concentration range in accordance with ICH Q2(R1).

2. Scope

This SOP applies to the Analytical Method Development (AMD) department and covers linearity studies performed for Active Pharmaceutical Ingredients (APIs) and drug products analyzed by UV-visible spectrophotometry.

3. Responsibilities

• Analytical Chemist: Prepares dilution series, measures absorbance, and performs calculations.
• Reviewer: Verifies calibration curve, linear regression output, and correlation coefficient.
• QA Officer: Audits raw data and ensures compliance with validation protocols.
• Head – AMD: Approves final linearity data for method validation or regulatory submission.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring that linearity studies are performed according to validated procedures and meet required statistical acceptance criteria.

5. Procedure

5.1 Preparation of Standard Stock Solution

1. Accurately weigh approximately 100 mg of the API.
2. Dissolve in a suitable solvent (e.g., water, methanol, ethanol) in a 100 mL volumetric flask.
3. Sonicate to dissolve completely and make up to volume. This yields a stock solution of 1 mg/mL (1000 µg/mL).
4. Label as Standard Stock Solution and document in Annexure-1: Preparation Log.

5.2 Preparation of Linearity Dilutions

1. From the stock solution, prepare at least five concentration levels (e.g., 50%, 75%, 100%, 125%, and 150% of test concentration).
2. Example for 100 µg/mL target concentration:
   • 50 µg/mL
   • 75 µg/mL
   • 100 µg/mL
   • 125 µg/mL
   • 150 µg/mL
3. Use the same diluent for all dilutions and store samples in amber glassware if photosensitive.
4. Log each dilution in Annexure-2: Linearity Dilution Table.

5.3 Measurement of Absorbance

1. Use UV-visible spectrophotometer calibrated and qualified as per instrument SOP.
2. Measure absorbance of each dilution in triplicate at the selected λ_max.
3. Use 1.0 cm matched quartz cuvettes and ensure instrument is zeroed using blank solvent.
4. Document readings in Annexure-3: Absorbance Data Sheet.

5.4 Plotting Calibration Curve

1. Plot absorbance values on the Y-axis and concentration on the X-axis.
2. Use Microsoft Excel or validated software to perform linear regression.
3. Obtain slope, intercept, correlation coefficient (r), and regression equation.
4. Example output: y = 0.0047x + 0.0032; R² = 0.9996
5. Paste graph and calculations into Annexure-4: Calibration Graph and Regression Output.

5.5 Evaluation Criteria

1. Acceptance Criteria:
   • Correlation coefficient (r) ≥ 0.995
   • Line passes through or near origin (intercept ≤ ±0.01)
   • Residuals randomly distributed (no systematic error)
2. Calculate %RSD at each concentration level. Acceptance limit: ≤2.0%
3. Document in Annexure-5: Linearity Summary Sheet.

5.6 Repeatability Check

1. Inject 100% concentration level six times.
2. Calculate %RSD of absorbance values. Acceptance limit: ≤2.0%
3. Include in final method validation report.

6. Abbreviations

• UV: Ultraviolet
• API: Active Pharmaceutical Ingredient
• RSD: Relative Standard Deviation
• SOP: Standard Operating Procedure
• r: Correlation Coefficient
• λ_max: Wavelength of Maximum Absorbance

7. Documents

1. Preparation Log – Annexure-1
2. Linearity Dilution Table – Annexure-2
3. Absorbance Data Sheet – Annexure-3
4. Calibration Graph and Regression Output – Annexure-4
5. Linearity Summary Sheet – Annexure-5

8. References

• ICH Q2(R1) – Validation of Analytical Procedures: Text and Methodology
• USP <857> – Ultraviolet-Visible Spectroscopy
• Pharmaceutical Method Validation Guidelines – FDA, EMA

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Preparation Log

Compound	Weight (mg)	Solvent	Volume (mL)	Prepared By
API-LIN-001	100.0	Methanol	100	Rajesh Kumar

Annexure-2: Linearity Dilution Table

Level (%)	Concentration (µg/mL)	Dilution Details
50%	50	5 mL stock in 100 mL
100%	100	10 mL stock in 100 mL

Annexure-3: Absorbance Data Sheet

Concentration (µg/mL)	Absorbance 1	Absorbance 2	Absorbance 3	Mean
50	0.238	0.240	0.239	0.239

Annexure-4: Calibration Graph and Regression Output

Attach printed graph and Excel regression table showing slope, intercept, and R².

Annexure-5: Linearity Summary Sheet

Parameter	Value	Acceptance	Status
Correlation Coefficient (R²)	0.9996	≥ 0.995	Pass
%RSD (100%)	1.2%	≤ 2.0%	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Updated acceptance criteria and added regression plot section	Annual SOP Review