Satish A. Patel, Kalpesh M. Prajapati*
Department of Quality Assurance, S. K. Patel College of Pharmaceutical Education and Research,
Ganpat University, Ganpat Vidyanagar – 384012, Mehsana, Gujarat, India.

A simple, sensitive, precise, accurate and rapid RP-HPLC method has been developed and validated for the simultaneous determination of Chlorzoxazone 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 (0.02 M KH2PO4, pH adjusted to 3 using orthophosphoric acid), acetonitrile and methanol (30: 30: 40, v/v/v) at a flow rate of 1.0 ml/min. The detection wavelength was set at 279 nm. The proposed method was validated for linearity, accuracy, precision, LOD and LOQ. The calibration curve was linear over the range of 2-50 μg/ml for Chlorzoxazone and 2-50 μg/ml for Diclofenac sodium. The retention times were 2.8 min for Chlorzoxazone and 6.3 min for Diclofenac sodium. The mean recoveries were 101.1 ± 0.47 and 100.8 ± 0.77 for Chlorzoxazone and Diclofenac sodium, respectively. The method has been successfully applied to determine the content of both drugs from the synthetic mixture. Hence, the method can be easily adopted for quality control analysis of both drugs in mixture.


Chlorzoxazone (CLR) is chemically, 5-chloro-2, 3-dihydro-1, 3 -benzoxazol-2-one (Figure 1), is a well known muscle relaxant drug1. It is official in United States Pharmacopoeia (USP). USP2 describe spectrophotometric method for its estimation. Literature survey reveals HPLC3 and UV4 method for estimation of Chlorzoxazone alone.Literature survey also reveals HPLC5-8, HPTLC9 and spectrophotometric10-12 method for estimation of chlorzoxazone with other drug combination. Diclofenac sodium (DIC) is chemically, 2-[2,6dichlorophenylamino] benzene acetic acid sodium salt13 (Figure 2). Diclofenac sodium (DIC) is official in Indian Pharmacopoeia (IP)and British Pharmacopoeia (BP). IP14 and BP15 describe liquid chromatography method for its estimation. Literature survey reveals HPLC16 and UV17 methods for determination of DIC in single dosage form. Literature survey also reveals HPLC18-19 and HPTLC20 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 estimation of CLR and DIC in mixture or dosage forms. Literature survey does not reveal any chromatographic method for estimation of CLR and DIC in synthetic mixture or dosage forms. The present communication describes simple, sensitive, rapid, accurate and precise RP-HPLC method for simultaneous estimation of both drugs in their combined synthetic mixture.


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) were used in the study.

Reagent and materials
Chlorzoxazone (CLR) and Diclofenac sodium (DIC) bulk powder was kindly gifted by Acme Pharmaceuticals Ltd., Ahmedabad, Gujarat, India.HPLC grade methanol (Merck Ltd., Mumbai, India), HPLC grade acetonitrile (Finar Chemicals Ltd.,Mumbai, India) and KH2PO4 and otho phosphoric acid (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
Phosphate buffer (0.02 M KH2PO4, pH 3) was prepared by dissolving 2.72 g potassium dihydrogen phosphate in 1000 ml HPLC-grade water and the pH adjusted to 3.0 by dilute ortho-phosphoric acid.

Preparation of standard stock solutions of CLR and DIC (100 μg/ml)
An accurately weighed standard CLR 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 CLR and DIC.

To optimize the RP-HPLC parameters, several mobile phase compositions were tried. A satisfactory separation and good peak symmetry for CLR and DIC was obtained with a mobile phase consisting of phosphate buffer (0.02 M KH2PO4, pH adjusted to 3 using orthophosphoric acid): acetonitrile: methanol (30: 30: 40, v/v/v)at a flow rate 1.0 ml/min to get better reproducibility and repeatability. Quantification was carried out at 279 nm based on peak area. Complete resolution of the peaks with clear baseline was obtained (Figure 3). System suitability test parameters for CLR and DIC for the proposed method are reported in Table 1. Overlain UV spectrum showed that both drugs showed good absorbance at 279 nm, hence the wavelength of 279 nm was selected for quantification of CLR and DIC (Figure 4). 

Table 1: System suitability parameters of chromatogram



(n = 6)


(n = 6)

Retention time (min)

2.833 ± 0.22

6.265 ± 0.53

Tailing factor

1.255 ± 0.39

1.139 ± 0.48

Theoretical plates

      3042 ± 0.85

5486 ± 1.22


12.62 ± 0.81

Figure 4: U.V. Spectrum of CLR and DIC

Validation of proposed method
The proposed method was validated according to the International Conference on Harmonization (ICH) guidelines21.

Calibration Curve (linearity)
Calibration curves were constructed by plotting peak areas Vs concentrations of CLR and DIC, and the regression equations were calculated. The calibration curves were plotted over the concentration range 2-50 µg/ml for CLR and DIC. Accurately measured standard working solutions of CLR and DIC (0.2, 0.4, 0.8, 1.6, 2.0, 3.0, 4.0 and 5.0 ml) were transferred to a series of 10ml of volumetric flasks and diluted to the mark with mobile phase. Aliquots (20 µl) of each solution were injected under the operating chromatographic conditions described above.

Method precision (Repeatability)
The precision of the instrument was checked by repeatedly injecting six sample solutions of CLR and DIC (8 μg/ml) under the same chromatographic condition and measurements of peak area, retention time and tailing factor. Percentage relative standard deviation (RSD) or coefficient of variation (CV) should not be more than 2 %. 

Intermediate precision (Reproducibility)
The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days over a period of 1 week for 3 different concentrations of sample solutions of CLR and DIC (4, 8, and 16 μg/ml). The results were reported in terms of relative standard deviation (RSD).

Limit of detection and Limit of quantification
LOD and LOQ of drugs can be calculated using the following equations designated by International Conference on Harmonization (ICH) guidelines21.

LOD = 3.3 × σ/S

LOQ = 10 × σ/S

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

Accuracy (Recovery study)
The accuracy of the method was determined by calculating recovery of CLR and DIC by the standard addition method. Known amounts of standard solutions of CLR and DIC (50%, 100%, 150%) were added to pre quantified sample solutions of CLR and DIC (20 + 2 µg/ml). The amounts of CLR and DIC were estimated by applying obtained values to the regression equation of the calibration curve.

Estimation of CLR and DIC from synthetic mixture
CLR (50 mg) and DIC (5 mg) standard drug powder were accurately weighed and then mixed with commonly used formulation excipients like starch, lactose, magnesium stearate and talc in appropriate proportion. The mixture was then transferred to 100 ml volumetric flask containing 50 ml methanol and sonicated for 20 min. The solution was filtered through Whatman filter paper No. 41 and the volume was adjusted up to the mark with methanol. The above solution (0.4 ml) was transferred to 10 ml volumetric flask and diluted up to mark with methanol to obtain 20 µg/ml CLR and 5 µg/ml DIC. Aliquot (20 µl) of sample solution was injected under the operating chromatographic condition as described above and peak area was determined for both drugs. From these area values, the concentrations of CLR and DIC were determined using respective calibration graph. The analysis procedure was repeated six times with synthetic mixture.



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