SOP for Gas Evolution Reaction-Based Analytical Method in AMD Laboratory

Department	Analytical Method Development
SOP No.	SOP/AMD/061/2025
Supersedes	SOP/AMD/061/2022
Page No.	Page 1 of 14
Issue Date	19/05/2025
Effective Date	20/05/2025
Review Date	19/05/2026

1. Purpose

The purpose of this SOP is to establish a procedure for the development and validation of analytical methods based on gas evolution reactions. These methods involve quantification of analytes that release a measurable gas (e.g., CO₂, O₂, H₂) as part of their chemical reaction with specific reagents.

2. Scope

This SOP is applicable to the Analytical Method Development (AMD) laboratory and covers classical chemical methods that rely on gas evolution (e.g., carbonate determination via CO₂ evolution, peroxide determination by O₂ release) for analysis of raw materials, intermediates, and drug products.

3. Responsibilities

• Analytical Chemist: Executes method development trials, prepares reaction vessels, performs measurements, and documents observations.
• Reviewer: Verifies calculations, consistency of results, and ensures the accuracy of validation records.
• QA Officer: Ensures adherence to GMP principles and verifies documentation practices.
• Head – AMD: Reviews and approves validated methods for official use.

4. Accountability

The Head of AMD is accountable for ensuring that all gas evolution methods developed are scientifically justified, properly validated, and regulatory compliant.

5. Procedure

5.1 Method Selection and Principles

1. Select suitable gas evolution reaction based on analyte functionality. Examples:
   • Sodium bicarbonate/carbonate → CO₂ upon acid addition
   • Peroxides → O₂ evolution with potassium iodide
   • Hydrazine or metal hydrides → H₂ release in aqueous medium
2. Detection may be gravimetric (absorption in lime), volumetric (displacement), or titrimetric (back-titration after gas trapping).
3. Document rationale in Annexure-1: Method Selection Justification Log.

5.2 Apparatus and Setup

1. Use suitable reaction vessels like Kjeldahl flasks, gas burettes, or closed titration apparatus fitted with gas traps (e.g., Ba(OH)₂ or NaOH).
2. Ensure all apparatus is airtight and fitted with drying tubes if required.
3. For volumetric gas measurement, calibrate gas burette using known gas volume (e.g., atmospheric pressure water displacement method).
4. Document apparatus setup in Annexure-2: Equipment Preparation Log.

5.3 Sample Preparation

1. Weigh an accurate quantity of sample containing the analyte (e.g., 250 mg sodium bicarbonate).
2. Dissolve or suspend in distilled water if required.
3. Record sample weight and preparation in Annexure-3: Sample Preparation Log.

5.4 Reaction and Gas Collection

1. Place the prepared sample in the reaction chamber.
2. Add excess standardized reagent (e.g., HCl for carbonate analysis) through dropping funnel or septum port.
3. Allow gas to evolve and capture it in a connected gas absorption tube or volume displacement chamber.
4. Continue reaction until no further gas bubbles are visible.
5. For gravimetric methods, weigh the BaCO₃ formed from CO₂ in Ba(OH)₂.
6. For volumetric methods, note the displaced volume in burette and correct for temperature and pressure.
7. Record all reaction observations in Annexure-4: Gas Evolution Log.

5.5 Calculation

For carbonate determination by CO₂ evolution (titration method):

% Carbonate = (V × N × 0.060 × 100) / W
Where:
V = Volume of HCl consumed (mL)
N = Normality of HCl
W = Weight of sample (g)
0.060 = Equivalent weight of carbonate per mL N HCl

For gravimetric gas capture:

% Analyte = (Weight of precipitate × Conversion Factor × 100) / Sample Weight

5.6 Method Optimization

1. Optimize reaction conditions such as:
   • Acid concentration and volume
   • Reaction temperature (room temp vs. heating)
   • Rate of reagent addition to control foaming or violent reactions
2. Ensure complete gas capture by verifying plateau in evolution volume.
3. Perform duplicate trials to verify reproducibility.
4. Document outcomes in Annexure-5: Method Optimization Report.

5.7 Method Validation

1. Specificity: Use placebo matrices and excipient blanks to confirm absence of interfering gases.
2. Precision: Intra- and inter-day precision RSD ≤ 2.0% for six replicates.
3. Accuracy: Spike recovery at 80%, 100%, and 120% of target analyte level should be 98–102%.
4. Linearity: Prepare samples across 50–150% range and plot vs. gas volume evolved or titration volume.
5. Robustness: Test method sensitivity to variations in temperature, acid concentration, and reaction time.
6. Summarize all findings in Annexure-6: Validation Summary Report.

6. Abbreviations

• SOP: Standard Operating Procedure
• AMD: Analytical Method Development
• RSD: Relative Standard Deviation
• CO₂: Carbon Dioxide
• HCl: Hydrochloric Acid

7. Documents

1. Method Selection Justification Log – Annexure-1
2. Equipment Preparation Log – Annexure-2
3. Sample Preparation Log – Annexure-3
4. Gas Evolution Log – Annexure-4
5. Method Optimization Report – Annexure-5
6. Validation Summary Report – Annexure-6

8. References

• USP General Chapters <541>, <721> – Titrimetry and Volumetric Analysis
• IP Appendix 3.4 – Carbonates and Bicarbonates
• ICH Q2(R1) – Validation of Analytical Procedures
• AOAC Methods for Gas Evolution Analysis

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Method Selection Justification Log

Analyte	Gas Evolved	Detection Method	Analyst
Sodium Bicarbonate	CO2	Titrimetric via BaCO3	Sunita Reddy

Annexure-2: Equipment Preparation Log

Apparatus	ID	Calibrated On	Next Due Date	Checked By
Gas Burette	GB-003	01/04/2025	01/10/2025	Ajay Mehra

Annexure-3: Sample Preparation Log

Sample	Weight	Solvent	Analyst
NaHCO3 Powder	250 mg	None (direct)	Rajesh Kumar

Annexure-4: Gas Evolution Log

Trial	Volume of HCl Used	Gas Volume Evolved	Reaction Time	% Assay
1	25.0 mL	24.3 mL (CO2)	12 mins	99.2%

Annexure-5: Method Optimization Report

Parameter	Change Applied	Observation	Conclusion
Acid Volume	+10%	Ensured full reaction	Accepted

Annexure-6: Validation Summary Report

Parameter	Criteria	Result	Status
Accuracy	98–102%	99.4%	Pass
Precision	RSD ≤ 2.0%	0.78%	Pass
Linearity	R² ≥ 0.999	0.9992	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added validation section and expanded gas detection modalities	Annual SOP Review