Learn the complete SOP for HPLC Analysis, including mobile phase preparation, system suitability, documentation, acceptance criteria, and chromatogram recording in compliance with Good Laboratory Practices.

SOP for HPLC Analysis and Documentation (GLP-Compliant Guide)

High-Performance Liquid Chromatography (HPLC) is one of the most critical analytical techniques used in pharmaceutical and quality control laboratories. A well-defined SOP for HPLC Analysis ensures data integrity, regulatory compliance, and consistent performance under Good Laboratory Practices (GLP).

This guide outlines the standard operating procedure for HPLC Analysis and documentation, covering preparation, system suitability, acceptance criteria, chromatogram handling, and calculations.

1. Objective

To describe the procedure for performing HPLC Analysis and documentation while ensuring full compliance with Good Laboratory Practices (GLP).

2. Scope

This SOP applies to all HPLC Analysis conducted in the Quality Control (QC) laboratory, including system setup, mobile phase preparation, standard preparation, injection sequence, chromatogram handling, and documentation.

3. Responsibility

Officer/Executive – Quality Control

Responsible for conducting HPLC Analysis as per the approved Standard Testing Procedure (STP) and ensuring accurate documentation.

4. Accountability

Manager – Quality Control

Accountable for oversight, review, and ensuring compliance with GLP and regulatory standards.

5. Procedure for HPLC Analysis

5.1 Pre-Analysis Requirements

Ensure the injection sequence is available before instrument startup.
Confirm that the method parameters align with the respective STP.
Verify instrument readiness and calibration status.

5.2 Mobile Phase Preparation

Proper mobile phase preparation is crucial for reliable HPLC Analysis.

5.2.1 Preparation

Prepare the mobile phase according to the composition specified in the respective STP.

5.2.2 Labeling Requirements

Each mobile phase container must include:

Mobile Phase Name
Product Name
Analysis Of
Prepared By
Use Before Date
Buffer weight(s)
Balance number
Observed pH

All preparation details must be recorded in the analysis record.

5.2.3 Validity

Do not use mobile phase beyond 7 days from the preparation date.
Discard immediately if haziness or precipitation is observed.

5.3 Resolution Solution

If a resolution solution is stored for extended use, its shelf life must be validated based on:

Daily system suitability requirements as per STP.
Main analyte peak area variation must not exceed 20% compared to limit area count.
Impurity peak area variation must not exceed 20% compared to the initial area count.

5.4 Standard Preparation

Prepare the standard solution as per STP.
Consider stability and storage requirements.
Use properly labeled and documented containers.

5.5 HPLC Analysis Procedure

5.5.1 System Suitability

Before sample analysis:

Establish system suitability as per STP.
System suitability is valid for a maximum of 24 hours.

After every 24 hours:

Re-establish system suitability.
For assay and related substances, inject standard solution in triplicate.
% RSD for triplicate injections must be NMT (Not More Than) 1%.

5.5.2 Injection Sequence (Guideline)

The following sequence may be followed unless otherwise specified in STP:

Blank → System Suitability → Blank → Placebo (if required) → Impurity Standard (if applicable) → Standard → Sample

All injection sequences must be captured and recorded from the system.

5.6 Acceptance Criteria in HPLC Analysis

5.6.1 Resolution Solution

Stored resolution solution must meet acceptance criteria every day of use. If not, prepare fresh solution.

5.6.2 Duplicate/Triplicate Injections

Area ratio between successive injections must be between 0.99 – 1.01 of the mean.
If STP does not specify RSD limits, this same area ratio range applies to standard injections.

5.6.3 Standard Consistency

The area ratio between intermittent standards and initial standards must lie between 0.99 – 1.01 of the mean.

5.6.4 Isocratic Runs

Flow rate must remain constant throughout the run.
If flow changes for more than 200 minutes, re-establish system suitability.

5.6.5 Gradient Runs

If sample injection is delayed beyond runtime, continue blank gradient runs.
Record blank chromatograms.

5.6.6 Non-Conformance Handling

If acceptance criteria are not met:

Identify affected data.
Supervisor review is mandatory.
Re-establish system suitability before proceeding.

5.7 Recording and Handling Chromatograms

Proper documentation is essential in regulated HPLC Analysis.

5.7.1 Documentation

Record all chromatograms from pre-system suitability through the full run.
Print necessary system-generated details.

5.7.2 Disregarded Chromatograms

If a chromatogram must be disregarded:

Submit to Supervisor/QAM for review.
Stamp as “DISREGARDED”.
Clearly state the reason on the chromatogram.

Common reasons include:

Area variation
Inconsistent integration
Baseline drift
Ghost peaks
Faulty integration

Disregarded chromatograms must be filed along with valid test chromatograms.

5.7.3 Integration Parameters

Document:

Peak width
Peak threshold
Minimum peak area
Peak height

5.7.4 Reprocessing

If reprocessing is required:

Document reason with date/time.
Obtain QAM certification.

5.8 Calculation

Perform calculations as per STP.
Use the area count from the initial standard injection.
Do not use intermediate standard area counts for final calculations.

Include calculations such as Relative Response Factor (RRF), where applicable.

6. Abbreviations

SOP – Standard Operating Procedure
HPLC – High Performance Liquid Chromatography
STP – Standard Testing Procedure
RSD – Relative Standard Deviation

Why a Structured SOP for HPLC Analysis Matters

A well-defined SOP for HPLC Analysis ensures:

Regulatory compliance (GLP/GMP)
Data integrity
Reproducibility of results
Audit readiness
Minimized analytical errors

By following standardized documentation and system suitability requirements, laboratories maintain consistent analytical performance and ensure reliable product quality assessment.

Frequently Asked Questions (FAQs)

1. What is the purpose of an SOP for HPLC Analysis?

An SOP for HPLC Analysis ensures that the analytical procedure is performed consistently, accurately, and in compliance with Good Laboratory Practices. It helps maintain data integrity and regulatory compliance.

2. How long can a mobile phase be used in HPLC Analysis?

Typically, a mobile phase should not be used beyond 7 days from the date of preparation. It must also be discarded immediately if any haziness or precipitation is observed.

3. What is system suitability in HPLC Analysis?

System suitability is a verification process performed before sample analysis to ensure the HPLC system is functioning correctly. It includes parameters such as %RSD, peak resolution, and retention time consistency.

4. When should system suitability be re-established?

System suitability must be re-established after 24 hours or if significant changes occur, such as flow rate variation exceeding specified limits or system interruptions.

5. What should be done if acceptance criteria are not met during HPLC Analysis?

If acceptance criteria fail, the affected data must be identified, reviewed by a supervisor, and system suitability must be re-established before continuing the analysis.