HPLC Calibration Procedure

1. Introduction

High-Performance Liquid Chromatography (HPLC) is a critical analytical technique in pharmaceutical quality control. Regular calibration ensures accuracy, precision, and compliance with regulatory requirements like USP <621>, ICH Q2(R1), and GMP.

2. Objective

To verify and document that the HPLC system meets the performance specifications for reliable analytical results.

3. Calibration Parameters

HPLC calibration typically involves the following checks:

1. Flow Rate Accuracy

   • Deliver water at a set flow rate (e.g., 1.0 mL/min) for a specific time (10 min).

   • Measure the actual volume delivered using a calibrated volumetric flask.

   • Acceptance criteria: ±2% of the set value.

2. Injector Precision (Repeatability)

   • Inject the same standard solution (e.g., caffeine) multiple times (≥6).

   • Calculate %RSD of peak areas.

   • Acceptance criteria: %RSD ≤ 1.0% (per pharmacopoeial guidance).

3. Detector Linearity

   • Prepare at least five concentration levels of a standard.

   • Plot concentration vs. peak area; determine correlation coefficient (r).

   • Acceptance criteria: r ≥ 0.999.

4. Wavelength Accuracy

   • Use a holmium oxide filter or solution (for UV detector).

   • Compare measured absorbance maxima with reference values.

   • Acceptance criteria: ±1 nm.

5. Column Efficiency (Theoretical Plates)

   • Inject a standard solution.

   • Calculate N using:

     N=16(tRWb)2N = 16 \left( \frac{t_R}{W_b} \right)^2N=16(Wb​tR​​)2

   • Acceptance criteria: As per USP method requirement.

6. Tailing Factor

   • Measure tailing from chromatogram.

   • Acceptance criteria: Tailing factor ≤ 2.0.

7. Resolution

   • Inject a mixture of two closely eluting compounds.

   • Acceptance criteria: Resolution ≥ 2.0.

4. Frequency

• Initially before first use.

• At least once every 6 months, or as per SOP.

• After major maintenance or repair.

5. Documentation

• Record instrument ID, calibration date, parameters tested, results, pass/fail status, and signatures.

• Retain records per GMP and 21 CFR Part 11.

Regulatory References

• USP <621> Chromatography

• ICH Q2(R1) – Validation of Analytical Procedures

• ISO/IEC 17025 – General Requirements for Competence of Testing and Calibration Laboratories