stability during quantitative and qualitative analysis.

2. Scope

This SOP applies to all HPLC-based analytical methods developed within the Analytical Method Development (AMD) department for testing raw materials, active pharmaceutical ingredients (APIs), intermediates, and finished pharmaceutical formulations.

3. Responsibilities

• Analytical Chemist: Executes solubility studies, prepares samples, selects appropriate diluents, and performs filtration.
• Reviewer: Verifies completeness and appropriateness of sample prep strategy including accuracy, recovery, and matrix compatibility.
• QA Officer: Ensures GMP-compliant recording of all sample prep parameters and logs.
• Head – AMD: Approves the finalized sample preparation method and its integration into the method development protocol.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring all sample preparation strategies are scientifically justified and documented as per regulatory standards (ICH Q2, WHO, USP).

5. Procedure

5.1 Pre-Preparation Evaluation

1. Perform visual inspection of the sample for homogeneity, color, and physical form.
2. Check COA or formulation composition to identify:
   • Expected concentration of API
   • Excipient types (e.g., binders, coatings, diluents)
   • Solubility or known stability issues
3. Record in Annexure-1: Sample Evaluation Sheet.

5.2 Solubility and Diluent Selection

1. Conduct preliminary solubility study:
   • Diluents to test: Water, Acetonitrile, Methanol, Buffer (pH 3–7), DMSO
   • Record qualitative observations (clear, turbid, precipitate)
2. Select diluent that:
   • Dissolves API completely at desired concentration
   • Does not interfere at analyte’s retention time
   • Is stable over analysis period (typically 24–48 hours)
3. Document findings in Annexure-2: Diluent Selection Log.

5.3 Sample Weighting and Preparation

1. Weigh target amount of sample (e.g., 50–200 mg) into a volumetric flask.
2. Add 70–80% of total diluent volume and sonicate for 10–15 minutes.
3. Make up to volume and mix thoroughly by inversion.
4. If needed, perform sample extraction using liquid-liquid or solid-liquid extraction methods.
5. Document in Annexure-3: Sample Preparation Log.

5.4 Filtration and Clarification

1. Filter sample using 0.45 µm syringe filter (PVDF or Nylon preferred).
2. Perform filter compatibility and adsorption check using:
   • Spiked recovery of standard (80%, 100%, 120%)
   • Compare with unfiltered sample
3. Ensure recovery is within 98–102%.
4. Record results in Annexure-4: Filtration Recovery Log.

5.5 Matrix Interference Check

1. Inject:
   • Blank diluent
   • Placebo sample (without API)
   • Standard
   • Prepared sample
2. Ensure no interference near analyte RT (±1 minute).
3. Evaluate for ghost peaks or overlapping signals.
4. Document chromatograms and results in Annexure-5: Matrix Interference Assessment Sheet.

5.6 Stability Study of Sample Solution

1. Keep aliquots of prepared sample at ambient and refrigerated conditions.
2. Re-inject after 4, 8, and 24 hours.
3. Compare peak area and retention time to time zero sample.
4. Acceptance criteria: % deviation ≤ 2.0% over 24 hours.
5. Summarize in Annexure-6: Sample Stability Summary.

5.7 Finalization of Sample Prep Strategy

1. Review all logs and chromatographic data.
2. Finalize:
   • Weighing range
   • Diluent and concentration
   • Extraction technique (if any)
   • Filtration method
3. Record finalized strategy in Annexure-7: Sample Preparation Strategy Summary.

6. Abbreviations

• API: Active Pharmaceutical Ingredient
• HPLC: High-Performance Liquid Chromatography
• RT: Retention Time
• RSD: Relative Standard Deviation
• SOP: Standard Operating Procedure

7. Documents

1. Sample Evaluation Sheet – Annexure-1
2. Diluent Selection Log – Annexure-2
3. Sample Preparation Log – Annexure-3
4. Filtration Recovery Log – Annexure-4
5. Matrix Interference Assessment Sheet – Annexure-5
6. Sample Stability Summary – Annexure-6
7. Sample Preparation Strategy Summary – Annexure-7

8. References

• ICH Q2(R1) – Validation of Analytical Procedures
• USP <621> – Chromatography
• FDA Guidance for Industry – 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: Sample Evaluation Sheet

Sample ID	Type	API Label Claim	Appearance	Known Interference
API-101	Tablet	500 mg	White coated	Sorbitol

Annexure-2: Diluent Selection Log

Diluent	Solubility	Comments
Methanol	Complete	No interference
Buffer pH 4.5	Partial	Cloudy on standing

Annexure-3: Sample Preparation Log

Sample ID	Weight (mg)	Volume (mL)	Diluent	Sonication (min)
SP-001	100.5	100	Methanol	10

Annexure-4: Filtration Recovery Log

Filter Type	Spiked Level (%)	Recovery (%)	Status
PVDF	100%	99.4%	Acceptable

Annexure-5: Matrix Interference Assessment Sheet

Injection	Interference at RT	Peak Area	Status
Placebo	No	0	Complies

Annexure-6: Sample Stability Summary

Time Point	Peak Area	% Change	Status
0 hr	104532	—	—
24 hr	103890	-0.6%	Complies

Annexure-7: Sample Preparation Strategy Summary

Sample ID	Weight Range	Diluent	Preparation Steps	Final Concentration
API-101	95–105 mg	Methanol	Sonicate & filter	1000 µg/mL

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added matrix interference and solution stability annexures	Annual SOP Review