATYAJIT P. SATHE1, DR. RAJSHEKAR M. CHIMKODE1, MRS. ARATI R. KAPASE1, MR.

AJINKYA B. CHAVAN1, MR. SACHIN R. PATIL2

1. Sant Gajanan Maharaj College of Pharmacy, Mahagaon.

2. Sant Gajanan Maharaj Rural Pharmacy College, Mahagaon.

Accepted Date: 16/07/2017; Published Date: 27/08/2017

Abstract: A new, straightforward, easy, exact, precise, reproducible and Cost effective simultaneous equation method was developed and validated for simultaneous estimation of Paracetamol and Eperisone Hydrochloride in pure and pharmaceutical dosage form. The method was based on the measurement of absorbance at two wavelengths 245 nm and 255 nm, 𝜆 max of Paracetamol and Eperisone Hydrochloride in Methanol correspondingly. Calibration curves of PAR and EPE were found to be linear in the concentration ranges of 2-20 𝜇g/mL respectively, with their correlation coefficient values (R2) 0.999. The Absorptivity values λ1 and λ2 were found to be 1111.88 and 254.11 for PAR and 265.96 and 1140.86 for EPE at 245.0 nm & 255.0 nm respectively. LOD and LOQ were 0.49𝜇g/mL & 0.14𝜇g/mL and 0.059𝜇g/mL & 0.18𝜇g/mL for Paracetamol at 245nm & 255 nm respectively. 0.075𝜇g/mL & 0.22 g/mL and 0.018𝜇g/mL & 0.054𝜇g/mL for Eperisone Hydrochloride at 245nm & 255 nm respectively. In the precision study, the % RSD value was found within limits (RSD < 2%).The percentage recovery at various concentration levels varied from 99.60 ± 16 for PAR and 99.51 ± 0.78 for EPE confirming that the projected method is accurate. It could be concluded from the results obtained in the present investigation that this method for simultaneous estimation of Paracetamol and Eperisone Hydrochloride was simple, accurate, exact, and Cost effective & successfully applied in pure and tablet dosage form.

Keywords: Paracetamol, Eperisone Hydrochloride, Simultaneous Equation Method, UV Spectrophotometric method, 𝜆 max, ICH.

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INTRODUCTION

Introduction to UV Spectroscopy:

Spectroscopy is the study of the interaction between substance and energy of radiation.

Ultraviolet-visible spectroscopy UV refers to absorption spectroscopy or reflectance

spectroscopy in the ultraviolet-visible spectral region. In this region of UV the molecules

undergo electronic transitions under the influence of electromagnetic field. This method is

corresponding to fluorescence spectroscopy in that in which fluorescence observed due to

transitions of electron from the excited state to the ground state, while absorption measures

transitions from the ground state to the excited state.

The pharmaceuticals preparations are nothing but type of chemicals and therefore not an

exception from the use of UV Visible spectroscopy for their qualitative as well as quantitative

studies. The most pharmaceutical formulations contain drugs which is a combination of one or

more drug. Hence these combinations and their simultaneous determination is an significant

part of apprehension for a pharmaceutical analyst. Absorption of light by solution of drug is one

of the oldest and still become one of the most powerful and useful instrumental methods. The

wavelength of light absorb by drug to that compound will absorb its characteristic of its

chemical structure.

Specific regions of the electromagnetic spectrum are absorbed by exciting specific types of

molecular structure, its functional group, characteristics presence of covalent, double, triple

bond and atomic motion to higher energy levels. Absorption of microwave radiation is possible

because of excitation of rotational motion molecule. Absorption of visible and UV radiation is

linked with excitation of electrons, in atoms and in molecules, to high energy states. The

molecules containing conjugated electron in their outer shell light in the UV visible region is

sufficient. If the amount of conjugation increases, the spectrum shifts to lower energy because

absorption spectra are characteristic of molecular structure which can be used to qualitative

and quantitative identification of atomic and molecular species.

Principle:

The UV-Spectrophotometry consists of a light source is usually a hydrogen or deuterium lamp

and junction of source for UV measurements and a tungsten lamp for visible spectra

measurements. The wavelengths of continuous light sources are select with a wavelength

separator such as a Prism or Grating Monochromatic. Spectra are obtained by scanning the

wavelength Separator and Quantitative Measurements can be finished from a spectrum or at a

single and one assigned wavelength.



The amount of light I, Transmitted through a solution of an absorbing chemical in a transparent

solvent can be related to its concentration by Beers Law:

-log I/I0 = A = ελbc……… (1.1)

Where,

I0 = the incident light intensity.

A = the absorbance (also referred to as the optical density)

b = the cell path length in cm

C = Concentration of solution in moles/liter and

ελ = is the molar absorptivity.

Schematic Diagram of Double Beam UV-VIS Spectrophotometer

The spectrophotometric assay of drugs infrequently involves the measurement of absorbance

of samples containing only one absorbing component. The pharmaceutical analyst most

commonly faces the state of affairs where the concentration of one or more substances is

requisite in samples which potentially interfere in the assay.

All the spectrophotometric techniques for single or multicomponent mixture of drug samples is

the property that at all wavelengths:

The absorbance of a solution = Sum of absorbance of the individual components



Or

The measured absorbance is the difference between the total absorbance of the

solution in the sample cell and that of the solution in the reference cell.

There are various spectrophotometric methods are accessible which can be used for the

analysis of single or multi combination of samples. Following methods can be used:

Simultaneous Equation Method

Derivative Spectroscopic Method

Dual Wavelength Method

Absorbance Ratio Method (Q-Absorption Method)

Area Under Curve Method

Difference Spectroscopic Method

Simultaneous equations method

If a sample contains two soluble drugs (X and Y) one of them which absorbs at the λmax of the

other (as shown in figure λ1and λ2), it may be possible to determine both drugs by simultaneous

equation.

In this method, the absorbances of the solutions are measured at the λmax of both the drugs.

The criteria are that the ratios [(A2/A1) / (ax2/ax1)] and [(ay2/ay1) / (A2/A1)] should lie outside the

range 0.1-2.0 Concentration of both drugs is calculated by solving the simultaneous equations 1

& 2.

Cx = (A2 aY1 – A1 aY2) / (aY1 aX2 - aY2 aX1) ----- (1.2)

Cy = (A1 aX2 - A2 aX1) / (aY1 aX2 - aY2 aX1) ------ (1.3)

Where,

A1, A2= Absorbances of mixture at λ1 & λ2 respectively,

ax1 = Absorpitivity of first drug at λ1,

ax2 = Absorpitivity of first drug at λ2,

ay1 = Absorpitivity of second drug at λ1,



ay2 = Absorpitivity of second drug at λ2.

Overlain UV absorption spectra of two drugs

Introduction to Text & Methodology of validation of analytical procedures:

ICH had provide the guideline for validation of analytical procedure under the title of

“Validation of Analytical Procedures: Text & Methodology” in the section Q2B (R1). This

document provides the characteristics for consideration of validation of the analytical

procedures. The aim of validation of an analytical measure is to make obvious that it is

appropriate for its planned purpose.

The four most common types of analytical procedures:

Identification test

Quantitative tests for impurity

Limit test for the control of impurity

Quantitative tests of the API in samples



Method and Validation Parameters as per ICH and USP.

Treatment of muscle spasm

Mechanism of Action

Chemical Class Example

Peripherally acting Muscle Relaxant

Depolarizing agent Curare alkaloids Tubocurarine, Dimethyltubocurarine

Non Depolarizing agent

4-Ammonium agents Atracurium, Cisatracurium, Gallamine

ACH Release Inhibitor

Choline derivative Succinylcholine

Centrally acting Muscle Relaxant

Carbamic esters Meprobamate, Methocarbamol, Tybamate

Benzodiazepines Diazepam, Lorazepam, Nitrazepam

Anticholinergics Orphenadrine

Method

Validation

Precision

Accuracy

Limit of Detection

Limit of Quantification

Specificity

Linearity

Robustness

Ruggedness



Piperidine derivative

Eperisone, Tolperisone

Others Thiocolchicoside, Quinine, Baclofen

Directly acting Muscle Relaxant

Dantrolene

Analgesics Pyroxicam, Lornoxicam, Diclofenac, Aceclofenac, Ibuprofen

DRUG PROFILE

Drug Profile of Paracetamol:

Structure of Paracetamol

IUPAC Name: N-(4-hydroxyphenyl) ethanamide or N-(4-hydroxyphenyl) acetamide

Molecular Formula: C8H9NO2 or HOC6H4NHCOCH3

Molecular Mass: 151.163 g/mol

Physical Properties: [48]

Nature: Colorless Crystalline Powder.

Odour : Odorless

Taste: Bitter Taste

Solubility:

Very slightly soluble in cold water,

https://pubchem.ncbi.nlm.nih.gov/compound/water



Soluble in boiling water, Freely soluble in alcohol;

Slightly soluble in ether; Soluble in Methanol, Ethanol, Diethyl formamide, Ethylene

dichloride, Acetone, ethyl acetate.

Slightly soluble in Ether.

Practically insoluble in petroleum ether, pentane, benzene.

Melting point: 1690C

Boiling Point: 4200C

Mechanism of Action:

The mechanism of action of Paracetamol which is not completely understands by scientist. As

like NSAID’s does inhibit cyclooxygenase (COX) enzyme outside the CNS.

Basically paracetamol is act in the CNS by increasing the pain threshold by inhibiting both

different forms of cyclooxygenase enzyme COX-1, COX-2, and COX-3 involved in prostaglandin

(PG) synthesis. Paracetamol indirectly block the action of COX and this blockage is unsuccessful

in the occurrence of peroxide.

The antipyretic action of paracetamol is produced due to direct effect on the heat regulating

center of the hypothalamus below the pituitary gland.

The analgesic effect is produced by inhibitory action on spinal nitric oxide and serotonin

pathways. Its action is also produced by blocking nerve endings. [49]

Pharmacodynamics of Paracetamol:

Paracetamol is a synthetic drug which is commonly used as analgesic and antipyretic. It has

same therapeutic effect as that of salicylates. Paracetamol is a NSAID drug with powerful

antipyretic and analgesic activity with low anti-inflammatory action. When it is taken orally it

reduces level of prostaglandin. [50]

Pharmacokinetics of Paracetamol:

Pharmacokinetics of Paracetamol.

Sr. No. Pharmacokinetic parameter Normal values

1 Absorption In duodenum non ionized form

https://pubchem.ncbi.nlm.nih.gov/compound/water

https://pubchem.ncbi.nlm.nih.gov/compound/pentane

https://pubchem.ncbi.nlm.nih.gov/compound/benzene



2 Distribution ratio 50 L

3 Metabolism site Liver

4 Elimination 0.24 – 0.98

5 Bioavailability of drug 60-70% (Dose dependable)

6 Half-life of the drugs 1-3 hrs

7 Excretion Renal Excretion

Drug Profile of Eperisone Hydrochloride:

Structure of Eperisone Hydrochloride.

IUPAC Name: (2RS)-1-(4-ehtylphenyl-2-methyl-3-(1-piperidyl) propan-1-one

Molecular Formula: C17H25NO

Molecular Mass: 259.387 g/mol

Physical Properties: [51]

Nature: White solid

Solubility: Methanol

Melting point: 170-1720C



Action: Eperisone is an Antispasmodic drug which relaxes both skeletal muscles and

vascular smooth muscles, and demonstrates a variety of effects such as reduction of

myotonia, improvement of circulation, and suppression of the pain reflex.

Mechanism of Action:

Eperisone produces its action at the level of spinal cord. It blocks sodium and calcium channels.

Eperisone exerts its spinal reflex inhibitory action mostly via a pre synaptic inhibition of

the transmitter release. It starts from the major afferent endings by combined action on

voltage-gated sodium and calcium channels; Eperisone increases the blood supply towards

skeletal stimulation which produces its local anesthetic effect. Eperisone produces muscle

relaxation by acting on CNS.

Pharmacokinetics of Eperisone hydrochloride:

Pharmacokinetics of Eperisone hydrochloride.

Sr. No. Pharmacokinetic parameter Normal values

1 Absorption At Muscle

2 Distribution ratio 3 L/Kg

3 Metabolism site Liver

4 Elimination 0.33-0.69

5 Bioavailability of drug 77%

6 Half-life of the drugs 1.6 - 1.8 hr.

7 Excretion Renal Excretion

List of the Marketed Formulations:

List of the Marketed Formulations

Brand Name Formulation Composition Company

Enzoril Tablet Eperisone 50 mg Alkem Labs

Myosone Tablet Eperisone 50 mg Macleods



Rapisoine SR Tablet Eperisone 50 mg Abbot

Skelact Tablet Eperisone 50 mg Sun Pharma

Eprisan Tablet Eperisone 50 mg Unichem

Myosone Plus Tablet Eperisone 50 mg + Paracetamol 325 mg

Macleods

MATERIAL & METHODS

Material:

Reagents and materials

Paracetamol (PAR) kindly supplied as a gift samples from Centaur Pharmaceuticals Pvt. Ltd.,

Tivim Industrial Estate, Karaswada, Mhapusa, Goa- 403426, India.

Eperisone (EPE) kindly supplied as a gift samples from Abbot India Ltd., L-18, Verna

Industrial Area, Verna Salcette, Goa- 403722, India.

Methanol AR grade as solvent (Lobal Chemie, Boisar, Palghar, Mumbai, India.)

Apparatus used

A double beam UV-visible Spectrophotometer (Agilent, Carry UV-60, Japan), attached to a

computer software UV probe 2.0, with a spectral width of 2 nm, wavelength accuracy of 0.5

nm and pair of 1 cm matched quartz cells.

Analytical balance (Adarsh, Ambala Cantt.)

Corning volumetric flasks, amber coloured volumetric flasks and pipettes of borosilicate

glass were used in the study.

Preparation of solutions

Preparation of standard stock solution

An accurately weighed PAR and EPE powder (10 mg) were transferred to 100 ml separate

volumetric flasks and dissolved in methanol. The flasks were shaken and volumes were made

up to mark with methanol to give a solution having concentration 100 μg/ml for both of drugs. [40]



Preparation of Sample Solution

Twenty tablets were weighed accurately and powdered. Quantity of the powder equivalent to

0.010 mg PAR & 0.010 mg EPE was transferred in 100 ml volumetric flask separately and

powder was dissolved in 10 ml of methanol with shaking having slight warming temperature to

dissolve drug as completely as possible. Then the volume was adjusted up to mark with

methanol. 1 ml PAR & 1 ml of EPE was taken in 10 ml volumetric flask from the stock solution.

This procedure was again repeated for third time & the volume was adjusted up to the mark

with methanol to get a final concentration of PAR (10 μg/ml) and EPE (10 μg/ml). [40]

Methods

Determination of wavelength

In simultaneous equation method the absorbance of the both drug solutions are measured at

the absorption maxima for determination of λ max of both the drugs. Standard solutions of

both the drugs were scanned in the range of 200 – 400 nm. From the overlay spectra of two

drugs, the absorption maxima (λ max) were selected. [5]

Method Validation

Linearity

Experimental Procedure for Linearity and range:

Calibration curve were plotted over a concentration range of 2-20 μg/ml for PAR and EPE

individually. Accurately measured standard working solution of PAR (0.1, 0.2, 0.4, 0.6, 0.8, 1.0,

1.2, 1.4, 1.6, 1.8, 2.0 ml) and EPE (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 ml) was

pipette out in to a separate series of 10 ml volumetric flask. The volume was adjusted to the

mark with methanol and the absorbance of the solutions was measured at 245.0 nm (λ max of

PAR) and 255.0 nm (λ max of EPE) against methanol as a blank. The calibration curve was

constructed by plotting absorbance Vs concentration.

Precision

Method precision (Repeatability)\

The precision of the method was checked by repeated scanning and measurement of

absorbance of solutions (n = 6) for PAR (10 μg/ml) and EPE (10 μg/ml) without changing the

parameter of the proposed Spectrophotometric method. The results are reported in terms of

relative standard deviation (% RSD)



Intermediate precision (Reproducibility)

The intraday and interday precision of the proposed method was determined by analyzing the

corresponding responses 6 times on the same day and on 3 different days over a period of 1

week for concentrations of standard solutions of PAR and EPE (10 μg/ml for each). The result

was reported in terms of relative standard deviation (% RSD).

Limit of detection (LOD) and quantification (LOQ)

The limit of detection (LOD) and limit of quantification (LOQ) of the method were calculated by

using the following equations.

LOD = 3.3 X σ/S

LOQ = 10 X σ/S

Where, σ = the standard deviation of the response

S = slope of the calibration curve

Accuracy (% Recovery study)

Procedure:

The accuracy of the method was determined by calculating recoveries of PAR and EPE by the

standard addition method. Known amounts of standard solution of PAR and EPE were added at

50 %, 100 % and 150 % levels to pre quantified sample solutions of PAR and EPE. [40]

Determination of Absorbtivity Value

The Absorbtivity value of PAR and EPE at 245.0 nm & 255.0 nm respectively from each solution

was calculated using the following formula and the results were shown in table 8.1 & 8.2

Absorbtivity = Absorbance/ Concentration (gm/ 100 ml)

Determination of Ruggedness:-

Procedure:

The ruggedness of proposed method was evaluated by applying the developed procedures to

assay of 10 μg/ml of PAR & EPE at both 245.0 nm & 255.0 nm using the same instrument by two

different analysts under the same optimized conditions at different days. The obtained results

were found to be reproducible, since there was no significance difference between analysts.



Thus the proposed methods could be considered rugged. The results were shown in

table……………….

Where, N = Slandered deviation of the reponce.

S = Slope of the corresponding calibration curve.

Analysis of Drugs in Tablet Dosage Form

Twenty tablets were weighed and powdered. A powder quantity equivalent to 10 mg PAR and

10 mg EPE was accurately weighed and transferred to volumetric flask of 10 ml capacity. 10 ml

of methanol was transferred to this volumetric flask. The solution was filtered through

whatman filter paper. From this solution 01 ml was transferred to volumetric flask of 10 ml

capacity and Volume was made up to the mark to give 100 μg/ml PAR and EPE. From this

solution 01 ml was transferred to volumetric flask of 10 ml capacity and Volume was made up

to the mark to give a solution containing 10 μg/ml of PAR and EPE. The resulting solution was

analysed by proposed methods. The absorbance of sample solution was measured against

methanol as blank at 245.0 and 255.0 nm for quantification of PAR and EPE. The amount of PAR

and EPE present in the sample solutions were determined by solving the equation 1 and 2

(shown in later section).

RESULTS AND DISCUSSION

Method Development:

In this method, the absorbances of the solutions were measured at the λ max of both the drugs.

The criteria are that the ratios [(A2/A1) / (ax2/ax1)] and [(ay2/ay1) / (A2/A1)] should lie outside the

range 0.1 to 2.0.

For this measurement, the standard solutions of PAR and EPE (100 μg/ml) were scanned

separately in the range of 200-400 nm against ethanol as a blank. Data were recorded at an

interval of 1 nm.

The absorbance and absorptivity values at the particular wavelengths were calculated and

substituted in the following equation to obtain the concentration

Cx = (A2 Ay1 – A1 Ay2) / (Ay1 Ax2 – Ay2 Ax1) ----- (8.1)

Cy = (A1 Ax2 – A2 Ax1) / (Ay1 Ax2 – Ay2 Ax1) ------ (8.2)

Where, A1, A2 = Absorbances of mixture at λ1 & λ2 respectively,

ax1 = Absorpitivity of PAR at 245.0 nm



ax2 = Absorpitivity of PAR at 255.0 nm

ay1= Absorpitivity of EPE at 245.0 nm

ay2 = Absorpitivity of EPE at 255.0 nm.

Table No. 8.1 & Table No. 8.2 indicates the absorptivity value for PAR & EPE for both

wavelengths respectively.

Absorption Spectra of Paracetamol (100 μg/ml each) in Methanol at 245.0 nm

λ max at 245 nm for PAR



Absorption Spectra of Eperisone hydrochloride (100 μg/ml each) in Methanol at 255.0 nm

Overlain Absorption Spectra of Paracetamol & Eperisone hydrochloride (100 μg/ml each) in

Methanol at 245.0 nm & 255.0 nm respectively.

λ max at 255 nm for

EPE

λ max at 245 nm for PAR

λ max at 255 nm for

EPE



Absorbtivity Values for Paracetamol:

The Absorptivity values λ1 and λ2 were found to be 1111.88 and 254.11 for Paracetamol at

245.0 nm & 255.0 nm respectively.

Absorbtivity Values for Eperisone hydrochloride: The Absorptivity values λ1 and λ2 were found

to be 265.96 and 1140.86 for Eperisone hydrochloride at 245.0 nm & 255.0 nm respectively.

Method validation

Linearit: Calibration range was observed in the concentration range of 2-20 μg/ml for PAR and

EPE each. The calibration curves at different wavelengths are shown in Figure. 8.4, 8.5, 8.6 &

8.7.

Calibration Curve of Paracetamol at 245.0 nm in Methanol.

y = 0.1115x - 0.0038R² = 0.9999

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25

Ab

sorb

an

ce

Concentration

Series1

Linear (Series1)



Calibration Curve of Paracetamol at 255.0 nm in Methanol.

Calibration Curve of Eperisone hydrochloride at 245.0 nm in Methanol.

y = 0.025x + 0.0041R² = 0.998

0

0.1

0.2

0.3

0.4

0.5

0.6

0 5 10 15 20 25

Ab

sorb

an

ce

Concentration

Series1

Linear (Series1)

y = 0.026x + 0.0062R² = 0.997

0

0.1

0.2

0.3

0.4

0.5

0.6

0 5 10 15 20 25

Ab

sorb

an

ce

Concentration

Series1

Linear (Series1)



Calibration Curve of Eperisone hydrochloride at 255.0 nm in Methanol.

Precision

Method precision (Repeatability)

The % RSD values of PAR and EPE were found to be 0.046 % and 0.21 % respectively at 245.0

nm. The % RSD value of PAR and EPE were found to be 0.24 % and 0.044 % at 255.0 nm. Low

value of % RSD indicates that proposed method is repeatable.

Intermediate precision (Reproducibility)

The SD & % RSD values of PAR for inter-day were (0.00026 – 0.023 %) and intra-day (0.0011 –

0.10 %) at 245.0 nm.

The SD & % RSD values of PAR for inter-day were (0.00051 – 0.21 %) and intra-day (0.00054 –

0.47 %) at 255.0 nm.

The SD & % RSD values of EPE for inter-day were (0.00042 – 0.16 %) and intra-day were (0.0011

– 0.45 %) at 245.0 nm.

The SD & % RSD values for inter-day were (0.00062 - 0.053 %) and intra- ay were (0.00054 -

0.47 %) at 255.0 nm.

y = 0.1181x - 0.0255R² = 0.9985

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25

Ab

sorb

an

ce

Concetration

Series1

Linear (Series1)



LOD and LOQ

LOD and LOQ values for PAR and EPE were found to be, 0.49 and 0.14 μg/ml and 0.075 and 0.22

μg/ml at 245.0 nm respectively.

While the, LOD and LOQ values for PAR and EPE were found to be 0.059 and 0.18 μg/ml, 0.018

and 0.054 μg/ml at 255.0 nm respectively. Low value of LOD & LOQ indicates that the method is

sensitive.

Accuracy

The mean recoveries were found to be 99.90 ± 0.16 and 99.51 ± 0.78 for PAR and EPE,

respectively. The recoveries results indicate that the proposed method is accurate.

Ruggedness

In the ruggedness study the SD & % RSD values of PAR for Analyst 1 were (0.00063 – 0.057 %)

and for Analyst 2 (0.00078 – 0.070 %) at 245.0 nm.

In the ruggedness study the SD & % RSD values of PAR for Analyst 1 were (0.00046 – 0.18 %)


Low value of Ruggedness indicates that the method is sensitive.

In the ruggedness study the SD & % RSD values of EPE for Analyst 1 were (0.00063 – 0.057 %)


In the ruggedness study the SD & % RSD values of EPE for Analyst 1 were (0.00046 – 0.18 %)

and for Analyst 2 (0.00063 – 0.25 %) at 255.0 nm. Table 8.10

Low value of Ruggedness indicates that the method is sensitive.

Assay

The proposed validated method was successfully applied to determine PAR and EPE in

combined tablet dosage form. Results are given in Table 8.11. No interference of the excipient

with the absorbance of analyte of interest has been seen hence the proposed method is

suitable for the routine analysis of PAR and EPE in combined dosage forms.



Summary Data of Validation Parameter

Parameters PAR EPE

At 245.0 nm At 255.0 nm At 245.0 nm At 255.0 nm

Beer’s Law Limit ((µg/ml)

2-20 µg/ml 2-20 µg/ml 2-20 µg/ml 2-20 µg/ml

Absorptivity 1111.88 254.11 265.96 1140.86

Regression Equation

(y= mx + C)

y = 0.111x -0.003

y = 0.025x + 0.004

y = 0.026x + 0.006

y = 0.118x – 0.025

Slope (m) 0.111 0.025 0.026 0.118

Intercept (c) 0.003 0.004 0.006 0.025

Correlation Coefficient (r2)

0.999 0.998 0.997 0.998

Metod Precision (Repeatability) (% RSD, n=6)

0.046 0.24 0.21 0.044

Interday (n=6)

% RSD

0.023 0.021 0.16 0.053

Intraday (n=6)

% RSD

0.10 0.49 0.45 0.47

LOD ((µg/ml) 0.49 0.059 0.075 0.018

LOQ ((µg/ml) 0.14 0.18 0.22 0.054

Recovery ±

% RSD (n=3)

99.60 ± 0.16 99.51 ± 0.78



Parameters PAR EPE

At 245.0 nm At 255.0 nm At 245.0 nm At 255.0 nm

Ruggedness

(% RSD, n= 6)

Analyst 1 0.057 0.18 0.14 0.029

Analyst 2 0.070 0.25 0.13 0.035

Assay ± S. D.

(n=3)

99.90 ± 0.24 99.98 ± 0.20

SUMMARY & CONCLUSION:

The method described for the simultaneous estimation of Paracetamol and Eperisone

hydrochloride was found to be simple, responsive, correct, reproducible, economical, accurate

and precise procedures for routine simultaneous estimation of two drugs. The values of

standard deviation and % RSD were satisfactorily low and recoveries studies indicate the

reproducibility and accuracy of the method. The developed UV visible spectroscopic methods

were validated for linearity, method precision, intra-day and inter-day precision, limit of

detection, limit of quantification, accuracy, ruggedness. The standard deviation and % RSD were

satisfactorily shows low values which indicates reproducibility of method. The method

development & method validation were performed according to ICH Harmonized Tripertile

guidelines. The result of the analysis of the tablet dosage form by this method is reproducible

and reliable and is in good agreement with label claim of the drugs. The additive present in the

tablet dosage form did not interfere in the analysis. So the method can be used for the routine

analysis of drugs in combined dosage form.

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1. Beckett AH, and Stenlake JB, Practical Pharmaceutical Chemistry, Part II, CBS Publications

and Distributors (1997); 4th edition, Pg. No. 275-300.

2. Chatwal GR and Ananad SK, Instrumental mehod of chemical analysis, Himalya publication

house, (2004); Pg. No. 1.1-1.5.

3. Watson DG, Pharmaeutical analysis, Churchill Livingstone, (2000); Pg. No. 237-276.



4. Saraf S, Various UV spectrophotometric Simultaneous estimation methods, (2006);

www.pharmainfo.net/reviews/various- UV spectrophotometric simultaneous estimation

methods, (Accessed- February 2017).

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