Good Manufacturing Practices (cGMP).

2. Scope

This SOP applies to all HPLC systems installed and used within the Analytical Method Development (AMD) department for quantitative and qualitative analyses.

3. Responsibilities

• Analyst: Performs daily and periodic calibration tasks and records results.
• QA Reviewer: Reviews calibration records for accuracy, completeness, and compliance.
• Instrument Engineer: Investigates calibration failures and performs corrective actions.

4. Accountability

The Department Head is accountable for ensuring that HPLC systems are calibrated according to the defined schedule and that records are maintained as per regulatory requirements.

5. Procedure

5.1 Calibration Frequency

1. Daily calibration check: wavelength accuracy and pump pressure consistency.
2. Monthly calibration: flow rate, injector precision, and detector linearity.
3. Annual calibration: comprehensive validation by service engineer or trained personnel.

5.2 Calibration Standards and Reagents

1. Use NIST-traceable caffeine or holmium oxide for wavelength accuracy.
2. Use acetone or benzoic acid standards for UV absorbance calibration.
3. Ensure all solutions are prepared fresh and stored under recommended conditions.

5.3 Calibration Steps

5.3.1 Wavelength Accuracy

1. Inject caffeine standard (e.g., 0.02% w/v) and record UV spectrum.
2. Verify absorbance maxima at 205, 245, and 273 nm for conformance.

5.3.2 Flow Rate Accuracy

1. Set flow rate at 1.0 mL/min using water as mobile phase.
2. Collect effluent for 5 minutes and measure volume.
3. Acceptable range: ±2% of set flow rate.

5.3.3 Injector Precision

1. Inject standard solution six times consecutively.
2. Calculate % RSD of peak areas; acceptance criteria: ≤1.0%.

5.3.4 Detector Linearity

1. Prepare standard solutions at 50%, 75%, 100%, 125%, and 150% of target concentration.
2. Inject and record responses; plot concentration vs. response.
3. Acceptable R² ≥ 0.998.

5.3.5 Column Oven and Autosampler Temperature

1. Verify setpoint vs. actual temperature using calibrated thermometer.
2. Acceptance: ±2°C of set temperature.

5.4 Documentation

1. Enter calibration data in the HPLC Calibration Logbook (Annexure-1).
2. Attach chromatograms, spectra, and calibration curves as supporting data.
3. In case of calibration failure, initiate deviation and perform re-calibration after troubleshooting.

6. Abbreviations

• HPLC: High-Performance Liquid Chromatography
• RSD: Relative Standard Deviation
• QA: Quality Assurance
• R²: Coefficient of Determination

7. Documents

1. Calibration Logbook – Annexure-1
2. Calibration Certificates – Annexure-2
3. Deviation Report Format – Annexure-3

8. References

• ICH Q2(R1): Validation of Analytical Procedures
• 21 CFR Part 211: Current Good Manufacturing Practices
• USP <1058>: Analytical Instrument Qualification

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Rahul Verma	Priya Deshmukh	Dr. Harshita Goyal
Designation	AMD Analyst	QA Reviewer	Head – Analytical Method Development
Department	Analytical Method Development	Quality Assurance	Analytical Method Development

11. Annexures

Annexure-1: HPLC Calibration Logbook Format

Date	Parameter	Result	Acceptance Criteria	Status
01/06/2025	Flow Rate Accuracy	0.98 mL/min	0.98–1.02 mL/min	Pass

Annexure-2: Calibration Certificates

Attach valid calibration certificates from external vendors for modules such as UV detector, pump, and temperature controller.

Annexure-3: Deviation Report

Deviation ID: DEV-HPLC-311-01
Issue: Injector RSD exceeded limits
Root Cause: Air bubble in loop
CAPA: Loop purged, SOP updated for preventive flushing

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
01/06/2025	2.0	Updated calibration parameters and annexure formats	Annual SOP Review	Dr. Harshita Goyal
01/04/2022	1.0	Initial Release	New SOP	QA Head