and regulatory compliance using validated GC parameters and techniques.

2. Scope

This procedure is applicable to the Analytical Method Development (AMD) department and is intended for APIs that are volatile at ambient or elevated temperatures and require GC-based analysis for assay determination.

3. Responsibilities

• Analytical Chemist: Executes method development, sample preparation, and chromatographic analysis.
• Method Reviewer: Ensures scientific and regulatory soundness of the developed GC assay method.
• QA Officer: Verifies all documentation, calibration, and suitability checks.
• Head – AMD: Approves finalized assay method for validation and routine use.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring that all GC assay methods for volatile APIs meet the validation requirements set by ICH Q2(R1) and are suitable for intended use.

5. Procedure

5.1 Selection of Analytical Technique

1. Identify APIs with volatility above 40°C or vapor pressure >10 mmHg.
2. Evaluate GC compatibility and select direct injection or headspace based on matrix and stability.
3. Document rationale in Annexure-1: Technique Justification Log.

5.2 Instrument Configuration

1. Instrument: GC with Flame Ionization Detector (FID).
2. Column: DB-1 or equivalent non-polar column, 30 m × 0.25 mm ID × 0.25 µm film thickness.
3. Carrier Gas: Helium at constant flow (1 mL/min).
4. Oven Program: 50°C (1 min) to 250°C at 10°C/min.
5. Detector and Injector Temperatures: 250°C and 220°C, respectively.
6. Record in Annexure-2: GC System Setup Log.

5.3 Sample and Standard Preparation

1. Standard: Prepare in DMSO, DMF, or IPA at 100% concentration level of label claim.
2. Sample: Weigh accurately (e.g., 100 mg) into a 10 mL volumetric flask, add diluent, and vortex.
3. If needed, analyze freshly due to evaporation risk.
4. Document all preparation in Annexure-3: Sample Preparation Log.

5.4 Calibration and Linearity

1. Prepare standard solutions at 50%, 80%, 100%, 120%, and 150% of target concentration.
2. Inject in duplicate and plot concentration vs. peak area.
3. Acceptance Criteria:
   • R² ≥ 0.995
   • %RSD ≤ 2% for replicate injections
4. Document in Annexure-4: Linearity and Calibration Report.

5.5 Assay Calculation

1. Calculate API assay using:

   Assay (%) = (Area_sample / Area_standard) × (Conc_standard / Conc_sample) × 100
   

2. Include dilution factors and response factors if applicable.
3. Summarize results in Annexure-5: GC Assay Calculation Sheet.

5.6 System Suitability and Specificity

1. Inject standard solution six times and calculate:
   • %RSD of peak area ≤ 2%
   • RT consistency (SD ≤ 0.05 min)
2. Perform blank and placebo injection to ensure no interfering peaks.
3. Overlay chromatograms for confirmation and record in Annexure-6: System Suitability Report.

5.7 Method Robustness

1. Assess method response by varying:
   • Oven ramp rate ±2°C/min
   • Injector temperature ±5°C
   • Split ratio (if applicable)
2. Acceptance: Assay results within ±2% of original.
3. Document findings in Annexure-7: Robustness Study Log.

6. Abbreviations

• API: Active Pharmaceutical Ingredient
• GC: Gas Chromatography
• FID: Flame Ionization Detector
• RT: Retention Time
• RSD: Relative Standard Deviation
• SOP: Standard Operating Procedure

7. Documents

1. Technique Justification Log – Annexure-1
2. GC System Setup Log – Annexure-2
3. Sample Preparation Log – Annexure-3
4. Linearity and Calibration Report – Annexure-4
5. GC Assay Calculation Sheet – Annexure-5
6. System Suitability Report – Annexure-6
7. Robustness Study Log – Annexure-7

8. References

• ICH Q2(R1) – Validation of Analytical Procedures
• USP <621> – Chromatography
• USP <731> – Loss on Drying (if applicable)
• FDA Guidance – Analytical Procedures and Methods Validation

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Technique Justification Log

API	Boiling Point (°C)	Justification for GC
Ethyl acetate	77	Volatile, suitable for FID detection

Annexure-2: GC System Setup Log

Instrument	Column	Detector	Carrier Gas	Oven Program
Agilent 7890	DB-1, 30 m	FID	He	50°C to 250°C @ 10°C/min

Annexure-3: Sample Preparation Log

ID	Sample Type	Weight (mg)	Diluent	Prepared By
API-001	Volatile API	100	DMSO	Rajesh Kumar

Annexure-4: Linearity and Calibration Report

Level (%)	Concentration (ppm)	Peak Area	Status
100%	1000	145623	Pass

Annexure-5: GC Assay Calculation Sheet

Sample ID	Area Sample	Area Standard	Result (%)
API-001	143220	145623	98.4%

Annexure-6: System Suitability Report

Injection No.	Peak Area	RT (min)
1	145623	5.47
2	145481	5.46

Annexure-7: Robustness Study Log

Parameter Changed	Value	Assay (%)	Deviation
Injector Temp	225°C	97.8%	-0.6%

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Integrated assay calculation logic and robustness annexures	Annual SOP Review