SOP for Total Ash and Acid Insoluble Ash Method Development in AMD Laboratory

Department	Analytical Method Development
SOP No.	SOP/AMD/049/2025
Supersedes	SOP/AMD/049/2022
Page No.	Page 1 of 14
Issue Date	19/05/2025
Effective Date	20/05/2025
Review Date	19/05/2026

1. Purpose

This SOP defines the procedure for developing and validating analytical methods for the determination of Total Ash and Acid Insoluble Ash in APIs, excipients, and pharmaceutical formulations using ignition and hydrochloric acid treatment, as per pharmacopeial specifications.

2. Scope

This SOP is applicable to the Analytical Method Development (AMD) department for the quantitative assessment of total inorganic residue and acid-insoluble matter in raw materials and formulations, using muffle furnace techniques in accordance with IP, USP, and Ph. Eur. standards.

3. Responsibilities

• Analytical Chemist: Carries out sample ignition, acid treatment, and records results.
• Reviewer: Verifies calculations and confirms repeatability of method.
• QA: Reviews validation data and ensures method compliance with compendial guidelines.
• Head – AMD: Approves method and ensures integration into development reports and regulatory submissions.

4. Accountability

The Head of AMD is accountable for ensuring that the Total Ash and Acid Insoluble Ash methods are validated and conducted according to regulatory and pharmacopeial requirements.

5. Procedure

5.1 Selection of Method Type

1. Total Ash: Determines all inorganic content after igniting the sample to constant weight.
2. Acid Insoluble Ash: Measures portion of ash that remains insoluble after treatment with dilute hydrochloric acid.
3. Refer to IP, USP <281>, or Ph. Eur. 2.4.16 for guidance.

5.2 Equipment and Crucible Preparation

1. Use a silica crucible cleaned, ignited, and cooled in a desiccator prior to use.
2. Ensure muffle furnace is calibrated and certified for 600 ± 25°C operations.
3. Record crucible tare weights in Annexure-1: Crucible Weight Record.

5.3 Total Ash Determination

1. Accurately weigh 2–3 g of the powdered sample into the prepared crucible.
2. Ignite slowly at first to avoid loss due to spattering. Then raise temperature gradually to 600 ± 25°C.
3. Continue heating until a light grey or white ash is obtained (no black particles).
4. Cool in a desiccator and weigh the residue.
5. Repeat heating until constant weight is achieved (weight difference ≤ 0.5 mg).
6. Calculate Total Ash (%) using:

   Total Ash (%) = (Weight of ash / Weight of sample) × 100

7. Document results in Annexure-2: Total Ash Worksheet.

5.4 Acid Insoluble Ash Determination

1. Add 25 mL of 2N HCl to crucible containing total ash.
2. Cover with watch glass and gently boil for 5 minutes.
3. Filter the mixture using ashless filter paper and hot water wash until neutral.
4. Transfer filter paper with residue back to the crucible and ignite at 600 ± 25°C.
5. Cool and weigh until constant mass.
6. Calculate Acid Insoluble Ash (%) using:

   Acid Insoluble Ash (%) = (Weight of acid-insoluble residue / Weight of sample) × 100

7. Record results in Annexure-3: Acid Insoluble Ash Worksheet.

5.5 Method Optimization

1. Conduct method trials to evaluate:
   • Sample weight variation (1 g, 2 g, 3 g)
   • Furnace holding time (2 vs 3 hours)
   • Volume and concentration of HCl used
2. Document findings in Annexure-4: Method Optimization Log.

5.6 Method Validation

1. Follow ICH Q2(R1) validation guidelines:
2. Precision: Triplicate determinations with RSD ≤ 2.0%.
3. Accuracy: Spike samples with known mineral content and recover within 98–102%.
4. Specificity: Ensure no interference from volatile or acid-soluble matter.
5. Robustness: Confirm results under slight variation in ignition temperature or acid volume.
6. Summarize in Annexure-5: Validation Summary Report.

6. Abbreviations

• AMD: Analytical Method Development
• RSD: Relative Standard Deviation
• USP: United States Pharmacopeia
• Ph. Eur.: European Pharmacopoeia
• QA: Quality Assurance

7. Documents

1. Crucible Weight Record – Annexure-1
2. Total Ash Worksheet – Annexure-2
3. Acid Insoluble Ash Worksheet – Annexure-3
4. Method Optimization Log – Annexure-4
5. Validation Summary Report – Annexure-5

8. References

• USP <281> – Residue on Ignition
• Ph. Eur. 2.4.16 – Ash Tests
• IP 2022 – Total and Acid Insoluble Ash
• ICH Q2(R1) – Validation of Analytical Procedures

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Crucible Weight Record

Date	Crucible ID	Empty Weight (g)	Prepared By
03/05/2025	C-101	32.128	Meenal Patil

Annexure-2: Total Ash Worksheet

Sample	Initial Weight (g)	Residue Weight (g)	Total Ash (%)	Analyst
Excipient-X	2.000	0.046	2.30	Ajay Mehra

Annexure-3: Acid Insoluble Ash Worksheet

Sample	Residue Weight (g)	Insoluble Residue (g)	Acid Insoluble Ash (%)	Analyst
Excipient-X	0.046	0.003	0.15	Sunita Reddy

Annexure-4: Method Optimization Log

Trial	Sample Size	Ignition Time	HCl Volume	Total Ash (%)	Conclusion
1	1 g	2 h	20 mL	2.78	Acceptable
2	2 g	3 h	25 mL	2.30	Optimized

Annexure-5: Validation Summary Report

Parameter	Acceptance Criteria	Result	Status
Precision	RSD ≤ 2%	1.5%	Pass
Accuracy	98–102%	99.3%	Pass
Robustness	±10°C variation	Consistent	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Included optimization data and annexure restructuring	Annual Review Update