Testing Procedure L1: Single-drug (Compound A) Tablet Dosage Form

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

(Using 20 Tablets, Each Containing 100 mg Compound A and Weighing 1.000 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 A in a tablet dosage form using Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), with methanol as the mobile phase, by comparing the sample with a certified reference standard.

2. Materials and Equipment

Chemicals

  • Compound A reference standard (purity ≥ 99.8%)

  • 20 tablets containing 100 mg Compound A per tablet

  • HPLC-grade methanol

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

  1. Accurately measure the required volume of HPLC-grade methanol for the analysis.
  2. Filter the methanol through a 0.45 μm membrane filter to remove particulate matter.
  3. Degas the filtered methanol by sonicating for 10–15 minutes to eliminate dissolved gases.
  4. Transfer the degassed methanol to a clean, labeled solvent reservoir and connect it to the HPLC system for use as the mobile phase.

3.2 Preparation of Solutions

3.2.1 Standard Solution (10 μg/mL)

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

  2. Add about 70 mL of HPLC-grade methanol, and dissolve completely.

  3. Make up to volume with methanol 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 methanol 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. Collect and accurately count 20 tablets containing Compound A, each containing 100 mg of active ingredient.

  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 A.

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

  6. Add approximately 70 mL of HPLC-grade methanol to the flask.

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

  8. Allow the solution to cool to room temperature.

  9. Dilute to the 100 mL mark with methanol to obtain a stock solution containing 100 μg/mL of Compound A.

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 methanol to obtain a working solution of 10 μg/mL of Compound A.

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

3.3 HPLC Conditions

Parameter

Value

Column

C18, 150 mm × 4.6 mm, 3.5 μm

Mobile Phase

Methanol

Flow Rate

1.2 mL/min

Injection Volume

20 μL

Detection Wavelength

254 nm

Column Temperature

30°C

Run Time

~10 minutes

Retention Time

~5 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 A in the tablet.

5. Calculation of Purity

  • Weight of standard used: 10.0 mg

  • Weight of sample (API): 100.0 mg (from 1.000 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 methanol 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.

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