DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF TOLPERISONE HYDROCHLORIDE AND DICLOFENAC SODIUM IN SYNTHETIC MIXTURE

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ABOUT AUTHORS
Satish A. Patel, Kaushik P Hariyani*
Department of Quality Assurance, S. K. Patel College of Pharmaceutical Education and Research,
Ganpat University, Ganpat Vidyanagar – 384012, Mehsana, Gujarat, India.
*hariyanikaushik@gmail.com

ABSTRACT
A simple, sensitive, accurate, precise and rapid reverse phase high performance liquid chromatographic method has been developed and validated for the simultaneous determination of Tolperisone hydrochloride and Diclofenac sodium from synthetic mixture. The chromatographic separation was performed on ACE 5 C18 column (150 mm × 4.6 mm i.d, 5 μm particle size). Mobile phase consisted of a mixture of phosphate buffer pH 6, acetonitrile and methanol in the ratio of 10: 50: 40, v/v/v at a flow rate of 0.7 ml/min. The detection wavelength was set at 267 nm. The proposed method was validated for linearity, accuracy, precision, LOD and LOQ. The calibration was linear over the concentration range of 2-30 μg/ml for Tolperisone hydrochloride and 2-30 μg/ml for Diclofenac sodium. The retention times were found to be 2.1 ± 0.14min for Diclofenac sodium and 4.7 ± 0.13min for Tolperisone hydrochloride. The mean recoveries were 100.5 ± 0.34 and 100.8 ± 0.80 for Tolperisone hydrochloride and Diclofenac sodium, respectively. The method can be easily adopted for quality control analysis.

REFERECNE ID: PHARMATUTOR-ART-1717

INTRODUCTION
Tolperisone (TOL) is chemically, 2-methyl-1-(4-methylphenyl)-3-(1-piperidyl) propan-1-one (Figure 1) is a well known antispasmodic drug1. It is official in Japanese Pharmacopoeia (JP). JP2 describe potentiometric method for its estimation. Literature survey reveals HPLC3 and UV4 methods for estimation of TOL in single dosage form. Literature survey also reveals HPLC5 and UV spectrophotometry6 methods for determination of TOL with other drugs in combination. Diclofenac sodium (DIC) is chemically, 2-[2,6dichlorophenylamino] benzene acetic acid sodium salt7 (Figure 2). Diclofenac sodium (DIC) is official in IPand BP. IP8 and BP9 describes liquid chromatography method for its estimation. Literature survey reveals HPLC10-11 and UV12 methods for determination of DIC in single dosage form. Literature survey also reveals HPLC13-15, UV spectrophotometry16 and HPTLC17 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 TOL and DIC in their combined dosage forms. Literature survey does not reveal any simple chromatographic method for simultaneous estimation of TOL and DIC in synthetic mixture or dosage forms. The present communication describes simple, sensitive, rapid, accurate, precise and cost effective RP-HPLC method for simultaneous estimation of both drugs in their combined synthetic mixture.

MATERIALS AND METHODS

Apparatus
RP-HPLC instrument (Shimadzu, LC-2010CHT, Japan) equipped with a UV-Visible detector and a photodiode array detector, auto sampler, ACE 5 C18 column (150 x 4.6 mm, 5 µ particle size) was used. Chromatograms were automatically obtained by LC-Solution system software. A Sartorius CP224S analytical balance (Gottingen, Germany), an ultrasonic bath (Frontline FS 4, Mumbai, India), Nylon 0.45 µm – 0.47 mm membrane filter (Gelman Laboratory, Mumbai, India), Whatman filter paper no. 41 (Millipore, USA), Digital pH meter (LI 712 pH analyzer, Elico Ltd., Ahemedabad) were used in the study.

Reagent and materials
TOL and DIC bulk powder was kindly gifted by Torrent Research Centre, Gandhinagar, India and Acme Pharmaceuticals Ltd., Ahmedabad, Gujarat, India respectively.HPLC grade methanol (Merck Ltd., Mumbai, India), HPLC grade acetonitrile (Finar Chemicals Ltd.,Mumbai, India), NaH2PO4 and Na2HPO4 (S. D. Fine Chemicals Ltd., Mumbai, India)were used in the study. Water for RP-HPLC was prepared by triple glass distillation and filtered through a nylon 0.45 µm – 47 mm membrane filter.

Preparation of phosphate buffer solution (pH 6.0)
Phosphate buffer (pH 6) was prepared by dissolving 1.56 g sodium dihydrogen ortho phosphate and 0.35 g disodium hydrogen phosphate in 1000 ml HPLC-grade water.

Preparation of standard stock solutions
An accurately weighed standard TOL and DIC powder (10 mg) were weighed and 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 containing 100 μg/ml of each TOL and DIC.

Preparation of synthetic mixture
Tolperisone (150 mg) and Diclofenac (50 mg) were taken and then both the drug were mixed with routinely used excipients like starch, lactose, magnesium stearate, and Talc in suitable proportion. Total 500 mg of mixture was prepared and used as synthetic mixture.

Methodology
To optimize the RP-HPLC parameters, several mobile phase compositions were tried. A satisfactory separation and good peak symmetry for TOL and DIC was obtained with a mobile phase consisting of phosphate buffer (pH 6): acetonitrile: methanol (10: 50: 40, v/v/v)at a flow rate 0.7 ml/min to get better reproducibility and repeatability. Quantification was carried out at 267 nm based on peak area. Complete resolution of the peaks with clear baseline was obtained (Figure 3). System suitability test parameters for TOL and DIC for the proposed method are reported in Table 1. Overlain UV spectrum showed that both drugs showed good absorbance at 267 nm, hence the wavelength of 267 nm was selected for quantification of TOL and DIC (Figure 4). 

Table 1: System suitability parameters of chromatogram

Parameters

TOL ± RSD

(n = 6)

DIC ± RSD

(n = 6)

Retention time (min)

5.748 ± 0.14

2.194 ± 0.13

Tailing factor

1.362 ± 0.67

1.115 ± 0.54

Theoretical plates

      4529 ± 0.46

2166 ± 0.79

Resolution

3.665 ± 0.63

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