INTRODUCTION:

Chlorzoxazone (5-chloro-2,3-dihydro-1,3-benzoxazol-2-one) belongs to benzoxazolones class. (Figure 1). It is a central muscle relaxant with sedative properties. It is claimed to inhibit muscle spasm by exerting an effect primarily at the level of the spinal cord and subcortical areas of the brain. It is official in United States Pharmacopoeia. Diclofenac sodium (2-[(2,6-dichlorophenyl)]amino]benzene acetic acid monosodium salt) belongs to dichlorobenzenes class (Figure 1). It is a non-steroidal anti-inflammatory agent (NSAID) with antipyretic and analgesic actions. It is available as sodium and potassium salts. It is official in United States Pharmacopoeia, British Pharmacopoeia and Indian Pharmacopoeia. Acetaminophen, also known as paracetamol (N-(4-hydroxyphenyl) acetamide) is commonly used for its analgesic and antipyretic effects (Figure 1).

The therapeutic effects are similar to salicylates, but lacks anti-inflammatory, gastric ulcerative and antiplatelet effects. Pawar et al. 2009¹ developed a RP-HPLC method to quantify aceclofenac, paracetamol and chlorzoxazone in tablet formulation. Badgujar et al. 2011² developed RP-HPLC method to quantify chlorzoxazone, diclofenac sodium and paracetamol in tablet formulation. Chaudhari and Prajapati 2014³ developed a stability-indicating RP-HPLC method for the simultaneous estimation of chlorzoxazone and paracetamol in tablet. Patel and Prajapati⁴ developed a RP-HPLC method to quantify diclofenac sodium and chlorzoxazone in tablet formulation. Kulkarni et al. 2012⁵ developed a stability-indicating HPTLC method for the simultaneous estimation of chlorzoxazone, diclofenac sodium and paracetamol in bulk drug.

Literature studies showed that analytical techniques viz. HPTLC and HPLC methods have been reported for simultaneous determination of paracetamol, chlorzoxazone and diclofenac sodium. To our understanding, there is no article related to Stability Indicating HPLC method to quantify Chlorzoxazone, Diclofenac Sodium and Paracetamol in bulk and tablet has ever been mentioned in literature studies. The objective of this work was to develop a specific, accurate and reproducible stability indicating HPLC method for estimation of Chlorzoxazone (CHL), Diclofenac Sodium (DIC) and Paracetamol (PCM) as per the ICH guidelines.

Figure 1: Structures of Chlorzoxazone, Diclofenac and Paracetamol

MATERIALS AND METHODS:

Chemicals and reagents:

All chemicals were of analytical grade. Chlorzoxazone, Diclofenac sodium and Paracetamol working standards were obtained from Yarrow Chem Product (Mumbai, India). Methanol (HPLC grade) and potassium hydroxide (analytical grade) were purchased from Finar Limited (Mumbai, India), acetonitrile (HPLC grade) was purchased from Spectrochem Pvt. Ltd. (Mumbai, India), and potassium dihydrogen phosphate (analytical grade) was purchased from Lobachem (Mumbai, India). The tablet formulation (Powergesic^®MR, Jenburkt Pharmaceuticals Ltd., Mumbai, India) containing 50mg of diclofenac sodium, 325mg of paracetamol and 500mg of chlorzoxazone was procured from the local market and used for analysis of marketed formulation.

Instrumentation:

The liquid chromatographic system used was Shimadzu LC 20 AD (Shimadzu, Kyoto, Japan) equipped with gradient pump, auto- injector and photo diode array detector. The chromatographic analysis was performed using LC solution software on a C₁₈ Vydac Monomeric 120A (250 x 4.6mm, 5mm).

Preparation of Buffer:

20mM phosphate buffer (pH 6.2± 0.02): 2.72grams of potassium dihydrogen ortho-phosphate was dissolved in 1000ml of milli-Q water and pH adjusted to 6.2±0.02 with potassium hydroxide solution. The resulting solution was filtered with a 0.45m filter using vacuum filtration and sonicated for 15mins.

Preparation of Stock solution:

The stock solution was prepared by accurately weighing 10mg each of chlorzoxazone, diclofenac sodium and paracetamol working standard separately into three 10ml volumetric flasks. They were then dissolved in methanol and the resulting solutions were sonicated and volume made upto the mark with methanol to obtain a solution of 1000mg/ml of each drug. 1ml was withdrawn from each standard stock solution, transferred into separate 10ml volumetric flask and the volume was made up to the mark with the mobile phase (100mg/ml- Stock solution A). 1ml was withdrawn from each stock solution A into separate 10ml volumetric flask and the volume was made up to the mark with the mobile phase (10mg/ml- Stock solution B). 4ml, 0.4ml and 2.6ml from stock solution B of chlorzoxazone, diclofenac sodium and paracetamol respectively were pipetted out into a 10ml volumetric flask and the volume was made upto the mark with the mobile phase (4mg/ml chlorzoxazone, 0.4mg/ml diclofenac sodium and 2.6mg/ml paracetamol).

The HPLC analysis was performed on reversed- phase high- performance liquid chromatographic system with isocratic elution mode using a mobile of acetonitrile: 20mM phosphate buffer, pH adjusted to 6.2 with potassium hydroxide (30:70 v/v) on C₁₈ Vydac Monomeric 120A (250 x 4.6mm, 5mm) with 1ml/min flow rate at 275nm using PDA detector.

Calibration curves for CHL, DIC and PCM:

Tablet formulation contains PCM:CHL:DIC in ratio of 6.5:10:1. Appropriate aliquots of PCM, CHL and DIC stock solutions were taken in 10ml volumetric flask and diluted up to the mark with mobile phase to obtain final concentrations of 0.65- 6.5µg/ml for paracetamol, 1-10 µg/ml for chlorzoxazone and 0.1-1µg/ml for diclofenac sodium. The volume solutions injected were 20ml and chromatograms were recorded. Calibration curves were constructed by plotting average peak areas versus concentrations and regression equations were computed for all three drugs (Table 1)

Table 1: Linear Regression data for Calibration curves

Parameters	Paracetamol	Chlorzoxazone	Diclofenac Sodium
Linearity range (μg/ml)	0.65- 6.5	1-10	0.1- 1
Correlation coefficient	0.999	0.9997	0.9991
Slope	20662	33696	44824
y- intercept	2089.8	1800.6	660.4

Analysis of Marketed Formulation:

Twenty tablets were weighed and their average weight was calculated. These tablets were powdered and weight equivalent to 10mg of diclofenac sodium was weighed and transferred into 10ml volumetric flask. Then the powder was dissolved in methanol and the resulting solution was sonicated and volume made upto the mark with methanol to obtain a solution of 1000mg/ml of diclofenac sodium. The resulting solution was filtered with a 0.45m filter using vacuum filtration. 1ml from the above solution was withdrawn and transferred to 10ml volumetric flask, volume was made up with mobile phase. 1ml from the above solution was withdrawn and transferred to 10ml volumetric flask, volume was made up with mobile phase. 0.4ml from the above solution was withdrawn and transferred to 10ml volumetric flask, volume was made up with mobile phase and injected in the HPLC system (4mg/ml CHL, 0.4mg/ml DIC and 2.6mg/ml PCM). A 20ml volume of above solution was injected into HPLC and peak areas were measured under optimized chromatographic conditions.

Method Validation:

The method of analysis was validated as per the recommendations of ICH Q2 (R1) for the parameters like accuracy, linearity, precision, detection limit, quantitation limit and robustness. The accuracy of the method was determined by calculating percentage recovery of CHL, DIC and PCM. The recovery studies were carried out by applying the method to drug sample to which known amount of CHL, DIC and PCM corresponding to 80, 100 and 120% of label claim had been added (standard addition method). At each level of the amount, three determinations were performed and the results obtained were compared.

Intraday and interday precision studies of CHL, DIC and PCM were carried out by estimating the corresponding responses 3 times on the same day and on 3 different days for the concentration of 4mg/ml CHL, 0.4mg/ml DIC and 2.6mg/ml PCM. The limit of detection (LOD) and limit of quantitation (LOQ) were calculated using following formulae: LOD= 3.3(SD)/S and LOQ= 10 (SD)/S, where SD=standard deviation of response (peak area) and S= average of the slope of the calibration curve.

System suitability tests are an integral part of chromatographic method which are used to verify reproducibility of the chromatographic system. To ascertain its effectiveness, certain system suitability test parameters were checked by repetitively injecting the drug solution at the concentration level 10mg/ml CHL, 1mg/ml DIC and 6.5mg/ml PCM to check the reproducibility of the system and the results are shown in Table 2.

For robustness evaluation of HPLC method, a few parameters like flow rate, column oven temperature, wavelength and pH of mobile phase were deliberately changed. One factor was changed at one time to estimate the effect. Each factor selected was changed at three levels (-2, 0, +2) with respect to optimized parameters. Robustness of the method was done at the concentration level of 4mg/ml CHL, 0.4mg/ml DIC and 2.6mg/ml PCM.

Table 2: Summary of System suitability and Validation Parameters

Parameters	Paracetamol	Chlorzo xazone	Diclofenac Sodium
Retention time ± allowable time (min)	3.28± 0.03	13.27 ± 0.3	15.61± 0.4
Theoretical Plates	4364	10673	9607
Tailing factor (asymmetry factor)	1.46	1.42	1.01
Linearity range (μg/ml)	0.65- 6.5	1-10	0.1- 1
Correlation coefficient	0.999	0.9997	0.9991
LOD (μg/ml)	0.221	0.176	0.034
LOQ (μg/ml)	0.663	0.534	0.102
Recovery (%)	100.64	101.25	100.31
Precision (%RSD)
Inter-day (n=6)	0.38	0.49	1.27
Intra-day (n=6)	0.5	0.51	0.95
Robustness	Robust	Robust	Robust

Forced degradation studies:

Forced degradation studies of bulk and tablet formulation were carried out under conditions of acid and alkali hydrolysis, thermal, oxidative and photolytic degradation. Forced degradation in acid media was performed by weighing 325mg of PCM, 500mg CHL and 50mg of DIC in first 50ml round bottom flask and tablet powder equivalent to 50mg of diclofenac sodium in other 50ml round bottom flask and then 50ml of 0.1M hydrochloric acid was added to both the flasks. These mixtures were kept at room temperature up to 8 hours. Similarly, forced degradation in alkali media was performed by adding 50ml of 0.1M sodium hydroxide and kept for 6 hours at room temperature. Degradation with hydrogen peroxide was performed by weighing 65mg of PCM, 100mg CHL and 10mg of DIC in separate 10 ml volumetric flasks and volume was made up with methanol. From each, 1ml was withdrawn and transferred into a 10ml volumetric flask and volume was made up with 30%v/v hydrogen peroxide solution (100mg/ml) for bulk drugs and for tablet, powder equivalent to 10mg of diclofenac sodium was accurately weighed and transferred into 100 ml volumetric flask and volume was made up with 30%v/v hydrogen peroxide solution (100mg/ml). These mixtures were kept at room temperature for 4 hours. For thermal and photolytic degradations, bulk drugs and tablet powder were placed in petri dish in oven for 4 hours at 100° and in direct sunlight for 24 hours, respectively.

For HPLC analysis, all the degraded samples solutions were diluted with mobile phase to obtain final concentration of 4mg/ml CHL, 0.4mg/ml DIC and 2.6mg/ml PCM. Then 20μl solution of above solutions were injected into HPLC system and analyzed under the chromatographic condition described earlier.

Table 3: Stress testing report of Standard and Tablet

Sr. No.	Type of degradation	Stress Conditions		% Degradation observed
Sr. No.	Type of degradation	Stress Conditions		Paracetamol	Chlorzoxazone	Diclofenac sodium
1	Acid hydrolysis	0.1M hydrochloric acid at room temperature for 8 hours	Standard	12.7	21.1	47.1
1	Acid hydrolysis	0.1M hydrochloric acid at room temperature for 8 hours	Tablet	22.8	52.9	32
2	Alkali hydrolysis	0.1 M sodium hydroxide at room temperature for 6 hours	Standard	16.6	24.7	53.6
2	Alkali hydrolysis	0.1 M sodium hydroxide at room temperature for 6 hours	Tablet	15.6	6.7	74
3	Oxidative degradation	30% v/v hydrogen peroxide at room temperature for 4 hours	Standard	4.9	51.3	89.2
3	Oxidative degradation	30% v/v hydrogen peroxide at room temperature for 4 hours	Tablet	11.9	26.2	20.2
4	Photolytic degradation	Exposed to direct sunlight for 24 hours	Standard	8.9	25.4	4.6
4	Photolytic degradation	Exposed to direct sunlight for 24 hours	Tablet	19.1	13	22
5	Thermal degradation	Hot air oven at 100°C for 4 hours	Standard	53.6	22.2	32.6
5	Thermal degradation	Hot air oven at 100°C for 4 hours	Tablet	11.3	24.1	76

Figure 2: Optimised Chromatogram of PCM, CHL and DIC (API mixture)

Figure 3: Chromatogram of tablet mixture

RESULTS AND DISCUSSION:

The mobile phase consists of acetonitrile: 20mM phosphate buffer, pH adjusted to 6.2 with potassium hydroxide (30:70 v/v), at 1ml/min flow rate was optimised and it gave sharp, well-resolved peaks with tailing factor less than 2 for CHL, DIC and PCM. (Figure 2). The retention times of PCM, CHL and DIC were 3.28 mins, 13.27 mins and 15.61 mins respectively. Since the concentration of DIC is less as compared to PCM and CHL in the tablet formulation, therefore the wavelength 275nm (l_max of DIC) was selected as the detection wavelength. The calibration curve for PCM, CHL and DIC was linear over the range of 0.65- 6.5µg/ml, 1-10µg/ml and 0.1-1µg/ml respectively. The data of the calibration curves is shown in Table 1. The given method was effectively applied to the estimation of PCM, CHL and DIC in the tablet dosage form. The results obtained from the combination were comparable with the corresponding labelled amounts. The developed method was able to separate other excipients present in tablet from the three drugs, hence it was found to be specific (Figure 3).

The Limit of Detection for PCM, CHL and DIC was found to be 0.221μg/ml, 0.176μg/ml and 0.034mg/ml, respectively, while Limit of Quantification was 0.663μg/ml, 0.534μg/ml and 0.102mg/ml, respectively. The validation report and system suitability test parameters report data are summarized in Table 2. The summary report of Stress testing of Standard and Tablet are presented in Table 3.

In acid hydrolysis, the percentage of degradation of paracetamol and chlorzoxazone were more in tablet than in API; while the degradation of diclofenac sodium was more in API than in tablet. In alkali hydrolysis, the percentage of degradation of paracetamol and chlorzoxazone were more in API than in tablet; while degradation of diclofenac sodium was more tablet than in API. In oxidative degradation, the percentage of degradation of chlorzoxazone and diclofenac sodium were more in API than in tablet; while degradation of paracetamol was more tablet than in API. In photolytic degradation, the percentage of degradation of chlorzoxazone was more in API than in tablet; while the degradation of paracetamol and diclofenac sodium were more in tablet than in API. In thermal degradation, the percentage of degradation of paracetamol was more in API than in tablet; while the degradation of chlorzoxazone and diclofenac sodium were more in tablet than in API. The higher or lower level of percentage of degradation in PCM, CHL and DIC may be due to interaction among the drugs, interaction with the excipients or may be due to the manufacturing process.

CONCLUSION:

In the proposed study, a simple, precise, accurate, sensitive and cost effective stability indicating assay method for simultaneous estimation of Chlorzoxazone, Diclofenac Sodium and Paracetamol in bulk drug and tablet by RP-HPLC was established. The results obtained by analysing the forced degraded samples depicts that there was no other co- eluting peaks of interference from excipients due to variable stress components with the main peaks and the method was specific for the determination of Paracetamol, Chlorzoxazone and Diclofenac sodium in presence of various degradants. The method can be used for stability indicating assay of paracetamol, chlorzoxazone and diclofenac sodium from its tablet dosage forms.

ACKNOWLEDGEMENTS:

The authors are thankful to Department of Pharmaceutical Quality Assurance, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal for providing the facilities to carry the research work.

REFERENCES:

1. Pawar UD, Naik AV, Sulebhavikar AV, Datar TA, Mangaonkar KM. Simultaneous Determination of Aceclofenac, Paracetamol and Chlorzoxazone by HPLC in Tablet Dose Form. E-Journal of Chemistry, 2009; 6(1): 289-294.

2. Badgujar MA, Pingale SG, Mangaonkar KV. Simultaneous Determination of Paracetamol, Chlorzoxazone and Diclofenac Sodium in Tablet Dosage Form by High Performance Liquid Chromatography. E-Journal of Chemistry, 2011; 8(3): 1206-1211

3. Chaudhari BG, Prajapati HR. Stability-Indicating RP-HPLC Method for Simultaneous Estimation of Chlorzoxazone and Paracetamol in Tablet Dosage Form. Int. J. Pharm. Anal. 2014; 2(5): 402-412

4. Patel SA, Prajapati KM. Development and Validation Of RP-HPLC Method for Simultaneous Estimation Of Chlorzoxazone And Diclofenac Sodium In Combination. Pharmatutor. Epub. Pharmatutor-Art-1715

5. Kulkarni MB, Dange PB, Walode SG. Stability indicating thin-layer chromatographic determination of chlorzoxazone, diclofenac sodium and paracetamol as bulk drug: Application to forced degradation study. Der Pharmacia Sinica 2012; 3(6): 643-652

Received on 28.05.2020 Modified on 11.07.2020

Research J. Pharm. and Tech. 2021; 14(9):5024-5028.

DOI: 10.52711/0974-360X.2021.00876