1. INTRODUCTION:

Eperisone (EPE) is chemically 4-ethyl-2-methyle-3-piperidinopropiopenone (Figure 1) [2] is a well known antispasmodic drugand is official in Japanese Pharmacopoeia (JP). JP [1] describes potentiometric method for its estimation. Literature survey reveals use of HPLC/MS, GC/MS, NMR, UV and IR to Identify Degredation product of eperisone hydrochloride in the tablets[10]. Literature survey also reveals UV spectrophotometry [11] and HPLC [12] method for the determination of EPE with other drugs in combination. Diclofenac sodium (DIC) is chemically 2-[2,6dichlorophenylamino] benzene acetic acid sodium salt(Figure 2) [3].

Diclofenac sodium (DIC) is official in IPand BP. IP[3] and BP[4] describes liquid chromatography method for its estimation. Literature survey reveals UV spectrophoto-metric[13], HPLC [14] and HPTLC[15] method for the determination of DIC with other drugs in combination. The combination of these two drugs is not official in any pharmacopoeia; hence no official method is available for the simultaneous estimation of EPE and DIC in their combined dosage forms. Literature survey reveal spectrophotometric method for simultaneous estimation of EPE and DIC in synthetic mixture[16]but no RP-HPLC method for dosage form. The present communication describes simple, sensitive, rapid, accurate, precise and cost effective spectrophotometric method based on Vierodts Method for simultaneous estimation of both drugs in their combined dosage form.

Figure 1:Structure of Eperisone Hydrochloride

Figure 2:Structure of Diclofenac sodium

2. EXPERIMENTAL WORK:

2.1. Apparatus:

A SYSTRONICS 119 spectrophotometer and Shimadzu 1609 spectrophotometer double beam UV/Visible spectrophotometer with spectral width of 2 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched quartz cell was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV-Probe 2.0 system software. A Wensar PGB 100 analytical balance, an ultrasonic bath (Frontline FS 4) was used in the study.

2.2. Reagents and Materials:

DIC bulk powder was kindly gifted by Dr. Reddys Laboratories, Hyderabad, Andhra Pradesh, India. EPE bulk powder was kindly gifted by Sun Pharmaceuticals Ltd., Mumbai, Distilled Water and Whatman filter paper no. 45 (Millipore, USA) were used in the study. Rapisone D SR capsules containing DIC (100mg) and EPE (150mg) were purchased from local pharmacy.

2.3. Preparation of standard stock solutions:

An accurately weighed quantity of standard EPE (15 mg) and DIC (10 mg) powder were weighed and transferred to 100 mL separate volumetric flasks and dissolved in distilled water. The flasks were shaken and volumes were made up to mark with distilled water to give a solution containing 150 and 100 μg/mL each of EPE and DIC.

2.3.1. Preparation of working standard solutions:

The working standard solutions of EPE and DIC were prepared separately in distilled water having concentration of 15 and10 μg/mL. They were scanned in the wavelength range of 200-400 nm against as distilled water blank. Maximum absorbance was obtained at 255 nm and 277 nm for EPE and DIC, respectively.

2.4. Validation of the proposed method[5-9]:

The proposed method was validated according to the International Conference on Harmonization (ICH) guidelines.

2.4.1. Linearity and range:

The calibration curves were plotted over a concentration range of 2-12 μg/mL for DIC and 3-18 μg/mL for EPE. Accurately measured standard solutions of DIC (0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mL) and EPE (0.3, 0.6, 0.9,1.2, 1.5, 1.8 mL) were transferred to a series of 10 mL of volumetric flasks and diluted to the mark with distilled water. The absorbances of the solutions were measured at 255nm for EPE and 277 nm for DIC against distilled water as blank. The calibration curves were constructed by plotting absorbances versus concentrations and the regression equations were calculated and from range was calculated from calibration graph.

2.4.2. Intra and Inter-Day Precision:

Intra and Inter-Day Precision was determined by analysing corresponding concentrations 10μg/mL for DIC and 15 μg/mL for EPE five times on same day and on five alternate days over a period of 10 days and the results are reported by calculating % RSD of EPE and DIC.

2.4.3. Accuracy:

The accuracy of the method was determined by calculating % recovery of EPE and DIC by analyzing 50, 100 and 150 % level sample solutions of EPE and DIC. Three trials were performed and results are obtained in % mean recovery.

2.4.4. Ruggedness:

Ruggedness performed by both analyst and instrument variation. A concentrations of 10µg/mL of Diclofenac Sodium and 15 µg/mL of Eperisone Hydrochloride were prepared from stock solution and analysed by using two different UV-Visible spectrophotometers namely UV- 1601 (Shimadzu) and UV-119(systronics) and the results are reported in the form of % recovery. The same above concentrations are prepared by two different analyst and the absorbances were recorded both at 255nm for eperisone hydrochloride and 277 nm for diclofenac sodium and the results are reported in the form of % recovery.

2.4.5.Limit of detection and Limit of quantification:

LOD and LOQ values decide about the sensitivity of the method. LOD is the lowest detectable concentration of the analyte, while LOQ is the lowest quantifiable concentration.The LOD and LOQ are calculated from the calibration curve by using the formulas (Eq..1 and Eq..2).

LOD = 3 × σ/S………………………Eq..1

LOQ = 10 × σ/S…………………..… Eq..2

Where, σ = the standard deviation and S = slope of the calibration curve

2.4.6.Analysis of Rapisone D SR capsules by Vierodts method [5,6]:

Take 20 RAPISONE D SR capsules and weigh them(w₁).Now remove the shells of capsules and weigh empty shells(w₂).Calculate the weight of the capsule contents by subtracting the empty shells weight from capsules weight w₃=(w₁-w₂).Crush the contents into powder form. Calculate a weight equivalent to 100mg of Diclofenac Sodium and 150 mg of Eperisone Hydrochloride and take into a 100mL volumetric flask.add 50mL of distilled water and sonicate for 10min.Filter through Whatman filter paper and makeup the volume. Aliqouts were prepared such that the final concentrations are 10µg/mL of Diclofenac Sodium and 15 µg/mL of Eperisone Hydrochloride. Measure the absorbance at both the λ_max(both at 255 nm and 277nm).The amounts of the EPE and DIC present in the sample solution were calculated by using Eq..3 and Eq..4.

Cx = (A2 aY1 – A1 aY2) / (aY1 aX2 - aY2 aX1)………………...Eq..3

Cy = (A1 aX2 - A2 aX1) / (aY1 aX2 - aY2 aX1)………………... Eq..4

A₁ and A₂are absorbances of mixture at 255 nm and 281 nm;

aX₁and aY₁ are absorptivities of EPE and DIC respectively at 255 nm;

aX₂ and aY₂are absorptivities of EPE and DIC respectively at 277 nm.

3. RESULTS AND DISCUSSION:

The standard solutions of EPE and DIC were scanned separately in the UV range and zero-order spectra for EPE and DIC were recorded. Maximum absorbance was obtained at 255 nm and 277 nm for EPE and DIC, respectively. Overlain absorption spectrum of EPE and DIC in distilled water is shown in (Figure 3). Linear correlation was obtained between absorbances and concentrations of EPE and DIC in the concentration ranges of 2-12 µg/mL and 3-18µg/mL, respectively. The % RSD values of EPE and DIC was found to be 1.12 and 0.34% for inter day precision and 0.53 and 0.92 % for intra-day precision respectively. Relative standard deviation was less than 2 %, which indicates that proposed method is precise. LOD values for DIC and EPE were found to be 0.801 and 0.156 µg/mL and LOQ values were found to be 2.428 and 0.475 µg/mL respectively. These data show that method is sensitive for the determination of EPE and DIC. The regression analysis data and summary of validation parameters for the proposed method is summarized in Table 1.The mean % recoveries were 99.3 and 100.3% for DIC and EPE, respectively. The results of recovery studies indicate that the proposed method is highly accurate. The proposed method was analysed by different analysts and by using different systems and the results in % recovery are given in table 2 indicates the method is rugged. The proposed validated method was successfully applied to determine EPE and DIC in their capsule dosage form. The results obtained for EPE and DIC were comparable with the corresponding labeled amounts (Table 3 and 4). No interference of the excipients with the absorbance of interest appeared; hence the proposed method is applicable for the routine simultaneous estimation of EPE and DIC in pharmaceutical dosage forms.

Fig.3.Overlapping Spectra of Diclofenac Sodium and Eperisone Hydrochloride

Fig.4.calibration graph of Eperisone hydrochloride

Fig.5. calibration graph of Diclofenac sodium

Table.1.Representation of Results of Various Parameters of EPE and DIC

Parameter		Eperione Hydrochloride	Diclofenac Sodium
Wavelength		255nm	277nm
Linearity and Range		3-18µg/ml	2-12 µg/ml
Regression equation		y = 0.040x + 0.012	y = 0.007x - 0.002
Correlation coeffiient		0.999	0.999
Precision (%RSD)	Interday	0.34	1.12
Precision (%RSD)	Intraday	0.92	0.53
Accuracy(%Recovery)		100.3%	99.3%
LOD		0.156	0.801
LOQ		0.475	2.428

Table 2.Results of Ruggedness (%Recovery)

Drug	Instrument Variation (%Recovery)		Analyst Variation(%Recovery)
Drug	Systronics	Shimadzu	Analyst 1	Analyst 2
DIC	99.1	99.3	99.1	99.09
EPE	100.4	101	100.4	100.06

Table:3.%Recovery Of Standard Drugs

DRUG

Amount taken

(mg)

Amount recovered (mg)

Mean (%W/W recovery)

Diclofenac sodium

100

99.1

Eperisone hydrochloride

150

151.8

100.4

Table:4 Analysis Of Capsules

Brand Name	Drug	Label Claim (mg)	Amount Recovered (mg)	Mean (%W/W Recovery)
Rapisone DSR Capsules	Diclofenac Sodium	100	99.6	99.6
Rapisone DSR Capsules	Eperisone Hydrochloride	150	150.60	101.2

CONCLUSION:

A Spectrophotometric method was developed for the simultaneous estimation of Diclofenac Sodium and Eperisone Hydrochloride in Rapisone D SR capsules and validated as per ICH guidelines. The developed method utilized distilled water as solvent. The method exhibited good results of linearity, accuracy and precison. The developed method is economical and has been successfully applied to the analysis of dosage form.

ACKNOWLEDGEMENT:

The authors are thankful to Sun Pharmaceutical Ltd, Mumbai, India and Dr. Reddys Laboratories, Hyderabad, India for providing gift sample of EPE and DIC, respectively for carry out the research work. The authors are highly thankful to Nalanda College of Pharmacy, Cherlapally, Nalgonda, Andhra Pradesh, India- 508000 for providing all the facilities to carry out the research work successfully.

REFERENCES:

[1] Japanese Pharmacopoeia, 15th edition, Society of Japanese Pharmacopeia 2006: 618

[2] http://www.chemspider.com/eperisone.drugprofile

[3] Indian Pharmacopoeia, Vol. II, The Controller Publication, Govt. of India, New Delhi 2010: 1199.

[4] British Pharmacopoeia, Vol. I, The British Pharmacopoeia Commission, London 2010: 672.

[5] Skoog, A.D. and West M.D., Principles of instrumental analysis, Saunders golden, Japan, 3^rd ed., 1985, 212-213.

[6] A.H.Beckett,J.B. Stenlake , Practical Pharmaceutical Chemistry, CBS Publishers and distributors, 1997; 4(2), 275-337.

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[8] Michael E, Schartz IS, Krull. Analytical method development and Validation. 2004, 25-46.

[9] The International Conference on Harmonization, Q2 (R1), Validation of Analytical Procedure, Text and Methodology, 2005.

[10] Ding L, Wang X, Yang Z, and Chen Y The use of HPLC/MS, GC/MS, NMR, UV and IR to Identify Degredation product of eperisone hydrochloride in the the tablets, J of pharm and Bio Anal, 2008, 46: 282-287.

[11] Nirav Uchadadiya, Falgun Mehta, Pinak Sanchaniya, “Development and validation of derivative spectrophotometric method for simultaneous estimation of Eperisone hydrochloride and Paracetamol in combined tablet dosage form”,Novus Int J of Chem, 2012, Vol. 1, No. 1, 33-40

[12] Priyanka Patel, Dhara Patel, Hardik Modi, Falguni Patel, Hardiksinh Baria, Sanjay Patel“development and validation of RP-HPLC methods for simultaneous estimation of eperisone hydrochloride and lornoxicam in bulk and their synthetic mixture” Inventi Rapid: Pharm Analysis and Quality Assurance , Vol. 2013 ,724

[13] MR Sengar, SV Gandhi , UP Patil, VS Rajmane “Simultaneous Determination of Diclofenac Sodium and Thiocolchicoside in Fixed Dose Combination by Spectrophotometry” Asian J of Pharm and Cli Res , Vol.3 Issue 2, April‐June 2010 ,pg.no.86-9

[14] Chakraborty Mithun , Ray Chaudhury Dipanjan, Basu Arindam, Das Debabrata and Chakraborty Susanto, “Simultaneous Determination of Paracetamol, Chlorzoxazone and Diclofenac Sodium in Tablet Dosage form by HPLC”, Int J Of Pharm Innovations, Volume 2, Issue 2, pg.no.34-

[15] D. S. Patel, A. D. Captain, P. P. Prajapati, H. G. Shah, “Development and Validation of HPTLC Method for Simultaneous Determination of Tolperisone Hydrochloride and Diclofenac Sodium in Combined Dosage Form” Int J of PharmTechand Res,Vol.5, No.1, pp 147-154

[16] J.Umang Patel, Patel Paresh.U,Patelo Sejal K “Spectrophotometric method for simultaneous estimation of eperisone hydrochloride and diclofenac sodium in synthetic mixture”, Int Res J of pharmacy ,2012,vol.3(9)pg.no.203-206

Received on 03.06.2013 Modified on 20.06.2013

Asian J. Research Chem. 6(7): July 2013; Page 683-686