Determination of Compound B Purity in Tablet Dosage Form Using RP-HPLC

(Using 20 Tablets, Each Containing 100 mg Compound B and Weighing 0.100 g)

Important Disclaimer:The following procedure is intended solely for learning and demonstration purposes. It is not validated or approved for actual laboratory use. This procedure should never be used in any real analytical or pharmaceutical setting. Values, volumes, and methods are simplified to help learners understand the basic workflow involved in HPLC sample preparation.

1. Objective

To determine the purity of Compound B in a tablet dosage form using Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), with Phosphate Buffer (pH 6.5) : Acetonitrile (50:50, v/v) as the mobile phase, by comparing the sample with a certified reference standard.

2. Materials and Equipment

Chemicals

• Compound B reference standard (purity ≥ 99.8%)

• 20 tablets containing 100 mg Compound B per tablet

• HPLC-grade Acetonitrile

Equipment

• HPLC system

• C18 column (150 mm × 4.6 mm, 3.5 μm)

• UV detector (254 nm)

• Analytical balance

• 10 mL, 100 mL, and 250 mL volumetric flasks

• Sonicator

• Mortar and pestle

• Syringes and 0.45 μm membrane filters

3. Methodology

3.1Mobile Phase Preparation Procedure,

3.1.1 Phosphate Buffer (pH 6.5):

1. 1. 1. 1. 1. 1. Weighing and Dissolving:

                  • • Accurately weigh 6.8 grams of potassium dihydrogen phosphate (KH₂PO₄).

                    • Transfer the salt into a beaker containing approximately 800 mL of distilled water.

                    • Stir the solution using a magnetic stirrer or glass rod until the KH₂PO₄ is completely dissolved.

               2. pH Adjustment:

                  • • Using a calibrated pH meter, monitor the pH of the solution.

                    • Adjust the pH to 6.5 by adding 1 M potassium hydroxide (KOH) solution dropwise while stirring continuously.

                    • After reaching the desired pH, transfer the solution to a 1000 mL volumetric flask and make up the volume to the mark with distilled water.

3.1.2 Final Mobile Phase:

1. 1. 1. 1. 1. 1. Mixing:

                  • • In a clean mixing container, combine 50 parts of the prepared phosphate buffer (pH 6.5) with 50 parts of HPLC-grade acetonitrile by volume (v/v).

                    • Mix thoroughly to ensure a homogeneous mobile phase.

               2. Degassing and Filtration:

                  • • Filter the mobile phase through a 0.22 µm or 0.45 µm membrane filter,

                    • Degas the mixture using an ultrasonic bath for at least 10–15 minutes to remove dissolved gases. To a labeled reservoir suitable for HPLC use.

3.1.3 Dilution Solvent (Water:Acetonitrile, 20:80 v/v),(used in standard/sample prep)

1. 1. 1. 1. Mix 200 mL of HPLC-grade water with 800 mL of HPLC-grade acetonitrile.

         2. Filter through a 0.45 μm membrane filter and degas by ultrasonication before use.

3.2 Preparation of Solutions

3.2.1 Standard Solution (10 μg/mL)

1. 1. 1. Accurately weigh 10.0 mg of Compound B reference standard and transfer into a 100 mL volumetric flask.

      2. Add about 70 mL of Dilution Solvent, and dissolve completely(Sonicate).

      3. Make up to volume with Dilution Solvent to obtain a 100 μg/mL stock solution.

      4. Pipette 1.0 mL of the stock solution into a 10 mL volumetric flask and dilute to volume with Dilution Solvent to obtain a 10 μg/mL working standard solution.

      5. Filter the solution through a 0.45 μm syringe filter to remove particulate matter.

3.2.2 Sample Solution (10 μg/mL)

Step 1: Preparation of Stock Solution

1. 1. 1. 1. 1. 1. Accurately count and weigh 20.000 g of 20 tablets.
               2. Pulverize the tablets using a clean mortar and pestle.
               3. Weigh 0.100 g of the tablet powder (equivalent to 10 mg of Compound B, based on label claim).
               4. Transfer the powder to a 100 mL volumetric flask
               5. Add approximately 70 mL of dilution solvent (Water:Acetonitrile (20:80)).
               6. Sonicate the mixture for 15–20 minutes to ensure complete extraction.
               7. Centrifuge at 4000–5000 rpm for 10 minutes to remove insoluble excipients.
               8. Transfer the clear supernatant and allow it to cool to room temperature.
               9. Make up the volume to 100 mL with dilution solvent (Water:Acetonitrile (20:80)) to obtain a 100 μg/mL stock solution.
               10. Filter using a 0.45 μm syringe filter before HPLC analysis

Step 2: Preparation of Working Solution

1. 1. 1. 1. 1. 1. Pipette 1.0 mL of the stock solution into a 10 mL volumetric flask.
               2. Dilute to the mark with dilution solvent (Water:Acetonitrile (20:80)) to prepare a 10 μg/mL working sample solution.

3.3 HPLC Conditions

4. Sample Analysis

1. Inject the standard and sample solutions under the specified HPLC conditions.

2. Record the retention times and peak areas.

3. Confirm identity by comparing sample retention time with that of the standard.

4. Calculate the % purity of Compound B in the tablet.

5. Calculation of Purity

• Weight of standard used: 10.0 mg

• Weight of sample (API): 100.0 mg (from 0.100 g powder)

• Acceptance Criteria: 98.5% – 101.5%

6. Reporting

Report must include:

• Tablet strength and total weight used (20.000 g)

• Sample preparation steps

• Chromatograms with retention times and peak areas

• Calculation details and % purity result

• Analyst and reviewer signatures

• Observations or deviations

7. Safety and Waste Disposal

• Wear appropriate PPE (gloves, goggles, lab coat)

• Handle Acetonitrile in a fume hood (flammable and toxic)

• Dispose of solvents and samples according to local safety and environmental regulations

 Important Disclaimer:The following procedure is intended solely for learning and demonstration purposes. It is not validated or approved for actual laboratory use. This procedure should never be used in any real analytical or pharmaceutical setting. Values, volumes, and methods are simplified to help learners understand the basic workflow involved in HPLC sample preparation.