Testing Procedure L3: Multi-drug tablet (Compound C and Compound D) Dosage Form

Determination of Compound C and Compound D Purity in Tablet Dosage Form Using RP-HPLC

(Using 20 Tablets, Each Containing 100 mg Compound C and 150 mg Compound D )

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 quantitatively determine the content of Compound C and Compound D in combination tablets using reverse-phase high-performance liquid chromatography (RP-HPLC), via a single injection providing two distinct peaks..

2. Materials and Equipment

Chemicals

    • Compound C reference standard (purity ≥ 99.8%)

    • Compound D reference standard (purity ≥ 99.8%)

    • Combination tablets (Each tablet contains 100 mg of Compound C and 150 mg of Compound D)

    • HPLC-grade acetonitrile

    • KH₂PO₄ (potassium dihydrogen phosphate)

    • 1 M KOH

    • Distilled water

Equipment

    • HPLC system with UV detector (254 nm)

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

    • pH meter

    • Analytical balance

    • 0.45 µm membrane filters

    • Volumetric flasks (10 mL, 100 mL, 200 mL)

    • Ultrasonicator

    • Centrifuge

    • Syringes

3. Methodology

3.1Mobile Phase Preparation Procedure

3.1.1 Phosphate Buffer (pH 6.5):

            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. Mixing:

                • In a clean mixing container, combine 40 parts of the prepared phosphate buffer (pH 6.5) with 60 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. Transfer to a labeled reservoir suitable for HPLC use.

3.2 Preparation of  Dissolving solvent 

        • Mix 20 mL distilled water with 80 mL acetonitrile (20:80 v/v)

3.3 Preparation of Solutions

3.3.1 Standard Solution 
            1. Accurately weigh 10.0 mg of Compound C and 15.0 mg of Compound D
            2. Transfer both to the same 100 mL volumetric flask
            3. Dissolve in Dissolving solvent (water:acetonitrile,20:80)
            4. Sonicate if necessary
            5. This is a 100 μg/mL Compound C + 150 μg/mL Compound D stock
            6. Pipette 1.0 mL of stock into a 10 mL volumetric flask
            7. Dilute with Dissolving solvent
            8. Final concentration: 10 μg/mL Compound C, 15 μg/mL Compound D,
            9. Filter using a 0.45 μm syringe filter before HPLC analysis
3.2.2 Sample Solution (10 μg/mL)

Step 1: Preparation of Stock Solution

          1. Collect and accurately count 20 tablets containing Compound C and Compound D.

          2. Weigh the 20 tablets .

          3. Pulverize the tablets into a fine, uniform powder using a clean mortar and pestle.

          4. Weigh 0.1 g of the powdered tablet mixture, equivalent to 10 mg of Compound C and 15 mg of Compound D.

          5. Transfer the 0.1 g powder into a 100 mL volumetric flask.

          6. Add approximately 70 mL of Dissolving solvent to the flask.

          7. Sonicate the mixture for 15–20 minutes to ensure complete extraction of Compounds.

          8. Allow the solution to cool to room temperature.

          9. Centrifuge at 4000–5000 rpm for 10 minutes (if needed)
          10. Collect the supernatant
          11. Make up to volume with the same dilution solvent

Step 2: Preparation of Working Solution

          1. Pipette 1.0 mL of the stock solution into a 10 mL volumetric flask.

          2. Dilute to the mark with Dissolving solvent to obtain a working solution of 10 μg/mL of Compound C and 15 μg/mL of Compound D.

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

3.4 HPLC Conditions

Parameter

Value

Column

C18, 150 mm × 4.6 mm, 3.5 μm

Mobile Phase

Phosphate Buffer : Acetonitrile (40:60, v/v)

Flow Rate

1.2 mL/min

Injection Volume

20 μL

Detection Wavelength

254 nm

Column Temperature

30°C

Run Time

~20 minutes

Retention Time

~Compound C – 6 minutes (expected),Compound D – 9 minutes (expected)

 

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 C and Compound D 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 organic solvents (e.g., acetonitrile) in a fume hood

  • 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.

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