Development and Validation of Metaxalone and Diclofenac Potassium in Bulk and Its Formulation using RP-HPLC Techniques

Diclofenac potassium (DICLO) exerts its anti-inflammatory, antipyretic, and analgesic effects by blocking prostaglandin production via cyclooxygenase (COX). By preventing bacterial DNA synthesis, it also seems to have bacteriostatic properties. Diclofenac potassium is chemically 2-[2-(2,6-dichloroanilino) phenyl] acetate, with a molecular mass of 334.2 g/mol and the molecular formula C₁₄H₁₀C_l2KNO₂.^1,2

Metaxalone helps to reduce pain from injury and other muscular-skeletal disorders by relaxing muscles.¹ The precise mechanism of action of metaxalone in humans is unclear, however, it may be associated with a widespread central nervous system depression. It has no direct impact on the motor end plate, nerve fiber, or striated muscle's contractile mechanism. The liver breaks down metaxalone, which is then eliminated in the urine as unknown metabolites.^3,4, The IUPAC name of Metaxalone is 5- [(3, 5-dimethyl phenoxy) methyl]-1,3-oxazolidin-2-one with molecular formula C₁₂H₁₅NO₃ and molecular mass 221.25 g/mol.¹ According to the literature survey, fewer RP-HPLC techniques have been documented to estimate these drugs both alone and in combination in pharmaceutical dosage forms. Other analytical techniques, including UV^5,6, HPLC^7,8, LCMS⁹, HPTLC³, RP-UPLC¹⁰, and stability-indicating RP-HPLC¹¹, have also been reported.

MATERIALS AND METHODS

Materials:

A standard Diclofenac Potassium and Metaxalone were obtained from Micro Lab Mumbai as a gift sample. While Diclofenac Potassium and Metaxalone Tablets purchase from local market.   Were as Acetonitrile, Potassium dihydrogen phosphate, and Orthophosphoric acid from Merk Laboratories Mumbai.

Instrument:

Table: 1 Instrument and Equipment used for the Analytical Method Development and Validation studies

Methods

Preparation of Phosphate Buffer

Potassium dihydrogen orthophosphate (2.72 g) was precisely weighed and diluted in 1000 millilitres of HPLC-grade water. Before being sonicated, the pH was adjusted to 4.2 using orthophosphoric acid.

Preparation of standard stock solution

1. Diclofenac Potassium

100 mg of Diclofenac potassium was precisely weighed and transferred into a 100 mL volumetric flask, diluting it with acetonitrile, and then sonicating it to the proper strength, resulting in a concentration of 1000 µg/mL. Acetonitrile was used to dilute 10 mL of the above standard stock solution to the necessary concentration in a 100 mL volumetric flask, resulting in a sub-stock solution with a concentration of 100 µg/mL.

2. Metaxalone

100 mg of Metaxalone was precisely weighed and transferred into a 100 mL volumetric flask, diluting it with acetonitrile, and then sonicating it to the proper strength, resulting in a concentration of 1000 µg/mL. Acetonitrile was used to dilute 10 mL of the above standard stock solution to the necessary concentration in a 100 mL volumetric flask, resulting in a sub-stock solution with a concentration of 100 µg/mL.

Preparation of sample stock solution

1. Diclofenac Potassium

Twenty tablets were carefully weighed and crushed into a fine powder. In a volumetric flask with a total volume of 100 mL, a volume equal to 100 mg was obtained, transferred, diluted with acetonitrile, and sonicated for 15 minutes. Whatman No. 1 filter paper was used to filter this mixture. 1 mL of the previously described sample stock solution was put into a 10 mL volumetric flask and diluted with acetonitrile to create the solution with a concentration of 100 µg/mL.

2. Metaxalone

Twenty tablets were carefully weighed and crushed into a fine powder. In a volumetric flask with a total volume of 100 mL, a volume equal to 100 mg was obtained, transferred, diluted with acetonitrile, and sonicated for 15 minutes. Whatman No. 1 filter paper was used to filter this mixture. 1 mL of the previously described sample stock solution was put into a 10 mL volumetric flask and diluted with acetonitrile to create the solution with a concentration of 100 µg/mL.

Method Development

Trial 1

Column Used: Eclipse plus C₁₈ (250 mm × 4.6 mm ID, 5?m)

Mobile Phase: Acetonitrile: Water (50:50)

Flow rate: 1 ml/min

Wavelength:280 nm

Temperature:30°C

Fig: 2 Chromatogram for Acetonitrile: Water (50:50)

Column Used: Eclipse plus C₁₈ (250 mm × 4.6 mm ID, 5?m)

Mobile Phase: Acetonitrile: Methanol (60:40)

Flow rate: 1 ml/min

Wavelength:280 nm

Temperature:30°C

Fig: 3 Chromatogram for Acetonitrile: Methanol (60:40)

Fig: 4 Chromatogram for Acetonitrile: Methanol (60:40)

Drugs were eluted with a good retention time, and all system parameters, including tailing factors and plate count, were within acceptable limits.

Column Used: Eclipse plus C₁₈ (250 mm × 4.6 mm ID, 5?m)

Mobile Phase: Phosphate Buffer (pH 4.2): Acetonitrile (30:70)

Flow rate: 1 ml/min

Wavelength:280 nm

Temperature:30°C

Fig: 5 Chromatogram for Phosphate Buffer (pH 4.2): Acetonitrile (30:70)

1. System suitability: According to ICH criteria, every system suitability parameter is within the acceptable range.

2. Linearity: A series of dilutions were made using the standard stock solution to evaluate the linearity of the medication Diclofenac Potassium and Metaxalone. The linearity range was 5–30 ?g/mL.

1. Diclofenac Potassium

Table: 3 Calibration data for Diclofenac Potassium

Fig: 8 Calibration Curve of Diclofenac Potassium

Fig: 9 Typical Chromatogram of Diclofenac Potassium

2. Metaxalone

Table: 4 Calibration data for Diclofenac Potassium

Fig: 10 Calibration Curve of Metaxalone

Fig: 11 Typical Chromatogram of Metaxalone

3. Precision

The intraday (Repeatability was tested by evaluating a standard solution on same day.) and inter-day (Repeatability was tested by evaluating a standard solution on three days.) methods were used to measure precision. To do precise examination, six injections of standard solutions were produced. Results are expressed as %RSD.

Table: 5 Intraday and Interday Precision data for Diclofenac Potassium

Fig:12 Chromatogram of Diclofenac Potassium for intraday precision

Fig:13 Chromatogram of Diclofenac Potassium for interday precision

Fig:14 Chromatogram of Metaxalone for intraday precision

Fig:15 Chromatogram of Metaxalone for interday precision

4. Accuracy

   Three distinct concentrations of the drug's percentage accuracy are 80%, 100%, and 120%. The mean recovery percentage was then calculated based on the results obtained from the recovery studies.

Fig:16 Chromatogram of Diclofenac Potassium for accuracy at 80%

Fig:17 Chromatogram of Diclofenac Potassium for accuracy at 100%

Fig:18 Chromatogram of Diclofenac Potassium for accuracy at 120%

Fig:19 Chromatogram of Metaxalone for accuracy at 80%

Fig: 20 Chromatogram of Diclofenac Potassium for accuracy at 100%

Fig: 21 Chromatogram of Metaxalone for accuracy at 120%

5. Robustness

Small deliberate adjustments were made to the flow rate, temperature, and wavelength, but the results were within the ICH standards and showed no significant variation.

1. Diclofenac Potassium

2. Metaxalone