procedure applies to the Analytical Method Development (AMD) laboratory and covers all UV spectrophotometric methods where determination of wavelength is necessary for quantitation, identification, or method validation of Active Pharmaceutical Ingredients (APIs) or finished products.

3. Responsibilities

• Analytical Chemist: Conducts scanning, identifies λ_max, and performs initial evaluations.
• Reviewer: Validates selection of λ_max and ensures absence of interference or anomalies.
• QA Officer: Ensures compliance of documentation and adherence to regulatory practices.
• Head – AMD: Approves final wavelength selection for use in method development.

4. Accountability

The Head of Analytical Method Development is accountable for confirming the scientific integrity of wavelength selection and proper documentation for regulatory and analytical compliance.

5. Procedure

5.1 Preparation of Standard Solution

1. Weigh 10 mg of API and dissolve in 100 mL of suitable solvent (e.g., water, methanol, ethanol).
2. Sonicate if required to ensure complete dissolution.
3. Further dilute to a concentration of 10–50 µg/mL for scanning range.
4. Label solution and record preparation in Annexure-1: Sample Preparation Log.

5.2 Instrument Setup and Blank Correction

1. Switch on the UV-visible spectrophotometer and allow warm-up (20–30 min).
2. Select scanning mode (full or selected range).
3. Set scan speed (typically 240 nm/min) and bandwidth (1 nm).
4. Run blank with same solvent in both reference and sample cell compartments.
5. Document baseline in Annexure-2: Instrument Blank Log.

5.3 Scanning Procedure

1. Fill sample cell with test solution (typically 1 cm quartz cuvette).
2. Scan from 200 to 400 nm or a narrower range depending on molecular structure.
3. Ensure no particulate matter is present in the sample that may affect the signal.
4. Record spectrum and save electronically with sample ID and date.

5.4 Identification and Selection of λ_max

1. Observe highest absorbance peak in the scanned spectrum. Designate it as λ_max.
2. Evaluate peak shape: should be symmetrical, sharp, and free from overlapping signals.
3. Absorbance at λ_max should lie within linear detector range (0.2–1.0 AU ideally).
4. If more than one peak is observed, select the most stable and reproducible wavelength for quantification.
5. Record final selected wavelength in Annexure-3: Wavelength Selection Report.

5.5 Specificity and Interference Check

1. Scan placebo and blank at same parameters to ensure no interference at selected λ_max.
2. Overlay placebo, blank, and sample spectra to confirm specificity.
3. Document overlay data in Annexure-4: Interference Evaluation Sheet.

5.6 Stability of λ_max and Repeatability

1. Re-scan standard solution after 30 minutes to confirm stability of λ_max.
2. Acceptance criteria: ±1 nm shift in λ_max is acceptable.
3. If shift is observed, investigate possible degradation, solvent interaction, or instrument drift.
4. Document repeat scans in Annexure-5: Stability Check Record.

6. Abbreviations

• UV: Ultraviolet
• API: Active Pharmaceutical Ingredient
• λ_max: Wavelength of Maximum Absorbance
• SOP: Standard Operating Procedure
• AU: Absorbance Unit

7. Documents

1. Sample Preparation Log – Annexure-1
2. Instrument Blank Log – Annexure-2
3. Wavelength Selection Report – Annexure-3
4. Interference Evaluation Sheet – Annexure-4
5. Stability Check Record – Annexure-5

8. References

• ICH Q2(R1) – Validation of Analytical Procedures
• USP <857> – Ultraviolet-Visible Spectrophotometry
• Pharmaceutical Analysis Textbooks
• Instrument Operation Manuals

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 Preparation Log

Sample ID	Weight (mg)	Solvent	Final Volume (mL)	Concentration (µg/mL)
WS-001	10	Methanol	100	100

Annexure-2: Instrument Blank Log

Date	Solvent Used	Absorbance Range	Remarks
19/05/2025	Methanol	0.000–0.002	Acceptable

Annexure-3: Wavelength Selection Report

Sample ID	λmax (nm)	Absorbance	Scan Date
WS-001	274	0.845	19/05/2025

Annexure-4: Interference Evaluation Sheet

Solution	λmax	Absorbance	Interference Observed
Placebo	274	0.003	No
Blank	274	0.001	No

Annexure-5: Stability Check Record

Time Interval	λmax (nm)	Absorbance	Status
0 min	274	0.845	Stable
30 min	274	0.842	Stable

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Enhanced specificity check and updated annexure structure	Annual SOP Review