SIMULTANEOUS SPECTROPHOTOMETRIC ESTIMATION OF DICLOFENAC SODIUM AND EPERISONE HYDROCHLORIDE USING ABSORBANCE RATIO METHOD IN CAPSULE DOSAGE FORM

ABOUT AUTHORS:
Lalit F. Raiyani*¹, Dharanant V. Borakhatariya², Bhargav D. Patel³, Kuldip R. Marwada⁴, Dr.Priti D. Trivedi⁵, Mr.Rajendra K. Patel^6
1Parul institute of Pharmacy, Vadodara
²B. K. Modi Government Pharmacy College, Rajkot
³Ramanbhai Patel College of Pharmacy, Changa
⁴R. K. College of Pharmacy, Rajkot
⁵Professor at K. B. Institute of Pharmaceutical Education and Research, Gandhinagar
⁶Lecturer at K. B. Institute of Pharmaceutical Education and Research, Gandhinagar
*lalitraiyani@gmail.com

ABSTRACT:
A simple, rapid, sensitive, precise and accurate UV-spectrophotometric method (absorbance ratio) was developed and validated for simultaneous estimation of Diclofenac sodium and Eperisone hydrochloride in pharmaceutical capsule dosage form. In absorbance ratio method absorbance measurement of sample at 239.2 nm (isoabsorbtive point, λ₁) and 256 nm, λ₂.The absorbance ratio method was developed using methanol as solvent. Developed method is linear between 4-12µg/ml and 5-15µg/ml for diclofenac sodium and eperisone hydrochloride respectively. The mean % recovery was found to be 99.68% & 99.14% for diclofenac sodium and eperisone hydrochloride respectively.

REFERENCE ID: PHARMATUTOR-ART-1754

1. INTRODUCTION
Diclofenac sodium (Sodium [o-(2, 6-dichloroanilino) phenyl] acetate)^[1][Figure 1a] is Nonsteroidal anti-inflammatory drug. It acts as nonselective inhibitors of the enzyme cyclo oxygenase (COX). COX catalyzes the formation of prostaglandins and thromboxane from arachidonic acid (itself derived from the cellular phospholipids bilayer by phospholipase A₂). Prostaglandins act (among other things) as messenger molecules in the process of inflammation^[2].Eperisone hydrochloride[(2RS)-1-(4-Ethylphenyl)-2-methyl-3-piperidin-1-ylpropan-1-one monohydrochloride][Figure 1b]^[3,4]iscentrally acting muscle relaxant drug used for lower back pain (LBP)Its peculiar mechanism of action is believed to blockade both sodium and calcium voltage-gated channels exerting its activity mainly on the spinal cord structures by reducing the gamma-efferent firing and inhibiting the spinal cord activities  Furthermore, eperisone (EPS) have also shown some vasodilator activity with a consequent increase in muscle blood perfusion , and it exerts an antinociceptive effect by antagonistic activity on P substance , Because of its mechanisms of action, EPS is almost devoid of the side effects on CNS that are often reported with other centrally acting muscle relaxants^[5]. Literature survey reveals that only one method is available for the simultaneous estimation of diclofenac sodium and eperisone hydrochloride by UV spectroscopy using simultaneous equation method in synthetic mixture prepared in laboratory^[6].  So aim of present study is to developedsimple, rapid, precise and accurate spectrophotometric method for simultaneous estimation of diclofenac sodium and eperisone hydrochloride in pharmaceutical capsule (Eperisan-d, SR) dosage form without any interference of excipient present in dosage form.

2. EXPERIMENTAL:
2.1 Materials and methods:
Chemical.
Diclofenac sodium was obtained as gift sample from Montage Laboratories, Himatnagar, and Gujarat, India. Eperisone hydrochloride was obtained as gift sample from Sun Pharma Pvt. Ltd., Jammu, India. Methanol AR grade is used as solvent.

Instrumentation.
The dual wavelength Spectrophotometric method was developed using A double beam UV-Visible Spectrophotometer, (SHIMADZU, Japan Model: 1800) having a pair of 10 mm matched quartz cuvettes, which was used to measure absorbance of the resulting solutions, spectra were automatically obtained by UV-Probe system software. An analytical balance (CP 124S, Sartorius, Germany) was used to weigh accurately the standard and test samples, an ultrasonic bath (Fast clean, Toshniwal process instrument Pvt. Ltd., Ajmer, India) were used in study. All the solutions were filtered using whatman filter paper no.41.

Preparation of standard stock solution.
Diclofenac Sodium standard stock solution (sss).
An accurately weighed quantity of Diclofenac Sodium (10 mg) was transferred in 10 mL volumetric flask, dissolved in sufficient quantity of methanol and volume was made up to the mark with methanol (concentration: 1000 µg/mL).

Eperisone hydrochloride standard stock solution (sss).
An  accurately weighed quantity of Eperisone hydrochloride (10 mg) was transferred in 10 mL volumetric flask, dissolved in sufficient quantity of methanol and volume was made up to the mark with methanol (concentration: 1000 µg/mL).

Preparation of working standard solution (wss).
1 ml of each sss was taken into 10 ml volumetric flask separately and diluted up to 10 ml using methanol to get 100 µg/mL (wss) of Diclofenac sodium and eperisone hydrochloride, then suitable dilutions were made to prepare a mixture containing a working range of 4-12 µg/mL of diclofenac sodium and 5-15 µg/mL of eperisone hydrochloride using methanol into 10 mL volumetric flask.

Selection of analytical wave length.
In the quantitative assay of two components in admixture by the absorbance ratio method, absorbances are measured at two wavelengths. One being the λmax of one of the component is λ₂ and other being a wavelength of equal absorption of two component known as isoabsorbtive point λ₁ ^[7].Appropriate dilution were made of each drug and scan the solution in the range of 400 nm to 200 nm. Over lay spectrum of the both drug(Figure 2) shows 239.2 nm is isoabsorbtive point (λ₁), and 256 nm λmax of eperisone (λ₂).

2.2 Assay procedure of Marketed formulationby absorbance ratio method:
An accurately weighed 20 capsule (Diclofenac sodium-100mg+Eperisone hydrochloride-150mg, Eperisan-d SR). Remove the content from capsule and triturate it. Now, equivalent quantity of powder to about 100 mg of diclofenac sodium and 150 mg of eperisone hydrochloride was transferred to 100ml volumetric flask add 70ml of methanol and sonicated for 25 min. The volume was made up to the mark using methanol as solvent. The resulting solution was filtered through Whatman filter paper no.41. Above filtrate was appropriately diluted to get concentration of 100 μg/ml of diclofnac sodium and 150 μg/ml of eperisone hydrochloride. From the above prepared solution, 7 ml was transferred in 10 ml volumetric flask; volume is made up to mark with methanol to get final concentration of Diclofenac sodium (7μg/ml) and eperisone hydrochloride (10.5μg/ml). The absorbance was measured at the selected wavelengths and concentrations were determined.

3. METHOD VALIDATION.^[8]

Linearity and range.
The linearity curve is obtained by plotting specific range of concentration against absorbance obtained. Statically linearity was calculated by regression co efficient.(Figure 3)

Precision.
Precision of the methods was determined by performing method repeatability studies, intraday precision, and interday precision. In repeatability study, mixture of both drugs (7μg/ml diclofenac sodium & 10.5μg/ml eperisone hydrochloride) was analyzed six times. In intraday precision were analyzed three times in a day for 2 hrs time interval. In interday precision were analyzed in three consecutive days.

Limits of detection (LOD) and Limit of quantification (LOQ).
The LOD and LOQ of this method were calculated by using following equations:

Limit of detection:

LOD = 3.3×S.D./S

Limit of Quantification:
LOQ = 10×S.D./S

Where, S.D. = the Standard deviation of the response,