1. What is an HPLC Assay?

• An HPLC (High Performance Liquid Chromatography) assay is a quantitative analytical method used to determine the concentration or purity of an active pharmaceutical ingredient (API) in a sample.
• It’s commonly used for quality control, stability studies, and method validation.

Basic HPLC Assay Calculation Formula

Components Explained:

• Area_sample: Peak area of analyte in sample chromatogram.
• Area_standard: Peak area of standard solution.
• Conc_standard: Known concentration of the standard solution (mg/mL).
• Conc_sample: Theoretical concentration of sample solution.
• Purity_standard: Purity of the reference standard (as a percentage).
• Dilution Factor: If any dilutions are made for either solution.

Scientific Tips for Accurate HPLC Assay Calculation

1. Use Purity-Corrected Standards

• Always account for the purity of your reference standard. A 98% pure standard will underestimate the sample concentration if assumed 100%.

2. Injection Volume Consistency

• Use the same injection volume for both sample and standard unless you’re correcting for it in your formula.

3. System Suitability Parameters

• Ensure the system passes suitability tests (e.g. resolution, theoretical plates, tailing factor) before using the result.

4. Use Linear Range of Calibration Curve

• Ensure the sample and standard areas fall within the validated linear range of the method.

5. Replicate Injections

• Use average peak area from replicate injections for better accuracy and precision.

Scientific References & Guidelines

You can cite or build around:

• ICH Q2(R2) – Validation of Analytical Procedures
• USP <621> – Chromatography
• ICH Q6A – Specifications: Test Procedures and Acceptance Criteria